[Evaluation of the anti-neuraminidase antibodies in clinical trials of the live influenza vaccine of the A(H5N2) subtype].

In the current study, we evaluated the neuraminidase-inhibition (NI) antibodies among volunteers during the phase I and phase II of the clinical trials of a monovalent live attenuated influenza vaccine (LAIV) A/17/duck/ Potsdam/86/92(H5N2). The reassortant influenza virus RN2/57-human A(H7N2) containing neuraminidase (NA) from the A/Leningrad/134/17/57(H2N2) was used in NI test. It was shown that two doses of the monovalent LAIV A(H5N2) led to a statistically significant increase in the NI antibodies to vaccine strain NA. More than twofold increase in antibodies was obtained among 19.5-33.3% of vaccinated. The microneutralization test and NI assay results coincidence in the same pairs of sera of the vaccinated volunteers was 73.2%, suggesting thus a statistically significant interdependence between the values of increase in antibodies revealed in both tests (p = 0.04).

[Study of biological properties of cold-adapted reassortant strain of influenza virus subtype H7N3].

For the development of live attenuated influenza vaccine (LAIV) against influenza virus strains with pandemic potential, method of classic genetic reassortment of donor of attenuation A/Leningrad/134/17/57 (H2N2) [Len/17] with avian apathogenic influenza viruses of different subtypes was used. Strain with genome formula 6:2, which contains HA and NA genes from avian apathogenic virus A/wild duck/Netherlands/12/00 (H7N3) [N7N3-wt] and 6 other genes--from Len/17, was studied. Reassortant strain A/17/ wild duck/Netherlands/00/84 (H7N3) [Lenl7/ H7] exhibited ts- and ca- phenotype specific for cold-adapted strains. Reassortant was identical on antigenic profile to parent avian virus H7N3-wt. Like cold-adapted donor strain Len/17, Len17/H7 was attenuated for chickens, whereas wild-type parent strain was lethal in 60% of birds after its intravenous challenge. Reassortant strain Len17/H7 was attenuated during intranasal inoculation of 6 EID50 to white mice, which was confirmed by absence of its isolation from the lungs, actively reproduced on nasal mucosa and stimulated specific systemic and local antibody response.

[The inoculative properties of cold-adapted reassortant A(H5N2) influenza strain during intranasal administration to mice].

Classical genetic reassortant techniques were used to have a cold-adapted (ca) reassortant A/17/Duck/Potsdam/86/92 (H5N2) that inherited the hemagglutinin (HA) gene from the nonpathogenic avian virus A/Duck/Potsdam/ 1402-6186 (H5N2) and the genes of neuraminidase (NA) and non-glycated proteins from the ca attenuation donor A/Leningrad/134/17/57 (H2N2). All experiments were performed under increased biological protection (BSV-3+). The reassortant and parent H5N2 virus were non-pathogenic to Balb/c mice, the reassortant replication in the murine nasal passages (3.5 Ig EID50/ml) being higher than that in the lung (2.1 lg EID50/ml). Intranasal inoculation of mice with reassortant A/17/Duck/Potsdam/86/92 caused an immune response to both homological H5N2 virus and antigenically differing variants of influenza A (H5N1) virus isolated from humans in 1997 and 2003. The mice intranasally immunized with the ca reassortant were protected against fatal infection with the highly pathogenic A/Hong Kong/483/9797 (H5N1) virus and against infection with A/Hong Kong/213/03(H5N1) virus (80 and 100%, respectively).

[Growth characteristics of single gene mutants of A/Leningrad/134/57(H2N2) influenza virus and cold-adapted mutant A/Leningrad/134/17/57(H2N2)].

The authors examined a role of some mutated A/Leningrad/134/17/57(H2N2) virus genes in the realization of growth characteristics. The latter of single gene reassortants (SGRs) (PB2, PB1, PA, M, and NS), epidemic virus and attenuation donor were assessed by infecting MDCK cells and hen embryos at a low inoculation index. Viral replication in the hen embryos and cultured tissue was compared at 34 degrees C. The viruses and reassortants tested showed a high growth capacity in the hen embryos (9.5-10.5 Ig TCID50). The growth curves of viruses were studied on the cultured MDCK cells at a low inoculation index indicated that Len/17 and the single gene reassortants M and NS had the highest growth capacity. At the same time the growth of both PB1 and PB2 SGRs was less extensive. The reproduction of PB2 SGR was 100-1000 times less than that of other viruses tested. M, NS, and PA gene mutations did not affect viral growth in hen embryos and cultured tissue while PB2 gene mutation and its constellations with other genes caused a reduction in viral growth in the cultured tissue.

[Production of early cytokines after infection with A/Leningrad/134/57 (H2N2) wide-type virus and cold-adapted attenuated vaccine viruses].

The production of proinflammatory cytokines was studied following experimental infection of BALB/c mice with influenza viruses that differed in virulence. The generation of TNF-alpha, IL-6, IL-12, and IFN-gamma was investigated in the lung homogenates in the early periods after intranasal infection of mice with A/Leningrad/134/57 (H2N2) wild-type virus and cold-adapted attenuated vaccine viruses: A/Leningrad/134/17157 (H2N2) and A/Leningrad/134/47/57 (H2N2). Wild-type virus induced substantially higher levels of proinflammatory cytokines: TNF-alpha, IL-6, IL-12, and IFN-gamma. After infection with the cold-adapted viruses, the levels of the cytokines were reduced as compared to those induced by the wild-type virus. The A/Leningrad/134/47/57 virus was marked by a noticeable production of IL-6 and IFN-gamma in the murine lung, but it was less than with wild-type virus infection. At the same time, the more attenuated strain A/Leningrad/134/47/57 induced TNF-alpha and IFN-gamma in the quantities similar to those in the control animals. Thus, a response of proinflammatory cytokines in early infection in the murine lung depended on the level of viral replication in the lower respiratory tract and on the attenuation of influenza virus strains.

[Immune response to live influenza vaccine]. / Immunnyi otvet na zhivuiu groppoznuiu vaktsinu.

Priority data on the induction, by using a Russian live cold-adapted reassortant influenza vaccine (LIV), of the cellular and humoral immunity with regard for attenuation and genetic reassortment of vaccine stains as well as with regard for the age of vaccinated persons and the production of Th1 (IFNY, IL-2) and Th2 (IL-4) cytokine markers in vitro are presented. It was demonstrated in vivo that a pathogenic virus of the A group by far more actively induced the lymphocyte apoptosis as compared with attenuated genetically reassorted stains. Unlike the influenza pathogenic virus, the genetically attenuated and reassorted strain did not produce any negative effects on the induction of cellular immunity. A comparative study of the LIV immunogenic properties in vaccinated persons showed an advantage of LIV over inactivated influenza vaccine (IIV) in stimulating the cellular and local immunity in the elderly. Unlike IIV, LIV induced an active and balanced immune response developing due to Th1 and Th2 activation. LIV was found to stimulate well enough the production of IFN and IL-2 in both young and old persons.

[B-cell and cytotoxic lymphocyte immune response to virulent, attenuated and reassortant influenza viruses]. / B-kletochnyi i tsitotoksicheskii limfotsitarnyi immunnyi otvet na patogennye, attenuirovannye i reassortantnye virusy grippa.

The quantitative estimation of the production of the virus specific IgA-, IgM-, IgG1, IgG2a, IgG2b and IgG3-antibody secreting B-cells (ASC) and extent of the immune response of the cytotoxic T-lymphocytes was carried out in mice after primary and secondary immunization with the parental cold adapted (ca) virulent epidemic wild-type (wt) master strain viruses and their reassortant variant (RV) with the incorporated in their genom different genes from ca master strain. The chick embryo derived ca master strain virus A/Len/134/47/57 (H2N2) reduced or eliminated the viral potency to induce the primary B-cellular response. Reassortant incorporation of wt derived HA and NA genes into the ca virus genome restored the virus immunogenic activity concerning the ASC production, but at the lower level than parental virulent virus. Reassortance of the viruses according to generally accepted genom formula 6/2 was associated with decrement of the functional activity of the cytotoxic T-lymphocytes memory and of the primary immune response especially. The data obtain demonstrate the necessity for the control of the immunogenicity of the reassortant viruses at the cloning stage.

[In vitro proliferative activity of lymphocytes from elderly persons after separate and combined immunization with live and inactivated flu vaccines]. / Proliferativnaia aktivnost' limfotsitov lits pozhilogo vozrasta in vitro pri razdel'noi i sochetannoi immunizatsii zhivoi i inaktivirovannoi grippoznymi vaktsinami.

Cellular (lymphocyte proliferation activity--LPA), humoral (serum antibodies), and secretory (IgA antibodies from the upper respiratory tract) immune responses were compared in 45 subjects aged 66-95 years, vaccinated with two influenza trivalent A(H1N1) + A(H3N2) + B vaccines: Russian live attenuated cold-adapted reassortant (LIV) and USA inactivated split-virus (IIV) vaccines. None of immunization protocols suppressed LPA after in vitro stimulation of cell culture with homologous virus antigens and nonspecific polyclonal mitogen (PHA). Simultaneous LIV + IIV vaccination was the most effective method of immunization, inducing humoral, secretory, and cellular immunity. LIV more actively than IIV stimulated the lymphoproliferative immune responses. Fluctuations in the mean values of cellular, humoral, and secretory immunity were in good correlation over the entire period of observation (19 weeks). Analysis of individual immune responses showed that a significant increase in quantitative parameters of LPA was observed only in 39-52% vaccinees.

[The immunogenic properties and prophylactic efficacy of a live polyvalent influenza vaccine in children 5 to 14 years old]. / Izuchenie immunogennykh svoistv i profilakticheskoi éffektivnosti zhivoi grippoznoi polivalentnoi vaktsiny u detei 5--14 let.

Production lots of a live influenza vaccine made of strains A/47/T (N1H1), A/47/6/2 (H3N2), and B/60/32 were used for vaccination of 3663 children aged from 5 to 14 years inoculated twice with monovaccines, a trivaccine made of the above strains, or placebo. Both mono- and polyvaccine were practically areactogenic. An average per cent of subjects with a significant rise in antibody titres to the respective three antigens was 60%. The efficacy of the vaccination was 31.0-42.8% for monopreparations and 36.3% for the trivaccine. The studies showed the possibility and expedience of using for children the live influenza vaccine in the form of a polyvalent preparation including current influenza type A and B viruses.

[Local immune response to live cold-adapted reassortant influenza vaccine in children, adults, and aged people]. / Mestnyi immunnyi otvet na zhivuiu kholodoadaptirvannuiu reassortantnuiu grippoznuiu vaktsinu u detei, vzroslykh i predstarelykh lits.

A study was conducted to compare the production of the serum and local IgA-antibodies in persons of different age groups (aged: 3-6, 7-14, 18-30, 65-89) after a single intranasal immunization with trivalent live cold-adapted reassortant of influenza vaccine (LIV). The geometric mean of titers of local IgA-antibodies increased, during post-vaccination period, against influenza viruses A(H1N1), A(H3N2) and B as much as people's age went up. It is noteworthy, that the parameters of the young and elderly did not virtually differ. As for the children, aged 3-6 and especially 7-14, an active local immune response developed in them to the LIV administration. Thus, no pronounced age-related immunologic insufficiency was found in children, aged 3-14, or in the elderly above 65 to the induced local response caused by LIV.

[Humoral and local immune response to the influenza vaccine in people of old and young ages]. / Gumoral'ny i mestnyi otvet na grippoznye vaktsiny u lits pozhilogo i molodogo vozrasta.

The specific features of the humoral and local immune responses to influenza vaccines were comparatively studied in people of different age groups. A total of 79 elderly people (aged 67-89) and 80 young people (aged 18-27) were immunized according to one of the four schemes: live cold-adapted reassortant trivalent influenza vaccine (LIV), administered intranasally; inactivated split trivalent influenza vaccine (IIV), administered parenterally; a combination of both above vaccines; and placebo. IIV was found, as compared to LIV, to stimulate more effectively the production of circulating antihaemagglutinins as well as of IgG,-, Ig1-, and Ig3-AT in young persons, while LIV has advantages before IIV in stimulating the synthesis of these immunoglobulins in the elderly. LIV has advantages before IIV in stimulating the synthesis of secretory IgA-AT irrespective of an age of the immunized persons. The combined immunization of the elderly by both vaccines increases the quantitative parameters of the humoral and local responses up to the level of intensity observed in young people. The obtained data are indicative of the possibility of correcting the immune response in the high-risk elderly in respect to influenza infection.

[Formation of humoral and secretory immunity in elderly persons with different schemes of immunization with flu vaccines]. / Formirovanie gumoral'nogo i sekretornogo immuniteta u lits pozhilogo vozrasta pri razlichnykh skhemakh immunizatsii grippoznymi vaktsinami.

The immunological efficacy of 5 protocols of immunization with two influenza vaccines are compared in 168 elderly subjects aged 64 to 87 years. Russian live cold-adapted reassortant trivalent (LCIV) and American inactivated cleaved trivalent (ICIV) influenza vaccines were used. The protocols of vaccination were as follows: 1) simultaneous vaccination with LCIV and ICIV and revaccination with LCIV after 1 months; 2) simultaneous vaccination with LCIV and ICIV and revaccination with placebo after 1 month; 3) vaccination and revaccination with LCIV; 4) vaccination with ICIV and revaccination with placebo; 5) vaccination and revaccination with placebo preparation (control); 6) vaccination with ICIV and revaccination with LCIV after 1 month. The incidence of significant increments and intensity of accumulation of serum (assessed by the hemagglutination inhibition test) and secretory (IgA) antibodies (assessed by enzyme immunoassay) was evaluated. For elderly subjects, simultancous vaccination with LCIV and ICIV followed by revaccination with LCIV is the most effective. After such vaccinations both secretory and humoral immune responses are characterized by the highest production of secretory IgA and serum antibodies. The quantitative parameters of both types of immune response in elderly subjects thus immunized are much higher than in young subjects vaccinated traditionally, that is, by LCIV or ICIV alone.

[Production of interleukin-2 in vitro and in vivo in elderly people, vaccinated with live and inactivated flu vaccines separately and combined]. / Produktsiia interleikina-2 in vitro i in vivo u pozhilykh liudei, privitykh razdel'no i sochetanno zhivoi i inaktivirovannoi grippoznymi vaktsinami.

Forty-three elderly individuals were immunized with Russian trivalent live cold-adapted influenza vaccine (LIV) and US trivalent influenza vaccine (IIV) administered separately or in combination. IL-2 production in vitro (in supernatants of cultures of lymphocytes stimulated with homologous viral antigens and PHA) and in vivo (in blood serum) and other factors of specific antiinfluenza immunity were compared. Vaccination of elderly subjects with commercial vaccines induced T-helper immunological memory, which manifests by increased secretion of IL-2 in vitro and in vivo. Simultaneous vaccination with LIV + IIV and revaccination (in 1 month) with LIV was the most effective method stimulating IL-2 production. The levels of IL-2 production in vitro were in good correlation with the secretion of this cytokin in vivo, lymph proliferation, and serum antibody production. No correlation between IL-2 production in vitro and the formation of local immune response (IgA in nasal swabs) was detected.

[Immune response of elderly persons depending on the number of annual seasonal immunizations of live and inactivated influenza vaccines]. / Immunnyi otvet lits pozhilogo vozrasta v zavisimosti ot chisla ezhgodnykh sezonnykh immunizatsii zhivoi i inaktivirovannoi grippoznymi vaktsinami.

A total of 159 subjects aged 65-87 years were immunized with live cold-adapted reassortant influenza vaccine (CRIV), inactivated influenza vaccine (IIV), and with both vaccines (CRIV + IIV) one year, two and three years running. The frequency and intensity of accumulation of postvaccinal secretory and humoral antibodies in elderly subjects depend on the scheme of immunization and history of vaccinations. Combination of the two vaccines effectively stimulated both components of immunity and ensured a longer persistence of postvaccinal antibodies in high concentrations. Immunization with CRIV + IIV for three years resulted in a gradual increase of the intensity of prevaccination secretory and humoral immunity. Before the third seasonal immunization the majority (63-75%) of vaccinees had antibodies in protective titers.

[The comparative characteristics of the safety, immunogenic activity and prophylactic potency of the adult and children types of live influenza vaccine in schoolchildren aged 7-14 years]. / Sravnitel'naia otsenka bezvrednosti, immunogennoi aktivnosti i profilakticheskoi éffektivnosti vzroslogo i detskogo variantov zhivoi grippoznoi vaktsiny u shkol'nikov 7-14 let.

In Russia for prevention of influenza in children, aged from 3 to 14 years, the children's live influenza vaccine (LIV), based upon A/Leningrad/134/47/57(H2N2) master strain (LIVI) is used. The need for double immunization appears to be one out of the faulties of this preparation. The study was aimed to comparing the safety, immunogenic activity and prevention of influenza by LIV for adults (LIVII) (A/Leningrad/134/17/57(H2N2 master strain) and LIVI in children aged from 7 to 17 years under similar administration schedule. The safety, the preventive efficacy, humoral and secretory immunity were studied. In total 2486 persons, including 539 children, twice inoculated with LIVI, 971 persons once inoculated with LIVII, and 840 treated by placebo were obserbed. From the data of the clinical observations during 7 days after immunization both vaccines appeared to be low reactogenic. The LIVII advantages in induction of the humoral and secretory antibodies in comparison with children's vaccine had been revealed. Both vaccines were highly efficacious, the efficiency of both preparations was more pronounced after serologic correction of the diagnosis. The results obtained permit to recommend the single immunization by the variant of LIV at the base on A/Leningrad/134/17/57/(H2N2) master strain for prevention of influenza in school children.

[The investigation of the safety, genetic stability and immunogenicity of live influenza vaccine for adults in vaccination of 3-6 years old children]. / Izuchenie bezvrednosti, geneticheskoi stabil'nosti i immunogennosti zhivoi grippoznoi vaktsiny dlia vzroslykh pri vaktsinatsii detei 3-6 let.

The study of the based on the A/Leningrad/134/17/57/(H2N2) attenuated adult live influenza vaccine (LIV) investigated features for immunization of the children, aged 3-6 years. During autumn, 1999, out of 256 children, aged 3-6 years, residents of the Leningrad region, who attended the kindergarten, 184 children were immunized with 1 or 2 doses of the live influenza vaccine, and 72 ones were given placebo. There were no any moderate or strong temperature reactions revealed after the inoculation. The LIV was shown to be genetically stable. After a single dose of the vaccine seroconversion to influenza type A virus and to influenza type B virus was observed respectively in 58% and in 39% of seronegative 3-6 year old vaccinees. The twofold LIV administration failed to give any advantages in stimulation of the immune response. During 6 months after immunization the morbidity rate in vaccinees did not exceed the morbidity rate in unvaccinated children. Thus LIV for adults proved safe and immunogenic and can be recommended for single dose immunization both of adults and children.

Characterization of an influenza A H5N2 reassortant as a candidate for live-attenuated and inactivated vaccines against highly pathogenic H5N1 viruses with pandemic potential.

We generated a high-growth 7:1 reassortant (Len17/H5) that contained the hemagglutinin (HA) gene from non-pathogenic A/Duck/Potsdam/1402-6/86 (H5N2) virus and other genes from the cold-adapted (ca) attenuated A/Leningrad/134/17/57 (H2H2) strain. Len17/H5 demonstrated an attenuated phenotype in mice and did not infect chickens. Mice administered Len17/H5 either as a live-attenuated intranasal vaccine or as an inactivated intramuscular vaccine were substantially protected from lethal challenge with highly pathogenic A/Hong Kong/483/97 (H5N1) virus and were protected from pulmonary infection with antigenically distinct A/Hong Kong/213/2003 (H5N1) virus. The cross-protective effect correlated with the levels of virus-specific mucosal IgA and/or serum IgG antibodies. Our results suggest a new strategy of using classical genetic reassortment between a high-growth ca H2N2 strain and antigenically related non-pathogenic avian viruses to prepare live-attenuated and inactivated vaccines for influenza pandemic.

Efficacy of live attenuated and inactivated influenza vaccines in schoolchildren and their unvaccinated contacts in Novgorod, Russia.

Children aged 7-14 years in Novgorod, Russia, were given Russian live cold-adapted or inactivated influenza vaccines or placebo over a 2-year period. Schools were randomly assigned as a whole to one of the preparations. In the first year, the vaccines were bivalent, containing types A (H3N2) and A (H1N1) components. In the second year, the vaccines also contained a type B component. In the first year, all viruses isolated were type A (H3N2); in the second, about three-quarters of the isolates were type B and the rest type A (H1N1). During both years, the vaccines protected the vaccinated children. Where significant differences existed, the live attenuated vaccine was more protective than the inactivated. Vaccination rates in schools in which live attenuated vaccines had been used were inversely related to illness rates of staff and unvaccinated children, suggesting that viral transmission had been reduced by the vaccine.