[Effect of Point Mutations in the Polymerase Genes of the Influenza A/PR/8/34 (H1N1) Virus on the Immune Response in a Mouse Model].

The vaccine strains for live attenuated influenza vaccines (LAIVs) have cold-adapted, temperature-sensitive, and attenuated phenotypes, which are guaranteed by the presence of specific mutations from the master donor virus in their internal genes. In this study, we used mutant viruses of the pathogenic A/Puerto Rico/8/34 (H1N1) that contained ts-mutations in PB1 (K265N, V591I), PB2 (V478L), and PA (L28P, V341L) genes along and/or in different combinations to evaluate the impact of these mutations in the immune responses. Sequential addition of tested mutations resulted in the stepwise decrease in virus-specific serum and, to a lesser extent, mucosal antibody levels. We demonstrated strong positive correlation between virus attenuation (virus titer in lung) and antibody titers. The ts-mutations in PB1, PB2, and PA genes are mostly involved in the modulation of the humoral immunity, but also have a moderate effect on the cellular adaptive immune response.

[Local antibody immune responses in influenza patients and persons vaccinated with seasonal, pre-pandemic, and pandemic live attenuated influenza vaccines].

Mucosal immunity is one of the most important factors of human anti-influenza defense. The data about local immune responses in influenza A (H3N2) patients and in persons vaccinated within 2000-2009 with different seasonal LAIVs, A (H1N1)pdm2009 LAIV, and A (H5N2) LAIV are discussed. The influenza infection resulted in the larger quantities of local IgA and IgG conversions than seasonal LAIV vaccination. 56% of young (18-21 y.o.) persons had high titers (> or = 1:64) of IgA to A (H1N1)pdm2009 virus before its circulation. 19% of persons had anti A (H5N2) IgA before vaccination. Two-fold vaccination with A (H1N1) pdm2009 and A (H5N2) LAIVs resulted in local antibody conversions in 54% and 27% of volunteers, respectively. Both these vaccines increased local IgA avidity. The number of antibody conversions after vaccination with seasonal LAIVs was in inverse dependence on their titers before vaccination. These results make it possible to conclude that the intensity of local antibody immune response to any LAIV depends on the state of local immunological memory, particularly on the presence of the crossreactive antibody-secreting B cells.

[Humoral and cell-mediated immune responses in humans to the A/California/07/ 2009 (H1N1) virus, A(H1N1)pdm2009].

During the twentieth century the world faced four influenza A pandemics: A (H1N1) in 1918, A (H2N2) in 1957, A (H3N2) in 1957 and A (H1N1) recirculation in 1977. In the beginning of 2009 the global spread of A(H1N1)pdm2009 virus was detected. In consideration of clinical evidences and genetic data analysis WHO declared as the novel pandemic of 21th century. However, the fact of exceedingly prolonged previous worldwide circulation of A (H1N1) influenza viruses was not taken into account. Further development showed epidemiological prognosis not to be accurate enough. The present work is an attempt to analyze this question from the immunological standpoint based on our studies of antibody and cellular immunity to A(H1N1)pdm2009 virus in vaccinated and non-vaccinated persons of different ages. The study results allow concluding that A(H1N1)pdm2009 is the drift variant of A (H1N1) viruses antigenically close to A/Swine/1976/1931 (H1N1). It was shown that the significant of persons have cross-reactive B and T cell immunological memory to A(H1N1)pdm2009 strain. This could be a reason of decreased A(H1N1)pdm2009 pandemic severity.

[Stimulation of homo- and heterologic T-cell immunological memory in volunteers inoculated with live influenza A (H5N2) reassortant vaccine].

The study deals with the ability of live attenuated reassortant influenza vaccine (LAIV) A (H5N2) to stimulate a CD4+ and CD8+ immunological memory T cell-mediated immune response in volunteers. These data were compared with the quantitative characteristics of a humoral immune response. A two-dose regimen of intranasal vaccination of avian influenza naïve people with A (H5N2) LAIV induced the production of circulating CD4+ and CD8+ memory cells specific to both A (H5N2) and seasonal A (H1N1) influenza strains. Some of the volunteers were not absolutely A (H5N2) influenza virus naïve since they had been found to have this virus-specific cross-reactive immunological memory T-cells in the prevaccination period. The content (%) of these cells varied significantly within the group. The quantitative values of postvaccination CD4+ and CD8+ memory cell accumulation were inversely related to their prevaccination level.

[Estimation of human and animal immunological memory by testing the trogocytosis of virus-specific T lymphocytes].

This study is the first attempt to evaluate the immunogenicity of Russian live attenuated influenza reassortant influenza vaccine (LAIV), by using a modified T-cell recognition of antigen presenting cells by protein capture (TRAP) method. Single vaccination of 18-20-year-old volunteers with LAIV causes an increase in the peripheral blood levels of virus-specific memory CD4+ T lymphocytes. Some (40-60%) LAIV-vaccination volunteers respond to immunization by showing a significant elevation in the peripheral blood level of memory CD4+ T cells without a systemic humoral immune response recorded in the passive hemagglutination test. Vaccination of mice with live attenuated A (H1N1) influenza reassortant virus stimulates the production of memory CD8+CD44hi T lymphocytes in the nasal-associated lymphoid tissue, the entry of infection, so does influenza infection. Vaccination with inactivated A (H1N1) influenza virus practically fails to induce these cells. A (H1N1) influenza virus-specific CD8+CD44hi T lymphocytes remain within at least 2 months (observation time). The authors' modified TRAP may be used to evaluate virus-specific immunological T-cell memory after vaccination.

Memory T-cell immune response in healthy young adults vaccinated with live attenuated influenza A (H5N2) vaccine.

Cellular immune responses of both CD4 and CD8 memory/effector T cells were evaluated in healthy young adults who received two doses of live attenuated influenza A (H5N2) vaccine. The vaccine was developed by reassortment of nonpathogenic avian A/Duck/Potsdam/1402-6/68 (H5N2) and cold-adapted A/Leningrad/134/17/57 (H2N2) viruses. T-cell responses were measured by standard methods of intracellular cytokine staining of gamma interferon (IFN-γ)-producing cells and a novel T-cell recognition of antigen-presenting cells by protein capture (TRAP) assay based on the trogocytosis phenomenon, namely, plasma membrane exchange between interacting immune cells. TRAP enables the detection of activated trogocytosis-positive T cells after virus stimulation. We showed that two doses of live attenuated influenza A (H5N2) vaccine promoted both CD4 and CD8 T-memory-cell responses in peripheral blood of healthy young subjects in the clinical study. Significant differences in geometric mean titers (GMTs) of influenza A (H5N2)-specific IFN-γ(+) cells were observed at day 42 following the second vaccination, while peak levels of trogocytosis(+) T cells were detected earlier, on the 21st day after the second vaccination. The inverse correlation of baseline levels compared to postvaccine fold changes in GMTs of influenza-specific CD4 and CD8 T cells demonstrated that baseline levels of these specific cells could be considered a predictive factor of vaccine immunogenicity.

[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.

[The temperature sensitivity of epidemic influenza A viruses as a marker of immunogenicity of reassortant vaccinal strains]. / Temperaturochuvstvitel'nost' épidemicheskikh virusov grippa A kak vozmozhnyi marker immunogennosti reassortantnykh vaktsinnykh shtammov.

The influence of ts-phenotype of epidemic viruses and of cold-adapted (CA) reassortant vaccines' strains, appropriately prepared, produced on the human immunogenicity was under investigation. A widespread variability of epidemic viruses' thermal sensitivity sign was established. It was shown that the CA reassortant vaccine strains, obtained through crossbreeding of attenuation donors and of thermally resistant epidemic viruses, are described by a higher immunogenicity. Therefore, the immunogenicity of live influenza vaccines (LIV) can be defined by the ts-phenotype of epidemic parent viruses, which must be sampled for the reassortant vaccine strains not only through searching for samples of antigenically actual viruses but also through search for non-ts-phenotype viruses.

[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.

[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.

[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.

[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.

[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.

[Immunoenzyme analysis of post-vaccination secretory immunity to influenza A and B viruses using a manufactured monoclonal immunoenzyme test system]. / Izuchenie v immunofermentnom analize postvaktsinal'nogo sekretornogo immuniteta k virusam grippa A i B s pomoshch'iu razrabotannoi monoklonal'noi immunofermentnoi test-sistemy.

The formation of postvaccinal secretory immunity to influenza A and B viruses was studied by a new monoclonal enzyme immunoassay test system for measuring specific secretory IgA in young people vaccinated with live cold-adapted vaccines (LCAV) intranasally and with inactivated commercial centrifuged influenza vaccine (IIV) parenterally, intranasally, and orally. Secretory IgA most intensively accumulated in subjects intranasally vaccinated with LCAV, less so in subjects vaccinated with IIV intranasally and orally, and just negligibly in those vaccinated with IIV parenterally. In vaccinees immunized with LCAV intranasally the intensity of immune secretory response depended on the initial concentrations of specific IgA before vaccination. Intranasal administration of LCAV in the presence of high concentrations of secretory IgA led in some subjects either to a decrease in the incidence of conversions or to a 2-8-fold drop of their initial titers. Parenteral injection of IIV caused the most expressed suppression of the immune response in the secretory immunity system. Use of biological stimulant adaptogen increased 2.2 times the incidence of conversions of secretory IgA in subjects intranasally vaccinated with LCAV.

[Monoclonal immunoenzyme test-system for evaluating secretory immunity to influenza A and B viruses]. / Monoklonal'naia immunofermentnaia test-sistema dlia otsenki sekretornogo immuniteta k virusam grippa A i B.

Enzyme immunoassay system has been developed for measuring the titers of secretory IgA in secretion from the upper respiratory airways (SURA). Russian ingredients are used. The conjugate includes monoclonal antibodies to heavy chains of human secretory IgA. The sensitivity and specificity of the kit was tested with SURA of normal subjects and patients with influenza. The initial level of specific secretory IgA antibodies detected by the kit clearly correlated with defense from natural influenza infection. The protective concentrations of the antibodies corresponded to at least 1:64 dilutions. The titers of serum and secretory antibodies in convalescents after influenza A or B were virtually the same. The level of secretory antibodies dropped sooner than that of serum antibodies.