[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.

[The impact of conservative and hypervariable immunodominant epitopes in internal proteins of the influenza A virus on cytotoxic T-cell immune responses].

The cytotoxic T-cell immune response plays an important role in the prevention of influenza infection and reducing of the illness severity. The knowledge about mechanisms of the virus-specific CD8+ T-cell induction in humans is necessary for better understanding of influenza epidemiology and vaccine development. Due to application of new immunological and genetic methods in last years, considerable amount of.data became available in the literature about CD8+ T-cell immune responses to different influenza A viruses. This review summarizes these data. The main attention is paid to (i) heterosubtypic CTL responses to conservative immunodominant sites; (ii) mechanisms of viral escape from the virus-specific CTLs by means of evolutional escape-mutations; (iii) influence of the HLA haplotype on CD8+ T-cell immune responses. The importance of these data for immunology and vaccinology is discussed.

[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.

[Heterosubtypic immunity to influenza A viruses: epidemiological data, involvement of different immunological factors, vaccination].

The fact that the spread of new pandemic influenza A strains poses a constant threat to public health attracts the particular attention of scientists to heterosubtypic immunity (HIS) against different subtypes of this pathogen. This review summarizes data from the world scientific literature on studies of HIS. It presents HIS-related data on the epidemiology of influenza, immunity factors, and groundwork for the design of a universal influenza vaccine.

[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.

[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.

[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.

[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.

[Specific immune response to vaccination with an inactivated flue vaccine depending on prevaccine status and age of the person vaccinated]. / Spetsificheskii immunnyi otvet na vaktsinatsiiu inaktivirovannoi grippoznoi vaktsinoi v zavisimosti ot prevaktsinal'nogo statusa i vozrasta vaktsiniruemykh.

Specific antibody immune response to vaccination with commercial inactivated trivalent vaccine A(H1N1) + A(H3N2) + B (IgA, IgG, IgG, subclasses G1, G3, G4, and accumulation of antiCD8) was studied in subjects aged 20-95 years. The initial immune status before vaccination is significant for a positive immune response to the vaccine. Subjects responding to immunization by an increment in specific IgG had a much lower prevaccination level of these antibodies than subjects without these Ig conversion. Antibody immune response to vaccination depended on patient's age. All vaccinees aged 20-25 years developed an increment in IgG to at least one of influenza antigens used. Specific postvaccinal immune response to inactivated influenza vaccine included accumulation of G1, G3, and A antibodies, but not G4 or E antibodies. This latter fact suggests the absence of sensitizing effect of vaccination. In elderly subjects an increment in G1, G3, and A antibodies may not involve an increase in the total level of IgG. In part of elderly subjects secretion of specific antibodies was observed in the presence of increased concentration of antilymphocytic antibodies (antiCD8), indicating a possibility of autoimmune reactions in subjects of this age after injection of inactivated influenza vaccine.

[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.

[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.

[The immunoprophylaxis of influenza among elderly persons]. / Immunoprofilaktika grippa sredi liudei pozhilogo vozrasta.

During three seasons at the period of 1992-1996 immunization of elderly persons, living in homes for old people, against influenza with inactivated influenza vaccine (IIV) was carried out. Altogether 856 persons were immunized intranasally, 581 persons constituting the control group. For comparison, 4,825 healthy young adults aged 18-24 years were immunized under similar conditions. The study revealed that the intranasal immunization of elderly persons with IIV, made in two administrations, was safe and stimulated sufficient humoral and secretory immunity: the level of seroconversions was 24.3-41.0% to type A(H1N1) influenza virus, 29.6-50.7% to type A(H3N2) influenza virus, 39.3-59.6% to type B influenza virus; the level of diagnostic IgA conversions was 31-38%. Immunization produced a pronounced prophylactic effect (the effectiveness index 1.6-1.7), as well as decreased the total mortality level by half. The tactics of the immunization of persons from high risk groups against influenza in medical practice is discussed.