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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.

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.

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.

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.

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.

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.

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.

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 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.

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 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.

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.

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.

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.

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 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 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.

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.

The functional activity of serum antibody had been shown previously to reflect the state of resistance to influenza more accurately than antibody concentration. The functional activity of antibodies in the vaccination process was studied. The immune response to influenza antigens was found to be accompanied by an increase in the functional activity of antibodies, the intensity of this increase was the highest in natural influenza infection, moderate after administration of live vaccines and the least after immunization with inactivated preparations. The vaccine strains may differ significantly in their capacity to stimulate antibody with high functional activity. The quantitative parameters of the functional activity of antibody are directly related to the antigen dose and frequency of antigenic stimulation.

In this work the reactogenic properties and antigenic potency of inactivated trivalent influenza vaccine, obtained by elution and centrifugation and containing up to 9-11 micrograms of hemagglutinin for influenza viruses A(H1N1) and A(H3N2) and up to 14 micrograms for influenza virus B, were studied. The reactogenicity of the preparation was found to correspond to the regulations. The immunogenic potency characteristics of individual batches of this trivaccine were higher than the immunogenicity of divaccines, but did not meet the requirements of technical specifications.

Indices of the functional activity of specific serum antibodies (IFAA) before and after outbreaks of influenza A (H3N2) and B were determined in 164 subjects of 18-20 years of age from the military communities observed in 1987-1988. The IFAA were calculated on the basis of kinetic HI tests followed by computer processing according to the program developed by the authors. The IFAA were found to indicate the protection of human subjects against influenza, reflecting the state of resistance to influenza more exactly than antibody titres. High initial IFAA correlated with lower severity of influenza infection. The IFAA to the same strain may differ significantly in individual subjects. In patients with influenza the time course of antibody titres did not coincide with dynamic changes in IFAA.

Parallel HI and virus-elution-from-erythrocytes-inhibition (a simplified method for titration of neuraminidase antibody) tests were used for examinations of 1117 blood serum specimens from 440 adults and children under study, 5250 single serum specimens from healthy subjects from birth to 65 years of age, 38 paired serum specimens from children who experienced influenza A/Texas/1/77 disease in the epidemic of 1979-1980, and 590 paired serum specimens from subjects immunized with influenza vaccines. In 7%-23% of influenza patients and immunized subjects antibody rise was observed to only one of the influenza A virus surface antigens, hemagglutinin or neuraminidase. The protective activity of antibody to influenza A virus neuraminidase was as good as that of antihemagglutinins. Both kinds of antibody interacted in protection against the disease. Antineuraminidase antibody was found to affect the decrease in severity of the infectious process in natural infection with influenza A. The formation of immunological memory in the system of synthesis of antihemagglutinins and antineuraminidase antibodies was shown to have features in common. The pattern of heterologous immune responses in immunized subjects and patients with influenza showed all antigenic varieties of neuraminidase N2 as well as neuraminidases N1 and N2 to share common cross-reacting determinants.

The work presents the evaluation of the effectiveness of mass immunization with inactivated chromatographic vaccine carried out in Leningrad during the influenza A (H3N2) epidemic of 1979--1980. These prophylactic measures were shown to cause a statistically significant decrease in morbidity rate among the vaccinees and the whole population (morbidity rate decreased 1.41 times among the whole population, 1.99 times among persons aged 8 years and over and 3.1 times among the vaccinees at industrial enterprises). The economic effect of 8.4 million roubles was achieved. To evaluate the apidemiological effectiveness of the above-mentioned antiepidemic measures, an original complex of techniques has been developed; such techniques can also be used for the evaluation of other similar measures.

The time course of the levels of circulating antihemagglutinins to influenza A (H1N1) virus in the populations of the towns in different geographical zones of the USSR was shown to be similar. The intensity of herd immunity in some areas differed considerably, however. The population of older age groups most of whom had no clinically manifest diseases during the influenza A (H1N1) epidemic was shown to experience immunological changes upon exposure to this agent. The intensity and the rate of changes in the immunological structure of this portion of the human population exceeded similar parameters among the subjects under 20 years of age who had experienced manifest forms of infection. Some people with immunological memory to influenza A (H1N1) virus developed antibody in response to infection or immunization with influenza A (H3N2) strains.

In 5 large cities of the USSR a four-year study of the population immunity to 3 antigens of influenza A virus (hemagglutinin, neuraminidase and ribonucleoprotein antigens) was carried out. The time course of immunity to these antigens had a similar character. The high level of population immunity to actual hemagglutinin and neuraminidase, observed for 2 years, in succession, indicated the possibility of shift-life changes in these surface antigens. Population immunity to ribonucleoproteid antigen had no influence on the epidemic process in influenza A. A method for the study of population immunity, based on the analysis of pooled serum samples from healthy persons, is proposed. This method considerably reduces the time of the test and the number of ingredients.

The capacity of live influenza type A (H3N2) vaccines to produce antihemagglutinins and antineuraminidase antibody to drift variants of a given serosubtype emerging later than the vaccine strain was studied. For this purpose, a wider set of antigens was used to examine retrospectively by the HI and virus elution from erythrocyte inhibition tests the paired sera from the subjects immunized in 1975 and 1976 with live vaccine virus strains similar to A/Port Chalmers/1/73 (H3N2) and A/Victoria/3/75. These vaccines were shown to actively stimulate antibody production in titres of 1:40 or higher to strains forestolling the vaccine strain by 1 (antihemagglutinins) and 2 (antineuraminidase antibody) degrees of the antigenic hierarchy. The intensity of production of both kinds of antibody to similar future strains depended on the intensity of immune response to the vaccine virus. By increasing the dose and frequency of administration of the virus serosubtype A (H3N2) to animals it was possible to intensify the production of antihemagglutinins and antineuraminidase antibodies to later drift variants of this agent with respect to the virus-immunogen. Volunteers immunized in 1983 with a commercial inactivated chromatographic bivaccine prepared from the strains similar to A/Bangkok/1/79 (H3N2) and A/Brazil/14/78 (H1N1) were found to intensively produce antihemagglutinins in titres of 1:40 or higher to viruses A/Philippines/2/84 (H3N2), A/Leningrad/167/83 (H3N2), A/Leningrad/3/82 (H1N1) but not to A/Dunedin/27/83 (H1N1) virus.

The spectrum of antibody production against hemagglutinin of all known influenza A virus serotypes after influenza A (H3N2) infection in persons of all age groups (from 1 month to 72 years) was analysed to investigated the regularities of accumulation of antibodies against influenza A viruses gone out of circulation. For the same purpose, a long-term (many years) longitudinal analysis of serum anti-HA antibody levels against "old" influenza A (HSW1N1), A (H0N1), A (H1N1), A (H2N2), and A (H3N2) viruses was carried out in children and adults of various age groups in 6 cities of different geographical regions of the country. Based on the results of the study, the concept of the "original antigenic sin" is analysed critically, and a new original concept on the regularities of antibody genesis against previously prevalent influenza A viruses and the factors affecting this process has been formulated.

Immunological investigations of the time course of serum anti-HA and anti-NA antibodies against influenza A and B viruses in the female and male population with blood groups 0(I), A(II), B(III), and AB(IV) were carried out for several months. The persons with the blood group AB (IV) were shown to be most sensitive to influenza A and B. They were affected by the epidemic virus earlier and more severely than those with the other blood groups. A special method is proposed for prediction of the etiology of the forthcoming epidemic. A correlation between blood groups and titres of anti-HA but not anti-NA antibodies was observed. The view on the lack of the genetic factor influence on the affliction with influenza and antibody response in persons of different sexes is substantiated.

The features of production and protective properties of antineuraminidase antibodies in subjects of various ages with influenza infection caused by currently important influenza A virus strains of H1N1 and H3N2 serosubtypes were studied. In children, antineuraminidase antibodies were found to accumulate less intensively than in adults and in infants under 1 year they were detected extremely rarely. In young infants, their titres after the disease decreased more rapidly than antihemagglutinin titres. As compared with adults, in younger children these antibodies have a higher protective role in modifying the severity of influenza infection. A direct correlation between the age of the children and the levels of antineuraminidase antibodies demonstrable in them was established. Within the same period of time, ratio of antihemagglutinins and antineuraminidase antibodies was shown to differ significantly in the population of different regions of the country. The results of trials in volunteers of 10 strains as candidates for a live influenza type A vaccine (H3N2) indicate considerable variability of the immunogenic potency of hemagglutinin and neuraminidase of these strains.

Paired specimens of blood sera, upper respiratory tract secretions, and saliva from healthy, influenza-infected, and live influenza trivaccine-immunized humans were tested in parallel by HI tests and enzyme immunoassay (EIA). The resulting comparative data suggest that determination by EIA of specific secretory IgA in the saliva may be recommended as an independent test for large-scale influenza diagnosis, evaluation of the immune status of people, and trials of influenza vaccines. By immunologic parameters, this method is not inferior to the standard ones. At the same time its advantages include simplicity and readily available large volumes of saliva even from children as well as no necessity to bleed subjects to be examined.