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This study addresses a current problem of vaccinal prevention—the development of approaches to increase the immunogenicity of influenza vaccines—and it is directed at studying the effect of mutations responsible for the degree of attenuation of influenza viruses on the formation of an immune response. We conducted the analysis of the humoral and cellular immune response in mice with the introduction of strains of the influenza A virus containing single and combined point mutations alike typical for the attenuation donor A/Leningrad/134/17/57 (H2N2), which is used at present for the preparation of domestic live influenza vaccine. In the study, 13 mutant strains were compared to the attenuation donor (containing all these mutations) and its “wild” predecessor (without mutations). It has been shown that the presence in internal genes of the “wild” virus of single mutations that are typical for the attenuation donor, as well as their combinations, can affect not only the quantitative indices of the humoral immune response, but also the rate of accumulation of virus-specific serum and secretory antibodies. A group of viruses with mutations in the M1 gene was isolated based on the ability to stimulate T cell response. A promising approach to development of ways to increase the immunogenic properties of live influenza vaccines will be a further search for a balanced combination of mutations in M1 and NS2 genes that enhance the stimulation of most of the immuneresponse factors studied, with mutations in genes of polymerase complex that impart attenuating properties to strains.

During the past decade, a number of H5 subtype influenza vaccines have been developed and tested in clinical trials, but most of them induced poor serum antibody responses prompting the evaluation of novel vaccination approaches. One of the most promising ones is a "prime-boost" strategy, which could result in the induction of prompt and robust immune responses to a booster influenza vaccine following priming with homologous or heterologous vaccine strains. In our study we evaluated immunogenicity of an adjuvanted A(H5N1) inactivated influenza vaccine (IIV) in healthy adult subjects who received A(H5N2) live attenuated influenza vaccine (LAIV) 1.5 years earlier and compared this with a group of naïve subjects. We found that priming with A(H5N2) LAIV induced a long-lasting B-cell immunological memory against influenza A(H5N1) virus, which was brought on by more prompt and vigorous antibody production to a single dose of A(H5N1) IIV in the primed group, compared to the naïve controls. Thus, by day 28 after the first booster dose, the hemagglutination inhibition and neutralizing (MN) antibody titer rises were 17.2 and 30.8 in the primed group, compared to 2.3 and 8.0 in the control group, respectively. The majority (79%) of the primed individuals achieved seroprotective MN antibody titers at 7 days after the first dose of the IIV. All LAIV-primed volunteers had MN titers ≥ 1:40 by Day 28 after one dose of IIV, whereas only 58% subjects from the naïve control group developed similar immune responses at this time point. The second A(H5N1) IIV dose did not increase the immune response in the LAIV-primed group, whereas 2 doses of IIV were required for naïve volunteers to develop significant immune responses. These findings were of special significance since Russian-based LAIV technology has been licensed to WHO, through whom the vaccine has been provided to vaccine manufacturers in India, China and Thailand - countries particularly vulnerable to a pandemic influenza. The results of our study will be useful to inform the development of vaccination strategies in these countries in the event of a pandemic.

H2N2 influenza viruses have not circulated in the human population since 1968, but they are still being regularly detected in the animal reservoir, suggesting their high pandemic potential. To prepare for a possible H2N2 pandemic, a number of H2N2 vaccine candidates have been generated and tested in preclinical and clinical studies. Here we describe the results of a randomized, double-blind placebo-controlled phase 1 clinical trial of an H2N2 live attenuated influenza vaccine (LAIV) candidate prepared from a human influenza virus isolated in 1966. The vaccine candidate was safe and well-tolerated by healthy adults, and did not cause serious adverse events or an increased rate of moderate or severe reactogenicities. The H2N2 vaccine virus was infectious for Humans. It was shed by 78.6% and 74.1% volunteers after the first and second dose, respectively, most probably due to the human origin of the virus. Importantly, no vaccine virus transmission to unvaccinated subjects was detected during the study. We employed multiple immunological tests to ensure the adequate assessment of the H2N2 pandemic LAIV candidate and demonstrated that the majority (92.6%) of the vaccinated subjects responded to the H2N2 LAIV in one or more immunological tests, including 85.2% of subjects with antibody responses and 55.6% volunteers with cell-mediated immune responses. In addition, we observed strong correlation between the H2N2 LAIV virus replication in the upper respiratory tract and the development of antibody responses.

Since conserved viral proteins of influenza virus, such as nucleoprotein (NP) and matrix 1 protein, are the main targets for virus-specific CD8+ cytotoxic T-lymphocytes (CTLs), we hypothesized that introduction of the NP gene of wild-type virus into the genome of vaccine reassortants could lead to better immunogenicity and afford better protection. This paper describes in vitro and in vivo preclinical studies of two new reassortants of pandemic H1N1 live attenuated influenza vaccine (LAIV) candidates. One had the hemagglutinin (HA) and neuraminidase (NA) genes from A/South Africa/3626/2013 H1N1 wild-type virus on the A/Leningrad/134/17/57 master donor virus backbone (6 : 2 formulation) while the second had the HA, NA, and NP genes of the wild-type virus on the same backbone (5 : 3 formulation). Although both LAIVs induced similar antibody immune responses, the 5 : 3 LAIV provoked greater production of virus-specific CTLs than the 6 : 2 variant. Furthermore, the 5 : 3 LAIV-induced CTLs had higher in vivo cytotoxic activity, compared to 6 : 2 LAIV. Finally, the 5 : 3 LAIV candidate afforded greater protection against infection and severe illness than the 6 : 2 LAIV. Inclusion in LAIV of the NP gene from wild-type influenza virus is a new approach to inducing cross-reactive cell-mediated immune responses and cross protection against pandemic influenza.

Please cite this paper as: Petukhova et al. (2011) B- and T-cell memory elicited by a seasonal live attenuated reassortant influenza vaccine: assessment of local antibody avidity and virus-specific memory T-cells using trogocytosis-based method. Influenza and Other Respiratory Viruses DOI: 10.1111/j.1750-2659.2011.00279.x. Purpose The main purpose of vaccination is to generate immunological memory providing enhanced immune responses against infectious pathogens. The standard and most commonly used assay for influenza vaccine immunogenicity evaluation is a hemagglutination inhibition assay (HAI). It is clear now that HAI assay is unable to properly assess the proven protective immunity elicited by live attenuated influenza vaccines (LAIV). New methods need to be developed for more accurate LAIV immunogenicity assessment and prediction of vaccine efficacy among target populations. Objective Randomized placebo-controlled study of memory B- and T-cell responses to intranasal LAIV in young adults. Methods A total of 56 healthy young adults 18–20 years old received seasonal monovalent LAIV. Mucosal memory B-cell responses were measured by IgA avidity assessment in nasal swabs. CD4 memory T cells in peripheral blood were examined by the expression of CD45RO marker and in functional test by the ability of virus-specific T cells to maintain the trogocytosis with antigen-loaded target cells. Results Intranasal LAIV immunization enhances mucosal IgA avidity even without reliable increases in antibody titers. At the day 21 after vaccination, up to 40% of subjects demonstrated significant increases in both total and virus-specific CD4 memory T cells that were observed regardless of seroconversion rate measured by HAI assay. Conclusion The data suggest that immunogenicity of LAIV vaccines should be evaluated on the mucosal and cellular immunity basis. The assays applied could be used to support influenza clinical trials through preliminary screening of volunteers and subsequent measurement of anti-influenza in immunity.

The first and most significant barrier against influenza infection is the mucosal-associated lymphoid tissue of the upper airways and rodent nasopharyngeal-associated lymphoid tissue (NALT) is considered equivalent to the lymphoid tissue of human Valdryer's ring. This study is the first attempt to analyze and compare local and systemic cellular and antibody immune responses in NALT and spleen in a mouse model of experimental influenza infection and intranasal vaccination with LAIV (live attenuated reassortant influenza vaccine). It was shown that the vaccine strain completely inherited the ability to induce high-grade local antibody responses (secretory IgA+IgG+IgM), local cellular lymphoproliferative activity, CD4(+), CD8(+) and CD19(+) lymphocyte and cytokine production responses from the virulent parental strain but it had less capacity to stimulate production of serum IgG, accumulation of CD8(+) cells and IFN-gamma production in the spleen. Primary non-complicated influenza infection and primary vaccination were accompanied by a short-term (24h) increase in the levels of lymphocyte apoptosis in both NALT and spleen. However, experimental data indicated that vaccination with LAIV and uncomplicated forms of influenza infection did not influence immune system apoptosis following a secondary immune response.

Background Asthmatics in particular have a need for influenza vaccines because influenza infection is a frequent cause of hospitalization of patients with bronchial asthma. Currently, only inactivated influenza vaccines are recommended for influenza prevention in asthma sufferers. Objective The aim of our study was to analyze and compare the effects of influenza infection and vaccination with live attenuated influenza vaccine (LAIV) on different phases of experimental murine allergic bronchial asthma (acute asthma and remission phase) and on subsequent exposure to allergen in sensitized animals. Methods Ovalbumin (OVA)-specific serum IgE levels, IL-4 production by spleen and lung lymphocytes, and histological changes in the lungs of mice infected with pathogenic virus or LAIV were studied at two phases of OVA-induced bronchial asthma (acute asthma and remission). Results Infection with pathogenic virus both in acute asthma and remission led to asthma exacerbation associated with the production of OVA-specific IgE, IL-4 and significant inflammatory infiltration in airways. Infection, even after complete virus clearance, induced the aggravation of lung inflammation and IgE production in asthmatic mice additionally exposed to OVA. Immunization with LAIV at remission did not enhance allergic inflammatory changes in the lung, OVA-specific IgE or IL-4 production. Then after additional OVA exposure, histological and immunological changes in these mice were the same as in the control group. Conclusions Influenza infection provokes asthma exacerbation regardless of the disease phase. Immunization with LAIV during the remission phase of bronchial asthma is safe and does not interfere upon subsequent contact of asthma sufferers with allergen.

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.

Introduction Live attenuated influenza vaccines (LAIVs) are being developed to protect humans against future epidemics and pandemics. This study describes the results of a double–blinded randomized placebo–controlled phase I clinical trial of cold–adapted and temperature sensitive H7N3 live attenuated influenza vaccine candidate in healthy seronegative adults. Objective The goal of the study was to evaluate the safety, tolerability, immunogenicity and potential shedding and transmission of H7N3 LAIV against H7 avian influenza virus of pandemic potential. Methods and Findings Two doses of H7N3 LAIV or placebo were administered to 40 randomly divided subjects (30 received vaccine and 10 placebo). The presence of influenza A virus RNA in nasal swabs was detected in 60.0% and 51.7% of subjects after the first and second vaccination, respectively. In addition, vaccine virus was not detected among placebo recipients demonstrating the absence of person–to–person transmission. The H7N3 live attenuated influenza vaccine demonstrated a good safety profile and was well tolerated. The two–dose immunization resulted in measurable serum and local antibody production and in generation of antigen–specific CD4+ and CD8+ memory T cells. Composite analysis of the immune response which included hemagglutinin inhibition assay, microneutralization tests, and measures of IgG and IgA and virus–specific T cells showed that the majority (86.2%) of vaccine recipients developed serum and/or local antibodies responses and generated CD4+ and CD8+ memory T cells. Conclusions The H7N3 LAIV was safe and well tolerated, immunogenic in healthy seronegative adults and elicited production of antibodies broadly reactive against the newly emerged H7N9 avian influenza virus. Trial registration ClinicalTrials.gov NCT01511419