Antigenic Characterization of Neuraminidase of Influenza A/H7N9 Viruses Isolated in Different Years.

Influenza outbreaks caused by A/H7N9 viruses have occurred since 2013. After 2016, A/H7N9 influenza viruses underwent evolutionary changes. In this study, we examined the antigenic properties of influenza neuraminidase (NA) of A/H7N9 viruses as part of a live influenza vaccine (LAIV). It was shown that neuraminidase inhibiting (NI) antibodies obtained after A/Anhui/1/2013(H7N9)-based LAIV vaccination did not inhibit A/Hong Kong/125/2017(H7N9) NA and vice versa. The A/Hong Kong/125/2017(H7N9)-based LAIV elicited higher levels of NI antibodies compared to the A/Anhui/1/2013(H7N9)-based LAIV after two doses. Thelow degree of coincidence of the antibody response to hemagglutinin (HA) and NA after LAIV vaccination allows us to consider an enzyme-linked lectin assay (ELLA) as an additional measure for assessing the immunogenicity of influenza vaccines. In mice, N9-reactive monoclonal antibodies (mABs) for the A/environment/Shanghai/RL01/2013(H7N9) influenza virus partially protected against lung infection from the A/Guangdong/17SF003/2016 IDCDC-RG56N(H7N9) virus, thus showing the cross-protective properties of monoclonal antibodies against the drift variant.

Cross-Reactivity of SARS-CoV-2 Nucleocapsid-Binding Antibodies and Its Implication for COVID-19 Serology Tests.

The emergence of the new coronavirus SARS-CoV-2 in late 2019 led to the global pandemic COVID-19, causing a profound socioeconomic crisis. Adequate diagnostic tools need to be developed to control the ongoing spread of infection. Virus-specific humoral immunity in COVID-19 patients and those vaccinated with specific vaccines has been characterized in numerous studies, mainly using Spike protein-based serology tests. However, Spike protein and specifically its receptor-binding domain (RBD) are mutation-prone, suggesting the reduced sensitivity of the validated serology tests in detecting antibodies raised to variants of concern (VOC). The viral nucleocapsid (N) protein is more conserved compared to Spike, but little is known about cross-reactivity of the N-specific antibodies between the ancestral B.1 virus and different VOCs. Here, we generated recombinant N phosphoproteins from different SARS-CoV-2 strains and analyzed the magnitude of N-specific antibodies in COVID-19 convalescent sera using an in-house N-based ELISA test system. We found a strong positive correlation in the magnitude of anti-N (B.1) antibodies and antibodies specific to various VOCs in COVID-19-recovered patients, suggesting that the N-binding antibodies are highly cross-reactive, and the most immunogenic epitopes within this protein are not under selective pressure. Overall, our study suggests that the RBD-based serology tests should be timely updated to reflect the constantly evolving nature of the SARS-CoV-2 Spike protein, whereas the validated N-based test systems can be used for the analysis of sera from COVID-19 patients regardless of the strain that caused the infection.

Study of Antibodies to Influenza Neuraminidase N2.

Humoral immunity to influenza neuraminidase (NA) was evaluated among different groups of people including patients with acute influenza infection and healthy people in different age groups using an enzyme linked lectin assay (ELLA). The amino acid composition of NA of seasonal influenza viruses A/Victoria/361/2011(H3N2) and A/Hong Kong/4801/2014(H3N2) differed by 2%, while cross-reacting neuraminidase-inhibiting (NI) antibodies to them in the same serum samples were detected in 10% of cases. Middle-aged patients born from 1977 to 2000 had a high level of hemagglutination-inhibiting (HI) antibodies to A/Hong Kong/4801/2014(H3N2), but almost no NI antibodies, which may indicate that in the case of a change in the hemagglutinin (HA) subtype, this age group will be susceptible to influenza A/H3N2 viruses. Therefore, it could mean there is a need for priority vaccination of this age group with a vaccine against the appropriate strain. It was shown that after intranasal administration of live influenza vaccine (LAIV) for the 2017-2018 season, serum antibody response was not lower compared to that during natural infection. In older people, antibodies to archival A/H2N2 viruses were detected more often than to modern A/H3N2. Since the conversion of antibodies to HA and NA often did not coincide, antibodies to NA can serve as an additional criterion for assessing the immunogenicity of influenza vaccines.

Universal Live-Attenuated Influenza Vaccine Candidates Expressing Multiple M2e Epitopes Protect Ferrets against a High-Dose Heterologous Virus Challenge.

The development of an influenza vaccine with broad protection and durability remains an attractive idea due to the high mutation rate of the influenza virus. An extracellular domain of Matrix 2 protein (M2e) is among the most attractive target for the universal influenza vaccine owing to its high conservancy rate. Here, we generated two recombinant live attenuated influenza vaccine (LAIV) candidates encoding four M2e epitopes representing consensus sequences of human, avian and swine influenza viruses, and studied them in a preclinical ferret model. Both LAIV+4M2e viruses induced higher levels of M2e-specific antibodies compared to the control LAIV strain, with the LAIV/HA+4M2e candidate being significantly more immunogenic than the LAIV/NS+4M2e counterpart. A high-dose heterosubtypic influenza virus challenge revealed the highest degree of protection after immunization with LAIV/HA+4M2e strain, followed by the NS-modified LAIV and the classical LAIV virus. Furthermore, only the immune sera from the LAIV/HA+4M2e-immunized ferrets protected mice from a panel of lethal influenza viruses encoding M genes of various origins. These data suggest that the improved cross-protection of the LAIV/HA+4M2e universal influenza vaccine candidate was mediated by the M2e-targeted antibodies. Taking into account the safety profile and improved cross-protective potential, the LAIV/HA+4M2e vaccine warrants its further evaluation in a phase I clinical trial.

Study of Neuraminidase-Inhibiting Antibodies in Clinical Trials of Live Influenza Vaccines.

BACKGROUND: Currently, the immunogenicity of influenza vaccines is assessed by detecting an increase of hemagglutination inhibition (HI) antibodies. As neuraminidase (NA)-based immunity may be significant in protecting against influenza infection, detection of neuraminidase inhibiting (NI) antibodies may improve the assessment of the immunogenicity of influenza vaccines. METHODS: We investigated the immune response to NA in people after immunization with live influenza vaccines (LAIVs). A number of A/H7NX or A/H6NX viruses were used to detect NI antibodies, using an enzyme-linked lectin assay (ELLA). RESULTS: Seasonal LAIV immunization stimulated an increase in NI antibodies not only to homologous A/H1N1 influenza, but also to A/H1N1pdm09 and A/H5N1 influenza. After A/17/California/09/38 (H1N1) pdm09 LAIV vaccination, there was no statistical relationship between post-vaccinated antibody seroconversion and two surface glycoproteins in serum samples obtained from the same individuals (p = 0.24). Vaccination with LAIV of H5N2, H2N2, H7N3, and H7N9 subtypes led to 7%-29.6% NI antibody seroconversions in the absence of HI antibody conversions. There was relatively low coordination of hemagglutinin (HA) and NA antibody responses (r = 0.24-0.59). CONCLUSIONS: The previously noted autonomy for HI and NI immune responses was confirmed when assessing the immunogenicity of LAIVs. Combining the traditional HI test with the detection of NI antibodies can provide a more complete assessment of LAIV immunogenicity.

A Phase 1 Randomized Placebo-Controlled Study to Assess the Safety, Immunogenicity and Genetic Stability of a New Potential Pandemic H7N9 Live Attenuated Influenza Vaccine in Healthy Adults.

This study describes a double-blind randomized placebo-controlled phase I clinical trial in healthy adults of a new potential pandemic H7N9 live attenuated influenza vaccine (LAIV) based on the human influenza virus of Yangtze River Delta hemagglutinin lineage (ClinicalTrials.gov Identifier: NCT03739229). Two doses of H7N9 LAIV or placebo were administered intranasally to 30 and 10 subjects, respectively. The vaccine was well-tolerated and not associated with increased rates of adverse events or with any serious adverse events. Vaccine virus was detected in nasal swabs during the 6 days after vaccination or revaccination. A lower frequency of shedding was observed after the second vaccination. Twenty-five clinical viral isolates obtained after the first and second doses of vaccine retained the temperature-sensitive and cold-adapted phenotypic characteristics of LAIV. There was no confirmed transmission of the vaccine strain from vaccinees to placebo recipients. After the two H7N9 LAIV doses, an immune response was observed in 96.6% of subjects in at least one of the assays conducted.

Sequential Immunization with Universal Live Attenuated Influenza Vaccine Candidates Protects Ferrets against a High-Dose Heterologous Virus Challenge.

The development of universal influenza vaccines has been a priority for more than 20 years. We conducted a preclinical study in ferrets of two sets of live attenuated influenza vaccines (LAIVs) expressing chimeric hemagglutinin (cHA). These vaccines contained the HA stalk domain from H1N1pdm09 virus but had antigenically unrelated globular head domains from avian influenza viruses H5N1, H8N4 and H9N2. The viral nucleoproteins (NPs) in the two sets of universal LAIV candidates were from different sources: one LAIV set contained NP from A/Leningrad/17 master donor virus (MDV), while in the other set this gene was from wild-type (WT) H1N1pdm09 virus, in order to better match the CD8 T-cell epitopes of currently circulating influenza A viruses. To avoid any difference in protective effect of the various anti-neuraminidase (NA) antibodies, all LAIVs were engineered to contain the NA gene of Len/17 MDV. Naïve ferrets were sequentially immunized with three doses of (i) classical LAIVs containing non-chimeric HA and NP from MDV (LAIVs (NP-MDV)); (ii) cHA-based LAIVs containing NP from MDV (cHA LAIVs (NP-MDV)); and (iii) cHA-based LAIVs containing NP from H1N1pdm09 virus (cHA LAIVs (NP-WT)). All vaccination regimens were safe, producing no significant increase in body temperature or weight loss, in comparison with the placebo group. The two groups of cHA-based vaccines induced a broadly reactive HA stalk-directed antibody, while classical LAIVs did not. A high-dose challenge with H1N1pdm09 virus induced significant pathology in the control, non-immunized ferrets, including high virus titers in respiratory tissues, clinical signs of disease and histopathological changes in nasal turbinates and lung tissues. All three vaccination regimens protected animals from clinical manifestations of disease: immunized ferrets did not lose weight or show clinical symptoms, and their fever was significantly lower than in the control group. Further analysis of virological and pathological data revealed the following hierarchy in the cross-protective efficacy of the vaccines: cHA LAIVs (NP-WT) > cHA LAIVs (NP-MDV) > LAIVs (NP-MDV). This ferret study showed that prototype universal cHA-based LAIVs are highly promising candidates for further clinical development.

Rationale for vaccination with trivalent or quadrivalent live attenuated influenza vaccines: Protective vaccine efficacy in the ferret model.

BACKGROUND AND AIM: The majority of seasonal influenza vaccines are trivalent, containing two A virus strains (H1N1 and H3N2) and one B virus strain. The co-circulation of two distinct lineages of B viruses can lead to mismatch between the influenza B virus strain recommended for the trivalent seasonal vaccine and the circulating B virus. This has led some manufacturers to produce quadrivalent influenza vaccines containing one strain from each B lineage in addition to H1N1 and H3N2 strains. However, it is also important to know whether vaccines containing a single influenza B strain can provide cross-protectivity against viruses of the antigenically distinct lineage. The aim of this study was to assess in naïve ferrets the potential cross-protective activity of trivalent live attenuated influenza vaccine (T-LAIV) against challenge with a heterologous wild-type influenza B virus belonging to the genetically different lineage and to compare this activity with effectiveness of quadrivalent LAIV (Q-LAIV) in the ferret model. METHODS AND RESULTS: Ferrets were vaccinated with either one dose of trivalent LAIV containing B/Victoria or B/Yamagata lineage virus, or quadrivalent LAIV (containing both B lineages), or placebo. They were then challenged with B/Victoria or B/Yamagata lineage wild-type virus 28 days after vaccination. The ferrets were monitored for clinical signs and morbidity. Nasal swabs and lung tissue samples were analyzed for the presence of challenge virus. Antibody response to vaccination was assessed by routine hemagglutination inhibition assay. All LAIVs tested were found to be safe and effective against wild-type influenza B viruses based on clinical signs, and virological and histological data. The absence of interference between vaccine strains in trivalent and quadrivalent vaccine formulations was confirmed. Trivalent LAIVs were shown to have the potential to be cross-protective against infection with genetically different influenza B/Victoria and B/Yamagata lineages. CONCLUSIONS: In this ferret model, quadrivalent vaccine provided higher protection to challenge against both B/Victoria and B/Yamagata lineage viruses. However, T-LAIV provided some cross-protection in the case of a mismatch between circulating and vaccine type B strains. Notably, B/Victoria-based T-LAIV was more protective compared to B/Yamagata-based T-LAIV.

Two Live Attenuated Vaccines against Recent Low⁻and Highly Pathogenic H7N9 Influenza Viruses Are Safe and Immunogenic in Ferrets.

Influenza H7N9 virus is a potentially pandemic subtype to which most people are immunologically naïve. To be better prepared for the potential occurrence of an H7N9 pandemic, in 2017 the World Health Organization recommended developing candidate vaccine viruses from two new H7N9 viruses, A/Guangdong/17SF003/2016 (A/GD) and A/Hong Kong/125/2017 (A/HK). This report describes the development of live attenuated influenza vaccine (LAIV) candidates against A/GD and A/HK viruses and study of their safety and immunogenicity in the ferret model in order to choose the most promising one for a phase I clinical trial. The A/HK-based vaccine candidate (A/17/HK) was developed by classical reassortment in eggs. The A/GD-based vaccine candidate (A/17/GD) was generated by reverse genetics. Ferrets were vaccinated with two doses of LAIV or phosphate-buffered saline. Both H7N9 LAIVs tested were safe for ferrets, as shown by absence of clinical signs, and by virological and histological data; they were immunogenic after a single vaccination. These results provide a compelling argument for further testing of these vaccines in volunteers. Since the A/HK virus represents the cluster that has caused the majority of human cases, and because the A/HK-based LAIV candidate was developed by classical reassortment, this is the preferred candidate for a phase I clinical trial.

Cross-Protective Efficacy of Monovalent Live Influenza B Vaccines against Genetically Different Lineages of B/Victoria and B/Yamagata in Ferrets.

BACKGROUND: Currently, two genetic lineages of influenza B virus, B/Victoria and B/Yamagata, are cocirculating in humans in various countries. This situation has raised a question regarding the possibility of cross-protection between B components of live attenuated influenza vaccine (LAIV) belonging to different lineages. This study aimed to assess in naïve ferrets the potential protective activity of monovalent B-LAIVs against challenge with homologous and heterologous wild-type (WT) influenza B viruses. METHODS: Groups of seronegative female ferrets 5-6 months of age were given one dose of monovalent LAIV based on B/Victoria or B/Yamagata lineage virus. Ferrets were challenged 21 days later with B/Victoria or B/Yamagata WT virus. Ferrets were monitored closely for clinical signs and morbidity outcomes including febrile response, body weight loss, nasal symptoms, and level of activity one week prior to vaccination and for three days following vaccination/challenge. Nasal washes were collected three days after vaccination/challenge. Samples of lung tissue were taken three days after challenge. All samples were analyzed for the presence of challenge virus by culturing in embryonated chicken eggs and real-time polymerase chain reaction. Antibody response to vaccination was assessed by routine hemagglutination inhibition assay and microneutralization test. RESULTS: Vaccination led to intensive production of specific neutralizing and antihemagglutinating antibodies to vaccine virus, protected ferrets from homologous challenge infection, and significantly reduced clinical signs and replication of homologous challenge virus. In contrast, cross-lineage serum antibodies were not detected. However, ferrets vaccinated with monovalent B-LAIV had a significantly lower level of heterologous challenge virus in the respiratory tract than those given challenge virus only. CONCLUSIONS: Monovalent B-LAIV has the potential to be cross-protective against infection with genetically different influenza lineages. Further studies are required to confirm this effect.

Broadly protective anti-hemagglutinin stalk antibodies induced by live attenuated influenza vaccine expressing chimeric hemagglutinin.

The development of influenza vaccines that can provide broad protection against all drifted seasonal virus variants, zoonotic infections and emerging pandemic strains, has been a priority for two decades. Here we propose a strategy of inducing broadly-reactive anti-stalk antibody by sequential immunizations with live attenuated influenza vaccines (LAIVs) expressing chimeric HAs (cHAs). These vaccines are designed to contain identical hemagglutinin stalk domains from H1N1 virus but antigenically unrelated globular head domains from avian influenza virus subtypes H5, H8 and H9. Mouse experiments demonstrated enhanced cross-protection of cHA-containing LAIVs compared to the relevant vaccine viruses expressing natural HAs, and this enhanced protection was driven by stalk-HA-reactive IgG antibodies. The establishment of fully functional cross-protective immunity after two doses of cHA LAIV vaccination in naïve animals suggests that a similar effect might be expected after a single cHA LAIV dose in primed individuals, or after two to three doses in naïve children.

Immunogenicity and Cross Protection in Mice Afforded by Pandemic H1N1 Live Attenuated Influenza Vaccine Containing Wild-Type Nucleoprotein.

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.

Comparative studies of infectivity, immunogenicity and cross-protective efficacy of live attenuated influenza vaccines containing nucleoprotein from cold-adapted or wild-type influenza virus in a mouse model.

This study sought to improve an existing live attenuated influenza vaccine (LAIV) by including nucleoprotein (NP) from wild-type virus rather than master donor virus (MDV). H7N9 LAIV reassortants with 6:2 (NP from MDV) and 5:3 (NP from wild-type virus) genome compositions were compared with regard to their growth characteristics, induction of humoral and cellular immune responses in mice, and ability to protect mice against homologous and heterologous challenge viruses. Although, in general, the 6:2 reassortant induced greater cell-mediated immunity in C57BL6 mice than the 5:3 vaccine, mice immunized with the 5:3 LAIV were better protected against heterologous challenge. The 5:3 LAIV-induced CTLs also had better in vivo killing activity against target cells loaded with the NP366 epitope of recent influenza viruses. Modification of the genome of reassortant vaccine viruses by incorporating the NP gene from wild-type viruses represents a simple strategy to improve the immunogenicity and cross-protection of influenza vaccines.

Assessment of immune responses to H5N1 inactivated influenza vaccine among individuals previously primed with H5N2 live attenuated influenza vaccine.

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.

Clinical testing of pre-pandemic live attenuated A/H5N2 influenza candidate vaccine in adult volunteers: results from a placebo-controlled, randomized double-blind phase I study.

BACKGROUND: This study describes a double-blinded randomized placebo-controlled phase I clinical trial of A/H5N2 live attenuated influenza vaccine in healthy volunteers. METHODS: Two doses of vaccine or placebo were administered intranasally to 30 and 10 subjects, respectively. Nasal swabs were examined for vaccine shedding and local antibody responses; serum samples were tested for binding, hemagglutinating and neutralizing antibodies and peripheral blood mononuclear cells were tested for cell-mediated immune responses. RESULTS: The vaccine was well tolerated and not associated with increased rates of adverse events or the occurrence of serious adverse events. Influenza virus was detected in nasal swabs on the first day in the majority of volunteers (93%), while 17% of volunteers tested positive on the second, none on the third day or later following the first vaccination; lower frequency of shedding was observed after the second vaccination. The vaccine was immunogenic as assessed four weeks after the second dose, with 37.9% and 48.3% of subjects seroconverting by hemagglutination inhibition and neutralization assays, respectively. An immune response was observed in 96.6% subjects that received A/H5N2 LAIV in at least one of the assays conducted. None of the placebo recipients exhibited a response in any of the assays. CONCLUSION: The A/H5N2 vaccine was safe, well tolerated, and immunogenic in healthy adults. TRIAL REGISTRATION: ClinicalTrials.gov NCT01719783.

Serum strain-specific or cross-reactive neuraminidase inhibiting antibodies against pandemic А/California/07/2009(H1N1) influenza in healthy volunteers.

BACKGROUND: Pre-existing antibodies to influenza virus neuraminidase may provide protection against infection influenza viruses containing novel hemagglutinin (HA). The aim of our study was to evaluate serum neuraminidase-inhibiting (NI) antibodies against А/California/07/2009(H1N1) [H1N1/2009pdm] and А/New Caledonia/20/1999(H1N1) [H1N1/1999] influenza viruses in relation with the age of participants and hemagglutination-inhibition (HI) antibody levels. Anti-H1N1/2009pdm neuraminidase and anti-H1N1/1999 neuraminidase antibody levels were measured in total 219 serum samples from Russian healthy peoples of various ages examined before and a year after pandemic strain appearance. We adjusted peroxidase-linked lectin micro-procedure to measure NI antibody titers using the reassortant A/H7N1 influenza viruses based on A/equine/Prague/1/56(H7N7). Also, HI antibody titers were estimated against H1N1/2009pdm, H1N1/1999 and a panel of seasonal A/H1N1 influenza viruses. RESULTS: In sera samples collected during the fall of 2010, mean titers of specific HI and NI antibodies to H1N1/2009pdm were 2-2.1 times lower than antibody levels against H1N1/1999. Of the 163 individuals examined, 58 (35.6%) had NI anti-H1N1/2009pdm antibody titers > 1:20, compared to 93 (57.1%) who had NI anti-H1N1/1999 antibody titers > 1:20. There were low correlations between HI and NI antibody levels against either H1N1/1999 or H1N1/2009pdm in the same serum samples. The 24 adults born between 1957 and 1977 expressed very low levels of NI antibodies to A/H1N1 influenza viruses. Persons with low HI anti-H1N1/2009pdm titers but positive to seasonal A/H1N1 demonstrated significantly higher NI anti-A/H1N1 antibody titers than unexposed subjects. In 2005 cross-reactive NI anti-H1N1/2009pdm antibody titers > 1:20 were detected among 7.1% of young people. CONCLUSIONS: Our study confirmed that contact with seasonal influenza viruses may have contributed to generating the cross-reacting anti-H1N1/2009pdm NI antibodies which were detected in the sera of 18-20 years old people examined before the pandemic virus active circulation. The lowest levels of antibodies to the neuraminidase of N1 subtype were in the group of participants born during the circulation of influenza A/H2N2 or A/H3N2 viruses. The low correlation between HI and NI antibody titers suggests that NI antibody detection can be used as an additional test to evaluate the immune response after influenza infections or immunizations.

H2N2 live attenuated influenza vaccine is safe and immunogenic for healthy adult volunteers.

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.

Assessment of human immune responses to H7 avian influenza virus of pandemic potential: results from a placebo-controlled, randomized double-blind phase I study of live attenuated H7N3 influenza vaccine.

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.

H7N3 live attenuated influenza vaccine has a potential to protect against new H7N9 avian influenza virus.

After recent emergence of new avian influenza A(H7N9) viruses in humans many people and Governments are asking about H7 influenza vaccine which could provide cross-protection against new viruses, until H7N9 vaccine is prepared from a relevant strain. Here we scientifically justify that available H7N3 live attenuated influenza vaccine (LAIV) can be protective against H7N9 viruses due to the presence of conserved immune epitopes in its hemagglutinin. We used Immune Epitope Database analysis resource to predict B-cell and CTL epitopes distributed across H7N3 HA molecule and assessed their identity with new H7N9 viruses at near 70% and 60% of the epitopes, respectively. In addition, we tested serum samples of volunteers participated in phase I clinical trial of H7N3 LAIV for the presence of anti-H7N9 hemagglutination-inhibition and neutralizing antibodies and found seroconversions in 44.8% of vaccinated persons, which suggests the potential of H7N3 LAIV to protect against new H7N9 avian influenza viruses.

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.

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.