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.

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).

Amino Acid Substitutions N123D and N149D in Hemagglutinin Molecule Enhance Immunigenicity of Live Attenuated Influenza H7N9 Vaccine Strain in Experiment.

We compared three cold-adapted live attenuated influenza vaccine strains prepared by reverse genetics methods on the basis of master donor virus A/Leningrad/134/17/57 and influenza H7N9 strains A/Anhui/1/2013 and A/Shanghai/1/2013. Two strains based on A/Anhui/1/2013 differed by amino acid positions 123 and 149 in HA1 (123N/149N; 123D/149D). All strains efficiently replicated in developing chicken embryos; A/Shanghai/1/2013-based strain and A/Anhui/1/2013-123N/149N variant were characterized by reduced replication in MDCK cells.

Safety, immunogenicity and protection of A(H3N2) live attenuated influenza vaccines containing wild-type nucleoprotein in a ferret model.

Live attenuated influenza vaccines (LAIVs) are promising tools for the induction of broad protection from influenza due to their ability to stimulate cross-reactive T cells against influenza pathogens. One of the major targets for cytotoxic T-cell immunity is viral nucleoprotein (NP), which is relatively conserved among antigenically distant influenza viruses. Nevertheless, a diversity of epitope composition has been found in the NP protein of different lineages of influenza A viruses.

Anti-neuraminidase antibodies against pandemic A/H1N1 influenza viruses in healthy and influenza-infected individuals.

The main objective of the study was to evaluate neuraminidase inhibiting (NI) antibodies against A/H1N1pdm09 influenza viruses in the community as a whole and after infection. We evaluated NI serum antibodies against A/California/07/09(H1N1)pdm and A/South Africa/3626/2013(H1N1)pdm in 134 blood donors of different ages using enzyme-linked lectin assay and in 15 paired sera from convalescents with laboratory confirmed influenza. The neuraminidase (NA) proteins of both A/H1N1pdm09 viruses had minimal genetic divergence, but demonstrated different enzymatic and antigenic properties.

Modeling of 3D Structure of Chimeric Constructs Based on Hemagglutinin of Influenza Virus and Immunogenic Epitopes of Streptococcus Agalactiae.

A project of an experimental recombinant vector vaccine for prevention of diseases caused by pathogenic streptococci based on ScaAB lipoprotein of Streptococcus agalactiae and a coldadapted strain of live influenza vaccine as a vector was developed. The sequence of ScaAB lipoprotein was analyzed and fragments forming immunodominant epitopes were determined. Chimeric molecules of influenza virus hemagglutinin H7 carrying insertions of bacterial origin were constructed.

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.

Live Attenuated Influenza Vaccines engineered to express the nucleoprotein of a recent isolate stimulate human influenza CD8 T cells more relevant to current infections.

Live attenuated influenza vaccines (LAIV) induce CD8 T lymphocyte responses that play an important role in killing virus-infected cells. Despite the relative conservation of internal influenza A proteins, the epitopes recognized by T cells can undergo drift under immune pressure. The internal proteins of Russian LAIVs are derived from the master donor virus A/Leningrad/134/17/57 (Len/17) isolated 60 years ago and as such, some CD8 T cell epitopes may vary between the vaccine and circulating wild-type strains.

Genotyping assay for differentiation of wild-type and vaccine viruses in subjects immunized with live attenuated influenza vaccine.

Live attenuated influenza vaccines (LAIVs) are considered as safe and effective tool to control influenza in different age groups, especially in young children. An important part of the LAIV safety evaluation is the detection of vaccine virus replication in the nasopharynx of the vaccinees, with special attention to a potential virus transmission to the unvaccinated close contacts. Conducting LAIV clinical trials in some geographical regions with year-round circulation of influenza viruses warrants the development of robust and reliable tools for differentiating vaccine viruses from wild-type influenza viruses in nasal pharyngeal wash (NPW) specimens of vaccinated subjects.

Prevention of Influenza A(H7N9) and Bacterial Infections in Mice Using Intranasal Immunization With Live Influenza Vaccine and the Group B Streptococcus Recombinant Polypeptides.

We investigate the protective effect of combined vaccination based on live attenuated influenza vaccine (LAIV) and group B streptococcus (GBS) recombinant polypeptides against potential pandemic H7N9 influenza infection followed by GBS burden. Mice were intranasally immunized using 107 50% egg infectious dose (EID) of H7N3 LAIV, the mix of the 4 GBS peptides (group B streptococcus vaccine [GBSV]), or combined LAIV + GBSV vaccine. The LAIV raised serum hemagglutination-inhibition antibodies against H7N9 in higher titers than against H7N3.

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).

Evaluation in Mouse Model of Combined Virus-bacterial Vaccine Based on Attenuated Influenza A(H7N3) Virus and the Group B Streptococcus Recombinant Polypeptides.

Background: Secondary bacterial influenza complications are a common cause of excesses morbidity and mortality, which determines the need to develop means for specific prophylaxis. Group B streptococcal infection is especially common cause of pneumonia among children and the elderly with underlying conditions. Here we investigate in a mouse model the effects of combined intranasal immunization using live attenuated influenza vaccine and recombinant polypeptides based on group B surface proteins.

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.

Analysis of Immune Epitopes of Respiratory Syncytial Virus for Designing of Vectored Vaccines Based on Influenza Virus Platform.

The immunoepitope database was used for analysis of experimentally detected epitopes of the respiratory syncytial virus (RSV) proteins and for selection of the epitope combinations for subsequent designing of recombinant vectored anti-RSV vaccines based on attenuated influenza viruses. Three cassettes containing the most promising B- and T-cell RSV epitopes were selected: peptide F (243-294) supporting the formation of humoral immunity in animals; fragment M2-1 (70-101+114-146) containing two MHC I epitopes (82-90 and 127-135); and MHC II-epitope (51-66). The selected constructions contained no neoepitopes causing undesirable effects of vaccination, such as immunotolerance or autoimmunity.

[DEVELOPMENT OF THE QUADRIVALENT LIVE ATTENUATED INFLUENZA VACCINE INCLUDING TWO INFLUENZA B LINEAGES--VICTORIA AND YAMAGATA].

This work is devoted to the research of the live attenuated influenza vaccine (LAIV) comprising two reassortant B/USSR/60/69-based vaccine influenza viruses Victoria and Yamagata. The intranasal immunization of the CBA mice with both Victoria and Yamagata strains induced 100% lung protection against the subsequent infection with the wild-type influenza B viruses of any antigen lineage. The quadrivalent LAIV (qLAIV) comprising both reassortant influenza B viruses Victoria and Yamagata were safe and areactogenic in adult volunteers.

H7N9 Live Attenuated Influenza Vaccine Is Highly Immunogenic, Prevents Virus Replication, and Protects Against Severe Bronchopneumonia in Ferrets.

Avian influenza viruses continue to cross the species barrier, and if such viruses become transmissible among humans, it would pose a great threat to public health. Since its emergence in China in 2013, H7N9 has caused considerable morbidity and mortality. In the absence of a universal influenza vaccine, preparedness includes development of subtype-specific vaccines.

[A preclinical trial of the reassortant influenza virus vaccine strain A/17/Quail/Hong Kong/97/84 (H9N2)].

In this work, we examined the reassortant influenza virus strain A/17/Quail/Hong Kong/97/84 (H9N2) prepared at the Virology Department, Institute of Experimental Medicine, Russian Academy of Medical Sciences. The A/ Leningrad/134/17 (H2N2)-based vaccine candidate contained hemagglutinin and the neuraminidase from the nonpathogenic avian influenza A virus A(H9N2) of the G1 antigenic lineage. The vaccine candidate showed the ts-properties and cold adaptation.

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.

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.

[Serodiagnosis in the surveillance of the influenza virus circulation during the development of the pandemic caused by the A (H1N1)pdm09].

The goal of this work was to present the data of the study of the peculiarities of the generation factors of humoral immunity in the response to the infection with the pandemic influenza A (HIN1) pdmO9 in patients with different epidemiological anamnesis. High ability of the influenza viruses to spread over closed communities and the transfer of the maternal antibodies to babies, including a pandemic strain of the influenza virus A (H1N1) pdm09, was confirmed. The results of this study showed that the immune response to the surface antigens of the influenza virus (hemagglutinin and neuraminidase) was formed during the natural infection with the pandemic strains of the influenza A (H1N1) pdm09 in more than a half of the cases simultaneously.

Development and pre-clinical evaluation of two LAIV strains against potentially pandemic H2N2 influenza virus.

H2N2 Influenza A caused the Asian flu pandemic in 1957, circulated for more than 10 years and disappeared from the human population after 1968. Given that people born after 1968 are naïve to H2N2, that the virus still circulates in wild birds and that this influenza subtype has a proven pandemic track record, H2N2 is regarded as a potential pandemic threat. To prepare for an H2N2 pandemic, here we developed and tested in mice and ferrets two live attenuated influenza vaccines based on the haemagglutinins of the two different H2N2 lineages that circulated at the end of the cycle, using the well characterized A/Leningrad/134/17/57 (H2N2) master donor virus as the backbone.

[Evaluation of the anti-neuraminidase antibodies in clinical trials of the live influenza vaccine of the A(H5N2) subtype].

In the current study, we evaluated the neuraminidase-inhibition (NI) antibodies among volunteers during the phase I and phase II of the clinical trials of a monovalent live attenuated influenza vaccine (LAIV) A/17/duck/ Potsdam/86/92(H5N2). The reassortant influenza virus RN2/57-human A(H7N2) containing neuraminidase (NA) from the A/Leningrad/134/17/57(H2N2) was used in NI test. It was shown that two doses of the monovalent LAIV A(H5N2) led to a statistically significant increase in the NI antibodies to vaccine strain NA.

[Formation of antibodies against neuraminidase of A/California/07/2009 (H1N1) influenza virus after immunization with live monovalent influenza vaccine].

Aim: Detection of antibodies against neuraminidase (NA) of A/California/07/2009 (H1N1) influenza virus in blood sera of volunteers after the immunization with live monovalent influenza vaccine (LIV).

Materials And Methods: Neuraminidase enzyme activity inhibition by antibodies test with reassortant strain A(H7N1) containing NA of pandemic strain was used. Anti-neuraminidase IgG antibodies against whole reassortant virus A(H7N1) and purified NA of A/California/07/2009 (H1N1) strains were determined by enzyme immunoassay (EIA).

[Study of biological properties of cold-adapted reassortant strain of influenza virus subtype H7N3].

For the development of live attenuated influenza vaccine (LAIV) against influenza virus strains with pandemic potential, method of classic genetic reassortment of donor of attenuation A/Leningrad/134/17/57 (H2N2) [Len/17] with avian apathogenic influenza viruses of different subtypes was used. Strain with genome formula 6:2, which contains HA and NA genes from avian apathogenic virus A/wild duck/Netherlands/12/00 (H7N3) [N7N3-wt] and 6 other genes--from Len/17, was studied. Reassortant strain A/17/ wild duck/Netherlands/00/84 (H7N3) [Lenl7/ H7] exhibited ts- and ca- phenotype specific for cold-adapted strains.