[Peculiarities of the influenza and ARVI viruses circulation during epidemic season 2019-2020 in some regions of Russia].

INTRODUCTION: The surveillance of influenza viruses in ARVI structure and study of their properties in epidemic season 2019-2020 in Russian Federation are actual for investigations due to tasks of Global Influenza Strategy initiated by WHO in 2019. MATERIAL AND METHODS: The data of epidemiological surveillance on influenza- and ARVI-associated morbidity and hospitalization in different age groups of population were analyzed; virological, genetic and statistical methods were used. RESULTS: Preschool children were involved in epidemic the most. Meanwhile, the highest rate of hospitalization was observed in patients of 18-40 years old. Influenza A(H1N1)pdm09 virus dominated in etiology of ARVI in hospitalized patients and pneumonia. The role of respiratory viruses in severe cases of pneumonia and bronchoalveolar syndrome in children was shown. The differences in spectrum of circulating viruses caused ARVI in different regions of Russia were found. Influenza A(H1N1)pdm09 and B/Victoria-like viruses were the main etiological agents that caused of epidemic; its activity among all ARVI was 7.3 and 8.0%, respectively. The differences in antigenic properties of influenza A(H3N2) and B epidemic strains compared to vaccine viruses were found. The populations of epidemic strains were presented by following dominant genetic groups: 6B1.A5/183P for A(H1N1)pdm09, 3С.2а1b+137F for A(H3N2) and V1A.3 line B/Victoria-like for B viruses. The good profile of epidemic strains susceptibility to anti-neuraminidase inhibitors has been saved. The most of the studied influenza strains had the receptor specificity characteristic of human influenza viruses. CONCLUSIONS: Obtained results identified the peculiarities of viruses caused the influenza and ARVI in epidemic season 2019-2020 in different regions of Russia. These results suggested the important role of influenza A(H1N1) pdm09 in severe cases and pneumonia in adults 18-40 years old. The continuing drift in influenza viruses was found, which, apparently, could not but affect the efficacy of vaccine prophylaxis and was also considered in the recommendations of WHO experts on the composition of influenza vaccines for the countries of the Northern Hemisphere in the 2020-2021 season.

Selective Capture and Determination of Receptor-Binding Hemagglutinin in Influenza Vaccine Preparations Using a Coupled Receptor-Binding/RP-HPLC Assay.

Influenza vaccine potency is determined by the quantification of immunologically active hemagglutinin capable of eliciting neutralizing antibodies upon immunization. Currently, the single radial immunodiffusion (SRID) method is the standard in vitro potency assay used for lot release of seasonal inactivated influenza vaccines. Despite the proven usage of SRID, significant limitations such as the time-consuming preparation of reagents and limited dynamic range warrant the need for the development of alternative potency assays. Such alternative approaches need to discriminate and quantify relevant hemagglutinin material, provide strain identity, and be independent of strain-specific and seasonal reagents. Herein, we present a proof of concept method that combines the capture of conformationally well-folded hemagglutinin via a sialic acid binding step with the resolving power of reversed-phase high-performance liquid chromatography for strain identity and determination. Details of the protocol for the selective capture of receptor-binding hemagglutinin, its release from the receptor, and its relative determination are presented. This approach was found to provide flexibility for the reagents to be used and was adaptable to varying strain compositions of influenza vaccines. This proof of concept approach was developed as an antibody-independent methodology.

Single-Step Detection of the Influenza Virus Hemagglutinin Using Bacterially-Produced Quenchbodies.

We have successfully generated a Quenchbody that enables the detection of the influenza virus hemagglutinin (HA), in a simple and convenient manner. By two-site labeling of the bacterially-produced anti-HA Fab with ATTO520, its fluorescence intensity was increased to 4.4-fold, in the presence of a nanomolar concentration of H1N1 HA. Our results indicate the potential use of this Quenchbody, as a sensor for the simple in situ detection of influenza A virus.

Transcriptional response to a prime/boost vaccination of chickens with three vaccine variants based on HA DNA and Pichia-produced HA protein.

Highly pathogenic avian influenza causes severe economic losses and is a potential threat to public health. Better knowledge of the mechanisms of chicken response to the novel types of vaccines against avian influenza might be helpful in their successful implementation into poultry vaccination programs in different countries. This work presents a comprehensive analysis of gene expression response elicited in chicken spleens by a combined DNA/recombinant protein prime/boost vaccination compared to DNA/DNA and protein/protein regimens. All groups of vaccinated chickens displayed changes in spleen transcriptomes in comparison to the control group with 423, 375 and 212 identified differentially expressed genes in protein/protein, DNA/DNA and DNA/protein group, respectively. Genes with most significantly changed expression belong to immune-related categories. Depending on a group, a fraction of 15-34% of up-regulated and a fraction of 15-42% of down-regulated immune-related genes are shared by all groups. Interestingly, the most upregulated genes encode ß-defensins, short peptides with antimicrobial activity and immunomodulatory functions. Microarray results were validated with RT-qPCR method, which confirmed differential regulation of the selected immune-related genes. Immune-related differentially expressed genes and metabolic pathways identified in this work are compared to the available literature data on gene expression changes in vaccinated and non-vaccinated chickens after influenza infection.

Electrochemical detection of influenza virus H9N2 based on both immunomagnetic extraction and gold catalysis using an immobilization-free screen printed carbon microelectrode.

Influenza is a viral infectious disease considered as a source of many health problems and enormous socioeconomic disruptions. Conventional methods are inadequate for in-field detection of the virus and generally suffer from being laborious and time-consuming. Thus, studies aiming to develop effective alternatives to conventional methods are urgently needed. In this work, we developed an approach for the isolation and detection of influenza A virus subtype H9N2. For this aim, two specific influenza receptors were used. The first, anti-matrix protein 2 (M2) antibody, was attached to iron magnetic nanoparticles (MNPs) and used for the isolation of the virus from allantoic fluid. The second biomolecule, Fetuin A, was attached to an electrochemical detectable label, gold nanoparticles (AuNPs), and used to detect the virus tacking advantage from fetuin-hemagglutinin interaction. The MNP-Influenza virus-AuNP formed complex was isolated and treated by an acid solution then the collected gold nanoparticles were deposited onto a screen printed carbon electrode. AuNPs catalyzes the hydrogen ions reduction in acidic medium while applying an appropriate potential, and the generated current signal was proportional to the virus titer. This approach allows the rapid detection of influenza virus A/H9N2 at a less than 16 HAU titer.

An Aptamer-Based Electrochemical Sensor That Can Distinguish Influenza Virus Subtype H1 from H5.

The surface protein hemagglutinin (HA) mediates the attachment of influenza virus to host cells containing sialic acid and thus facilitates viral infection. Therefore, HA is considered as a good target for the development of diagnostic tools for influenza virus. Previously, we reported the isolation of single-stranded aptamers that can distinguish influenza subtype H1 from H5. In this study, we describe a method for the selective electrical detection of H1 using the isolated aptamer as a molecular probe. After immobilization of the aptamer on Si wafer, enzyme-linked immunosorbent assay (ELISA) and field emission scanning electron microscopy (FE-SEM) showed that the immobilized aptamer bound specifically to the H1 subtype but not to the H5 subtype. Assessment by cyclic voltammetry (CV) also demonstrated that the immobilized aptamer on the indium thin oxide-coated surface was specifically bound to the H1 subtype only, which was consistent with the ELISA and FE-SEM results. Further measurement of CV using various amounts of H1 subtype provided the detection limit of the immobilized aptamer, which showed that a nanomolar scale of target protein was sufficient to produce the signal. These results indicated that the selected aptamer can be an effective probe for distinguishing the subtypes of influenza viruses by monitoring current changes.

Evolution of H5 highly pathogenic avian influenza: sequence data indicate stepwise changes in the cleavage site.

The genetic composition of an H5 subtype hemagglutinin gene quasispecies, obtained from ostrich tissues that had been infected with H5 subtype influenza virus was analysed using a next generation sequencing approach. The first evidence for the reiterative copying of a poly (U) stretch in the connecting peptide region in the haemagglutinin cleavage site (HACS) by the viral RNA-dependent RNA polymerase (RdRp) is provided. Multiple non-consensus species of RNA were detected in the infected host, corresponding to likely intermediate sequences between the putative low pathogenic precursor nucleotide sequence of the H5 influenza strain and the highly pathogenic avian influenza virus gene sequence. In silico analysis of the identified RNA sequences predicted that the intermediary H5 sequence PQREKRGLF plays an important role in subsequent mutational events that relocate the HACS coding region from stable base-paired RNA regions to a single-stranded bulge, thereby priming the connecting peptide coding region for RdRp slippage.

Purification and immunogenicity of hemagglutinin from highly pathogenic avian influenza virus H5N1 expressed in Nicotiana benthamiana.

Highly pathogenic avian influenza (HPAI) H5N1 is an ongoing global health concern due to its severe sporadic outbreaks in Asia, Africa and Europe, which poses a potential pandemic threat. The development of safe and cost-effective vaccine candidates for HPAI is considered the best strategy for managing the disease and addressing the pandemic preparedness. The most potential vaccine candidate is the antigenic determinant of influenza A virus, hemagglutinin (HA). The present research was aimed at developing optimized expression in Nicotiana benthamiana and protein purification process for HA from the Malaysian isolate of H5N1 as a vaccine antigen for HPAI H5N1. Expression of HA from the Malaysian isolate of HPAI in N. benthamiana was confirmed, and more soluble protein was expressed as truncated HA, the HA1 domain over the entire ectodomain of HA. Two different purification processes were evaluated for efficiency in terms of purity and yield. Due to the reduced yield, protein degradation and length of the 3-column purification process, the 2-column method was chosen for target purification. Purified HA1 was found immunogenic in mice inducing H5 HA-specific IgG and a hemagglutination inhibition antibody. This paper offers an alternative production system of a vaccine candidate against a locally circulating HPAI, which has a regional significance.

Versatility of a localized surface plasmon resonance-based gold nanoparticle-alloyed quantum dot nanobiosensor for immunofluorescence detection of viruses.

Flu infection, caused by the influenza virus, constitutes a serious threat to human lives worldwide. A rapid, sensitive and specific diagnosis is urgently needed for point-of-care treatment and to control the rapid spread of this disease. In this study, an ultrasensitive, rapid and specific localized surface plasmon resonance (LSPR)-induced immunofluorescence nanobiosensor has been developed for the influenza virus based on a gold nanoparticle (AuNP)-induced quantum dot (QD) fluorescence signal. Alloyed quaternary CdSeTeS QDs were synthesized via the hot-injection organometallic route and were subsequently capped with l-cysteine via a ligand exchange reaction. AuNPs were synthesized in HEPES buffer and thiolated with l-cysteine. The concept of the biosensor involves the conjugation of anti-neuraminidase (NA) antibody (anti-NA Ab) to thiolated AuNPs and the conjugation of anti-hemagglutinin (HA) antibody (anti-HA Ab) to alloyed quaternary l-cysteine-capped CdSeTeS QDs. Interaction of the antigens displaying on the surface of the influenza virus target with anti-NA Ab-conjugated AuNPs and anti-HA Ab-conjugated QDs induces an LSPR signal from adjacent AuNPs to trigger fluorescence-enhancement changes in the QDs in proportion to the concentration of the target virus. The detection limit for influenza H1N1 virus was 0.03pg/mL in deionized water and 0.4pg/mL in human serum; while, for the clinically isolated H3N2, the detection limit was 10PFU/mL. The detection of influenza virus H1N1 was accomplished with high sensitivity. The versatility of the biosensor was demonstrated for the detection of clinically isolated influenza virus H3N2 and norovirus-like particles (NoV-LPs).

Trypsin pre-treatment corrects SRID over-estimation of immunologically active, pre-fusion HA caused by mixed immunoprecipitin rings.

Influenza vaccines are the primary intervention to prevent the substantial health burden of seasonal and pandemic influenza. Subunit and split influenza vaccines are formulated, released for clinical use, and tested for stability based on their content of immunologically active (capable of eliciting functional antibodies) hemagglutinin (HA). Single-radial immunodiffusion (SRID), the standard in vitro potency assay in the field, is believed to specifically detect immunologically active HA. We confirmed that, with conformationally homogeneous HA preparations, SRID specifically detected native, pre-fusion HA, which elicited influenza neutralizing and hemagglutination inhibiting (HI) antibodies in mice, and it did not detect low-pH stressed, post-fusion HA, which was selectively removed from the SRID gel during a blotting step and was significantly less immunologically active. This selective detection was due to the SRID format, not a conformational specificity of the sheep antiserum used in the SRID, as the same antiserum detected non-stressed and low-pH stressed HA similarly when used in an ELISA format. However, when low-pH stressed HA was mixed with non-stressed HA, SRID detected both forms in mixed immunoprecipitin rings, leading to over-quantification of pre-fusion HA. We previously reported that trypsin digestion of antigen samples selectively degrade stressed HA, so that an otherwise conformationally insensitive biophysical quantification technique, reversed-phase high pressure liquid chromatography (RP-HPLC), can specifically quantify trypsin-resistant, immunologically active, pre-fusion HA. Here, we report that trypsin digestion can also improve the specificity of SRID so that it can quantify immunologically active, pre-fusion HA when it is mixed with less immunologically active, post-fusion HA.

Characterization of Influenza Vaccine Hemagglutinin Complexes by Cryo-Electron Microscopy and Image Analyses Reveals Structural Polymorphisms.

Influenza virus afflicts millions of people worldwide on an annual basis. There is an ever-present risk that animal viruses will cross the species barrier to cause epidemics and pandemics resulting in great morbidity and mortality. Zoonosis outbreaks, such as the H7N9 outbreak, underscore the need to better understand the molecular organization of viral immunogens, such as recombinant influenza virus hemagglutinin (HA) proteins, used in influenza virus subunit vaccines in order to optimize vaccine efficacy. Here, using cryo-electron microscopy and image analysis, we show that recombinant H7 HA in vaccines formed macromolecular complexes consisting of variable numbers of HA subunits (range, 6 to 8). In addition, HA complexes were distributed across at least four distinct structural classes (polymorphisms). Three-dimensional (3D) reconstruction and molecular modeling indicated that HA was in the prefusion state and suggested that the oligomerization and the structural polymorphisms observed were due to hydrophobic interactions involving the transmembrane regions. These experiments suggest that characterization of the molecular structures of influenza virus HA complexes used in subunit vaccines will lead to better understanding of the differences in vaccine efficacy and to the optimization of subunit vaccines to prevent influenza virus infection.

A novel immunochromatographic system for easy-to-use detection of group 1 avian influenza viruses with acquired human-type receptor binding specificity.

A switch of viral hemagglutinin receptor binding specificity from bird-type α2,3- to human-type α2,6-linked sialic acid is necessary for an avian influenza virus to become a pandemic virus. In this study, an easy-to-use strip test to detect receptor binding specificity of influenza virus was developed. A biotinylated anti-hemagglutinin antibody that bound a broad range of group 1 influenza A viruses and latex-conjugated α2,3 (blue) and α2,6 (red) sialylglycopolymers were used in an immunochromatographic strip test, with avidin and lectin immobilized on a nitrocellulose membrane at test and control lines, respectively. Accumulation of a sialylglycopolymer-virus-antibody complex at the test line was visualized by eye. The strip test could be completed in 30min and did not require special equipment or skills, thereby avoiding some disadvantages of current methods for analyzing receptor binding specificity of influenza virus. The strip test could detect the receptor binding specificity of a wide range of influenza viruses, as well as small increases in the binding affinity of variant H5N1 viruses to α2,6 sialylglycans at viral titers >128 hemagglutination units. The strip test results were in agreement with those of ELISA virus binding assays, with correlations >0.95. In conclusion, the immunochromatographic strip test developed in this study should be useful for monitoring potential changes in the receptor binding specificity of group 1 influenza A viruses in the field.

A fluorescent aptasensor for H5N1 influenza virus detection based-on the core-shell nanoparticles metal-enhanced fluorescence (MEF).

A fluorescent aptasensor system has been designed for the sensitive detection of recombinant hemagglutinin (rHA) protein of the H5N1 influenza virus in human serum. Guanine-richen anti-rHA aptamers by SELEX were immobilized on the surface of the Ag@SiO2 nanoparticles which performed as a metal-enhanced fluorescence (MEF) sensing platform. Thiazole orange (TO) was used as fluorescent tag which reported to the G-quadruplex secondary structural induced by aptamer-rHA binding event. In the absence of rHA protein, TO was free in the solution with almost no fluorescence emission. When rHA protein was added to the solution, the aptamer strand bound rHA protein to form a stable G-quadruplex complex, which can bind TO and excite the fluorescence emission of TO. Moreover, the excited-state TO captured by the G-quadruplex complex was forced to the surface of the Ag@SiO2 nanoparticles and could experience a surface plasmon resonance enhancement which can be transformed into more efficient fluorescence emission signals, therefore, the fluorescence signal of TO can be amplified largely. This system does not require covalent labeling with fluorophores to the aptamer and the background noise is very low. The detection of rHA protein of the H5N1 influenza virus could be operated both in aqueous buffer and human serum with the detection limit of 2 and 3.5ng/mL respectively. More important, the whole detection process can be finished in a PE tube within 30min, which makes it suitable as a self-contained diagnostic kit for H5N1 influenza virus point-of-care (POC) diagnostic.

Expression, purification and characterization of glycosylated influenza H5N1 hemagglutinin produced in Pichia pastoris.

The A/swan/Poland/305-135V08/2006 (H5N1-subtype) hemagglutinin (HA) gene was cloned and expressed in yeast Pichia pastoris (P. pastoris). The HA cDNA lacking the C-terminal transmembrane anchor-coding sequence was fused to an α-factor leader peptide and placed under control of the methanol-inducible P. pastoris alcohol oxidase 1 (AOX1) promoter. Two P. pastoris strains: SMD 1168 and KM 71 were used for protein expression. Recombinant HA protein was secreted into the culture medium reaching an approximately 15 mg/L (KM 71 strain). Fusion protein with a His6 tag was purified to homogeneity in one step affinity chromatography. SDS-PAGE and MS/MS analysis indicated that the protein is cleaved into HA1 and HA2 domains linked by a disulfide bond. Analysis of the N-linked glycans revealed that the overexpressed HA is fully glycosylated at the same sites as the native HA in the vaccine strain. Immunological activity of the hemagglutinin protein was tested in mice, where rHA elicited a high immune response.

Technology transfer and scale-up of the Flublok recombinant hemagglutinin (HA) influenza vaccine manufacturing process.

Multiple different hemagglutinin (HA) protein antigens have been reproducibly manufactured at the 650L scale by Protein Sciences Corporation (PSC) based on an insect cell culture with baculovirus infection. Significantly, these HA protein antigens were produced by the same Universal Manufacturing process as described in the biological license application (BLA) for the first recombinant influenza vaccine approved by the FDA (Flublok). The technology is uniquely designed so that a change in vaccine composition can be readily accommodated from one HA protein antigen to another one. Here we present a vaccine candidate to combat the recently emerged H7N9 virus as an example starting with the genetic sequence for the required HA, creation of the baculovirus and ending with purified protein antigen (or vaccine component) at the 10L scale accomplished within 38 days under GMP conditions. The same process performance is being achieved at the 2L, 10L, 100L, 650L and 2500L scale. An illustration is given of how the technology was transferred from the benchmark 650L scale facility to a retrofitted microbial facility at the 2500L scale within 100 days which includes the time for facility engineering changes. The successful development, technology transfer and scale-up of the Flublok process has major implications for being ready to make vaccine rapidly on a worldwide scale as a defense against pandemic influenza. The technology described does not have the same vulnerability to mutations in the egg adapted strain, and resulting loss in vaccine efficacy, faced by egg based manufacture.

Broad cross-reactive epitopes of the H5N1 influenza virus identified by murine antibodies against the A/Vietnam/1194/2004 hemagglutinin.

There is an urgent need for a rapid diagnostic system to detect the H5 subtype of the influenza A virus. We previously developed monoclonal antibodies (mAbs) against the H5 hemagglutinin (HA) for use in a rapid diagnostic kit. In this study, we determined the epitopes of the anti-H5 HA murine mAbs OM-b, AY-2C2, and YH-1A1. Binding assays of the mAbs to different strains of H5 HAs indicated that OM-b and AY-2C2 cross-reacted with HAs from clades 1, 2.1.3.2, 2.2, and 2.3.4, whereas YH-1A1 failed to bind to those of clades 2.1.3.2 and 2.3.4. HA chimeras revealed that the epitopes for each of the mAbs were in the HA1 region. Analysis of escape mutants revealed that OM-b and AY-2C2 mAbs interacted mainly with amino acid residues D43 and G46, and the YH-1A1 mAb interacted with G139 and K or R140 of H5 HA. Multiple alignments of H5 HA protein sequences showed that D43 and G46 were very conserved among H5N1 HAs, except those in clade 2.2.1 and clade 7 (88.7%). The epitope for YH-1A1 mAb was highly variable in the HAs of H5N1, although it was well conserved in those of H5N2-N9. The OM-b and AY-2C2 mAbs could bind to the HAs of clades 1.1 and 2.3.2.1 that are currently epidemic in Asia, and we conclude that these would be effective for the detection of H5N1 infections in this region.

Ultrasensitive electrochemical immunoassay for avian influenza subtype H5 using nanocomposite.

We report a novel electrochemical immunosensor that can sensitively detect avian influenza virus H5 subtype (AIV H5) captured by graphene oxide-H5-polychonal antibodies-bovine serum albumin (GO-PAb-BSA) nanocomposite. The graphene oxide (GO) carried H5-polychonal antibody (PAb) were used as signal amplification materials. Upon signal amplification, the immunosensor showed a 256-fold increase in detection sensitivity compared to the immunosensor without GO-PAb-BSA. We designed a PAb labeling GO strategy and signal amplification procedure that allow ultrasensitive and selective detection of AIV H5. The established method responded to 2(-15) HA unit/50 µL H5, with a linear calibration range from 2(-15) to 2(-8) HA unit/50 µL. In summary, we demonstrated that the immunosenser has a high specificity and sensitivity for AIV H5, and the established assay could be potentially applied in the rapid detection of other pathogenic microorganisms.

Cold adaptation improves the growth of seasonal influenza B vaccine viruses.

Gene reassortment has proved useful in improving yields of influenza A antigens of egg-based inactivated vaccines, but similar approaches have been difficult with influenza B antigens. Current regulations for influenza vaccine seed viruses limit the number of egg passages and as a result resultant yields from influenza B vaccine seed viruses are frequently inconsistent. Therefore, reliable approaches to enhance yields of influenza B vaccine seed viruses are required for efficient vaccine manufacture. In the present study three stable cold-adapted (ca) mutants, caF, caM and caB derived from seasonal epidemic strains, B/Florida/4/2006, B/Malaysia/2506/2004 and B/Brisbane/60/2008 were prepared, which produced high hemagglutinin antigen yields and also increased viral yields of reassortants possessing the desired 6:2 gene constellation. The results demonstrate that consistent improvements in yields of influenza B viruses can be obtained by cold adaptation following extended passage. Taken together, the three ca viruses were shown to have potential as donor viruses for the preparation of high-yielding influenza B vaccine viruses by reassortment.

Characterization of pandemic influenza A (H1N1) virus hemagglutinin specific polyclonal antibodies for biosensor applications.

In this study, recombinant hemagglutinin protein (rH1N1HA) of Pandemic influenza virus and polyclonal antibodies against it for biosensor applications have been characterized. For rapid and high sensitive detection of H1N1 virus or its antibodies, PCR-free and label free detection method based on a surface plasmon resonance technique has been proposed. The glycosylated H1N1HA protein was expressed in yeast and the authenticity of the expressed protein was confirmed by Western blotting. Rabbit polyclonal antibodies developed against rH1N1HA protein were evaluated for their ability to neutralize H1N1 virus through plaque reduction neutralization test and indirect ELISA. Affinity purified anti-H1N1HA IgG were characterized further for their specificity, affinity of interaction, the association and dissociation rates at which they interact through surface plasmon resonance technique. The equilibrium constant and maximum binding capacity of analyte was found to be 49.7 nM and 47.28m°, respectively. The assay could detect a lowest IgG of 0.5 ng on a rH1N1HA coated chip. Combined with the high sensitivity of surface plasmon resonance technique and specificity of the reagents, it is possible to develop a rapid detection assay for monitoring influenza infections.