Influenza Virus Antigens Research Articles

Potentiating lung-localized immune responses to conserved influenza virus antigen through a novel vaccine platform

Abstract Current influenza vaccines are primarily delivered at peripheral sites and provide immunity characterized by neutralizing antibody responses. However, this strategy often fails to provide broadly-protective immune capacity. Numerous studies have implicated lung localized T cells as key mediators of broadly protective immunity to influenza infection. The goal of our current research is to expand virus-specific CD4 T cells via the respiratory route in order to understand how this mode of priming poises the host for future protective immunity to infection. We have explored a novel nanolipoprotein (NLP) vaccine conjugated to influenza nucleocapsid protein (NP) to boost lung localized immunity. ELISA assays revealed that NLP immunization boosted anti-NP antibody titers, which have been shown to contribute to heterosubtypic immunity. Importantly, immunization with NLP elicited NP-specific CD4 and CD8 T cell responses in the lung, draining lymph node, and spleen. Intravascular labeling and flow cytometry were performed to assess the localization and phenotype of CD4 T cells in the lung. Analyses revealed that intranasal immunization with NLP elicited an increased abundance of lung parenchyma-localized CD4 T cells expressing surface markers associated with tissue residency and cytolytic potential. Using a novel pMHC class II tetramer, we observed that the majority of antigen-specific CD4 T cells were localized to the lung tissue and can rapidly produce antiviral cytokines upon restimulation. These studies will improve our understanding of vaccine strategies deigned to elicit effector T cells at mucousal sites and demonstrate the potential of tissue resident cells to mediate broadly protective immune responses to influenza.

Construction and Immunogenicity of a Novel Multivalent Vaccine Prototype Based on Conserved Influenza Virus Antigens.

Influenza, an acute, highly contagious respiratory disease, remains a significant threat to public health. More effective vaccination strategies aimed at inducing broad cross-protection not only against seasonal influenza variants, but also zoonotic and emerging pandemic influenza strains are urgently needed. A number of conserved protein targets to elicit such cross-protective immunity have been under investigation, with long alpha-helix (LAH) from hemagglutinin stalk and ectodomain of matrix protein 2 ion channel (M2e) being the most studied ones. Recently, we have reported the three-dimensional structure and some practical applications of LAH expressed in Escherichia coli system (referred to as tri-stalk protein). In the present study, we investigated the immunogenicity and efficacy of a panel of broadly protective influenza vaccine prototypes based on both influenza tri-stalk and triple M2e (3M2e) antigens integrated into phage AP205 virus-like particles (VLPs). While VLPs containing the 3M2e alone induced protection against standard homologous and heterologous virus challenge in mice, only the combination of both conserved influenza antigens into a single VLP fully protected mice from a high-dose homologous H1N1 influenza infection. We propose that a combination of genetic fusion and chemical coupling techniques to expose two different foreign influenza antigens on a single particle is a perspective approach for generation of a broadly-effective vaccine candidate that could protect against the constantly emerging influenza virus strains.

A Multi-Targeting, Nucleoside-Modified mRNA Influenza Virus Vaccine Provides Broad Protection in Mice

Influenza viruses are respiratory pathogens of public health concern worldwide with up to 650,000 deaths occurring each year. Seasonal influenza virus vaccines are employed to prevent disease, but with limited effectiveness. Development of a universal influenza virus vaccine with the potential to elicit long-lasting, broadly cross-reactive immune responses is necessary for reducing influenza virus prevalence. In this study, we have utilized lipid nanoparticle-encapsulated, nucleoside-modified mRNA vaccines to intradermally deliver a combination of conserved influenza virus antigens (hemagglutinin stalk, neuraminidase, matrix-2 ion channel, and nucleoprotein) and induce strong immune responses with substantial breadth and potency in a murine model. The immunity conferred by nucleoside-modified mRNA-lipid nanoparticle vaccines provided protection from challenge with pandemic H1N1 virus at 500 times the median lethal dose after administration of a single immunization, and the combination vaccine protected from morbidity at a dose of 50ng per antigen. The broad protective potential of a single dose of combination vaccine was confirmed by challenge with a panel of group 1 influenza A viruses. These findings support the advancement of nucleoside-modified mRNA-lipid nanoparticle vaccines expressing multiple conserved antigens as universal influenza virus vaccine candidates.

Production and identification of monoclonal antibodies and development of a sandwich ELISA for detection of the H3-subtype avian influenza virus antigen

The H3 subtype of avian influenza virus (AIV) is widespread in avian species and is frequently isolated in surveillance projects; thus, we have developed a more effective diagnostic approach of a monoclonal antibody (mAb)-based sandwich ELISA for the H3 AIV detection. First, we have produced the essential reagent of mAb against AIV H3 strains with the development of an mAb-Mouse immunization with a purified H3-subtype AIV strain and cell fusion to generate hybridoma cells. These cells were screened with hemagglutination inhibition (HI) tests, and optimal cells were subcloned. We chose a hybridoma cell line that steadily secreted a specific H3-subtype AIV mAb, designated 9F12, that belongs to the IgG1 subclass and has a K-type light chain. 9F12 was shown to bind specifically to the H3-subtype AIV antigen by both immunofluorescence assay and Western blot analysis. Finally, a 9F12-based sandwich ELISA was successfully developed and used to specifically test for this antigen. The sandwich ELISA conditions were optimized, and the specificity and sensitivity were validated. The results for clinical sample detection were consistent with viral isolation. Consequently, the 9F12-based sandwich ELISA is a specific, sensitive, robust, rapid and versatile diagnostic tool for H3-subtype AIV and provides a promising strategy for effective influenza virus prevention and control.

Development and characterization of standard reagents for cell-based prepandemic influenza vaccine products

ABSTRACT Outbreaks of infection by novel avian influenza virus strains in humans cause public health issues worldwide, and the development of vaccines against such novel strains is the most effective method for the prevention of these virus outbreaks. All types of vaccines must be tested for potency before use; thus, quantitative potency assays are needed for influenza vaccines. The single radial immunodiffusion (SRID) assay is considered the gold standard for quantification of influenza virus antigens, and the SRID reference reagents are essential for the determination of vaccine potency. However, it remains debatable whether reference reagents derived from egg-based vaccine platforms can be used to precisely quantify non-egg-derived vaccines; thus, influenza vaccine production using cell-based platforms has attracted increasing attention. To evaluate the utility of reference reagents derived from a cell-based influenza vaccine platform, we prepared cell-based reference reagents from MDCK cell-grown viruses and compared them with egg-derived reference reagents. A primary liquid standard (PLS) was purified from cell-derived candidate influenza vaccine viruses, and hemagglutinin (HA) antigen content was determined by a densitometric method. The produced PLS could be stored at 4°C for more than 10 months. We also established a simple HA protein purification method for goat antiserum preparation, and the performance of the resulting antiserum was compared to that of standard reagents obtained using different production platforms. The results of this study indicate that these reference reagents can be used for both cell-based and egg-based production platforms and that the differences between these two types of platforms are negligible.

Poly(I:C) augments inactivated influenza virus-chitosan nanovaccine induced cell mediated immune response in pigs vaccinated intranasally

To improve the innate and adaptive immune responses elicited by a killed/inactivated swine influenza virus antigen (KAg)-loaded chitosan nanoparticles (CS NPs-KAg), we used the adjuvant, poly(I:C). The formulated CS NPs-KAg and CS NPs-poly(I:C) had a net surface charge of +30.7 mV and +25.1 mV, respectively. The CS NPs-KAg was coadministered with CS NPs-poly(I:C) (chitosan nanovaccine) as intranasal mist. Vaccinations enhanced homologous (H1N2-OH10) and heterologous (H1N1-OH7) hemagglutination inhibition (HI) titers in both vaccinated and virus-challenged animals compared to the control soluble poly(I:C) vaccinated pigs. In addition, the chitosan nanovaccine induced the proliferation of antigen-specific IFNγ secreting T-helper/memory and γδ T cells compared to control poly(I:C) group; and an increased Th1 (IFNγ, IL-6 and IL-2) and Th2 (IL-10 and IL-13) cytokines mRNA expression in the tracheobronchial lymph nodes compared to lymphoid tissues obtained from pigs given commercial influenza vaccine. The virus load in nasal passages and microscopic lung lesions were partially reduced by both chitosan nanovaccine and commercial vaccine. The HA gene homology between the vaccine and challenge viruses indicated that the chitosan nanovaccine induced a cross-protective immune response. In conclusion, coadministration of CS NPs-poly(I:C) with CS NPs-KAg augmented the cross-reactive specific HI titers and the cell-mediated immune responses in pigs.

Strain specificities in influence of ageing on germinal centre reaction to inactivated influenza virus antigens in mice: Sex-based differences

Considering variability in vaccine responsiveness across human populations, in respect to magnitude and quality, and importance of vaccines in the elderly, the influence of recipient genetic background on the kinetics of age-related changes in the serum IgG antibody responses to seasonal trivalent inactivated split-virus influenza bulk (TIV) was studied in BALB/c and C57BL/6 mice showing quantitative and qualitative differences in this responses in young adult ages. With ageing the total serum IgG response to influenza viruses declined, in a strain-specific manner, so the strain disparity observed in young adult mice (the greater magnitude of IgG response in BALB/c mice) disappeared in aged mice. However, the sexual dimorphisms in this response (more prominent in females of both strains) remained in aged ones. The strain-specific differences in age-related decline in the magnitude of IgG response to TIV correlated with the number of germinal centre (GC) B splenocytes. The age-related decline in GC B cell number was consistent with the decrease in the proliferation of B cells and CD4+ cells in splenocyte cultures upon restimulation with TIV. Additionally, the age-related decrease in the magnitude of IgG response correlated with the increase in follicular T regulatory (fTreg)/follicular T helper (fTh) and fTreg/GC B splenocyte ratios (reflecting decrease in fTh and GC B numbers without changes in fTreg number), and the frequency of CD4+ splenocytes producing IL-21, a key factor in balancing the B cell and fTreg cell activity. With ageing the avidity of virus influenza-specific antibody increased in females of both strains. Moreover, ageing affected IgG2a/IgG1 and IgG2c/IgG1 ratios (reflecting Th1/Th2 balance) in male BALB/c mice and female C57BL/6 mice, respectively. Consequently, differently from young mice exhibiting the similar ratios in male and female mice, in aged female mice of both strains IgG2a(c)/IgG1 ratios were shifted towards a less effective IgG1 response (stimulated by IL-4 cytokines) compared with males. The age-related alterations in IgG subclass profiles in both strains correlated with those in IFN-γ/IL-4 production level ratio in splenocyte cultures restimulated with TIV. These findings stimulate further research to formulate sex-specific strategies to improve efficacy of influenza vaccine in the elderly.

Avian Influenza Virus Sample Types, Collection, and Handling.

Successful detection of avian influenza (AI) virus, viral antigen, nucleic acid, or antibody is dependent upon the collection of the appropriate sample type, the quality of the sample, and the proper storage and handling of the sample. The diagnostic tests to be performed should be considered prior to sample collection. Sera are acceptable samples for ELISA or agar gel immunodiffusion tests, but not for real-time RT-PCR. Likewise, swabs and/or tissues are acceptable for real-time RT-PCR and virus isolation. The sample type will also depend on the type of birds that are being tested; oropharyngeal swabs from gallinaceous poultry and cloacal swabs from waterfowl are the preferred specimens for most diagnostic tests, although it is optimal to collect swabs from both locations, if possible. In addition to collecting the appropriate sample for the tests to be performed, selecting the right materials for sample collection (i.e., type of swab) is very important. This chapter will outline the collection of different specimen types and procedures for proper specimen handling.

Incidence and Distribution of Respiratory Microorganisms Causing Acute Respiratory Infections at the University Hospital of Korea.

Acute respiratory infection caused by respiratory microorganisms including various kinds of viruses and bacteria is the most common infectious disease. When managing patients, it is crucial to detect these microorganisms rapidly and monitor their occurrence and tendency. Recently, the methods of detecting them have been implemented by molecular diagnostics. The authors intended to investigate their incidence and distribution and identify the significance of the molecular diagnosis for their detection. The retrospective study was conducted to investigate the incidence and distribution of respiratory microorganisms according to the age, gender, month, season, and the detection method and to analyze their co-infections from July 2016 to December 2019. In addition, the four types of turn-around time (TAT) for each detec-tion method were also analyzed. The overall incidence for at least one respiratory microorganism was 23.1% (3,645/15,808). The highest incidence was identified in age group 2 (1 - 3 months), 38.5%. The incidence rates by multiplex PCR using Anyplex and Allplex, FilmArray method, and influenza virus (flu) antigen detection test were 44.2% (718/1,625), 63.1% (1,198/1,899), and 14.1% (1,729/12,284), respectively. The overall incidence between male and female patients showed no statistically significant difference (p = 0.980), except for the flu antigen detection test (p = 0.000). Influenza A viruses (flu A) accounted for the highest percentage (34.9%), followed by rhinovirus/enterovirus (20.5%), RSV (12.8%), flu B (8.3%), and adenovirus (7.6%). These microorganisms showed characteristic distribution patterns according to season and month. Flu A and flu B predominated in winter and accounted for an increasing proportion as age increased according to the age groups. The overall co-infection rate was 22.5% (432/1,916). The average TATs of the FilmArray method were significantly much faster than multiplex PCR using Anyplex and Allplex (p = 0.000). The information on the incidence and distribution of respiratory microorganisms and their expeditious detection are considered critical to the management of the elderly, immunocompromised patients, and children. The rapid molecular-based diagnosis of respiratory infections would be beneficial in medical decision and prevention of their propagation.

Adsorptive mutation and N-linked glycosylation modulate influenza virus antigenicity and fitness

ABSTRACT Influenza viruses have an error-prone polymerase complex that facilitates a mutagenic environment. Antigenic mutants swiftly arise from this environment with the capacity to persist in both humans and economically important livestock even in the face of vaccination. Furthermore, influenza viruses can adjust the antigenicity of the haemagglutinin (HA) protein, the primary influenza immunogen, using one of four molecular mechanisms. Two prominent mechanisms are: (1) enhancing binding avidity of HA toward cellular receptors to outcompete antibody binding and (2) amino acid substitutions that introduce an N-linked glycan on HA that sterically block antibody binding. In this study we investigate the impact that adsorptive mutation and N-linked glycosylation have on receptor-binding, viral fitness, and antigenicity. We utilize the H9N2 A/chicken/Pakistan/SKP-827/16 virus which naturally contains HA residue T180 that we have previously shown to be an adsorptive mutant relative to virus with T180A. We find that the addition of N-linked glycans can be beneficial or deleterious to virus replication depending on the background receptor binding avidity. We also find that in some cases, an N-linked glycan can trump the effect of an avidity enhancing substitution with respect to antigenicity. Taken together these data shed light on a potential route to the generation of a virus which is “fit” and able to overcome vaccine pressure.

Immunohistochemical Staining of Influenza Virus in Tissues.

Immunohistochemical methods are commonly used for studying the pathogenesis of influenza A virus by allowing the identification of sites of replication of the virus in infected tissues and the correlation with the histopathological changes observed. In this chapter, the materials and procedures for performing immunohistochemical detection of influenza virus antigens in tissues using a biotin-streptavidin detection method are provided. The technique involves the following steps: heat-induced antigen retrieval, binding of a primary antibody to the virus antigen, antibody-antigen complex binding by a biotinylated secondary antibody, and binding of an enzyme-streptavidin conjugate. The enzyme is then visualized by application of the substrate chromogen solution to produce a colorimetric reaction with a product that can be visualized. Visualization of influenza virus antigen in tissues is based on chromogen deposition in the nucleus and cytoplasm of infected cells.

Antigenic Site Variation in the Hemagglutinin of Pandemic Influenza A(H1N1)pdm09 Viruses between 2009-2017 in Ukraine.

The hemagglutinin (HA) is a major influenza virus antigen, which, once recognized by antibodies and substitutions in HA genes, helps virus in escaping the human immune response. It is therefore critical to perform genetic and phylogenetic analysis of HA in circulating influenza viruses. We performed phylogenetic and genetic analysis of isolates from Ukraine, the vaccine strain and reference strains were used to phylogenetically identify trends in mutation locations and substitutions. Ukrainian isolates were collected between 2009–2017 and clustered in the influenza genetic groups 2, 6, 7, and 8. Genetic changes were observed in each of the antigenic sites: Sa – S162T, K163Q, K163I; Sb – S185T, A186T, S190G, S190R; Ca1 – S203T, R205K, E235V, E235D, S236P; Ca2 – P137H, H138R, A141T, D222G, D222N; Cb – A73S, S74R, S74N. In spite of detected mutations in antigenic sites, Ukrainian isolates retained similarity to the vaccine strain A/California/07/09 circulated during 2009–2017. However, WHO recommended a new vaccine strain A/Michigan/45/2015 for the Southern Hemisphere after the emergence of the new genetic groups 6B.1 and 6B.2. Our study demonstrated genetic variability of HA protein of A(H1N1)pdm09 viruses isolated in 2009–2017 in Ukraine. Influenza surveillance is very important for understanding epidemiological situations.

Generation of Stable Influenza Virus Hemagglutinin through Structure-Guided Recombination.

Hemagglutinin (HA) is the major surface antigen of influenza virus and the most promising influenza vaccine immunogen. In 2013, the devastating H7N9 influenza virus was identified in China, which induced high mortality. The HA of this virus (H7) is relatively unstable, making it challenging to produce an effective vaccine. To improve the stability of HA protein from H7N9 influenza virus for better vaccine antigens without impairing immunogenicity, we recombined the HA from H7N9 (H7) with a more stable HA from H3N2 (H3) by structure-guided recombination, resulting in six chimeric HAs, FrA-FrF. Two of these chimeric HAs, FrB and FrC, exhibited proper hemagglutination activity and presented improved thermal stability compared to the original H7. Mice immunized with FrB and FrC elicited H7-specific antibodies comparable to those induced by parental H7, and the antisera collected from these immunized mice successfully inhibited H7N9 infection in a microneutralization assay. These results suggest that our structural-recombination approach can create stabilizing chimeric antigens while maintaining proper immunogenicity, which may not only benefit the construction of more stable HA vaccines to fight against H7N9 infection, but also facilitate effective vaccine improvements for other influenza viruses or infectious pathogens. In addition, this study also demonstrates the potential for better engineering of multimeric protein complexes like HA to achieve improved function, which are often immunologically or pharmaceutically important but difficult to modify.

Detection of influenza virus by agglutination using nanoparticles conjugated with a sialic acid-mimic peptide

Influenza virus (IFV) detection in the early phase of disease is critical for effective anti-influenza therapy using neuraminidase inhibitors. Sialyloligosaccharide receptors on the surface of respiratory cells are recognized by IFV hemagglutinin (HA) in the infection. Here, we show that agglutination of IFV is detected using poly(glycidyl methacrylate) (PGMA)-coated polystyrene nanoparticles conjugated with a sialic acid-mimic peptide. The azido peptide was immobilized onto the surface of the PGMA-coated nanoparticles by click chemistry. The distribution of particle size, determined by dynamic light scattering, indicated that the peptide-conjugated nanoparticles were agglutinated in the presence of HA and IFV. Nanoparticles conjugated with the receptor-mimic peptide may be a useful alternative to red blood cells in the global surveillance and clinical diagnosis of influenza. This study examines the use of poly(glycidyl methacrylate) (PGMA)-coated polystyrene functionalized with peptides that mimic receptors found on red blood cells for detection of the influenza virus. Although the current method, the hemagglutination inhibition test, utilized by the World Health Organization, is simple, it has some limitations. We found that our peptide dimers attached to beads were efficiently agglutinated, leading us to detect the presence of the influenza virus antigens. We believe that our study makes a significant contribution to the fight against influenza because we provide an alternative method for the important task of influenza surveillance and diagnosis.

Vaccination with viral vectors expressing NP, M1 and chimeric hemagglutinin induces broad protection against influenza virus challenge in mice

Seasonal influenza virus infections cause significant morbidity and mortality every year. Annual influenza virus vaccines are effective but only when well matched with circulating strains. Therefore, there is an urgent need for better vaccines that induce broad protection against drifted seasonal and emerging pandemic influenza viruses. One approach to design such vaccines is based on targeting conserved regions of the influenza virus hemagglutinin. Sequential vaccination with chimeric hemagglutinin constructs can refocus antibody responses towards the conserved immunosubdominant stalk domain of the hemagglutinin, rather than the variable immunodominant head. A complementary approach for a universal influenza A virus vaccine is to induce T-cell responses to conserved internal influenza virus antigens. For this purpose, replication deficient recombinant viral vectors based on Chimpanzee Adenovirus Oxford 1 and Modified Vaccinia Ankara virus are used to express the viral nucleoprotein and the matrix protein 1. In this study, we combined these two strategies and evaluated the efficacy of viral vectors expressing both chimeric hemagglutinin and nucleoprotein plus matrix protein 1 in a mouse model against challenge with group 2 influenza viruses including H3N2, H7N9 and H10N8. We found that vectored vaccines expressing both sets of antigens provided enhanced protection against H3N2 virus challenge when compared to vaccination with viral vectors expressing only one set of antigens. Vaccine induced antibody responses against divergent group 2 hemagglutinins, nucleoprotein and matrix protein 1 as well as robust T-cell responses to the nucleoprotein and matrix protein 1 were detected. Of note, it was observed that while antibodies to the H3 stalk were already boosted to high levels after two vaccinations with chimeric hemagglutinins (cHAs), three exposures were required to induce strong reactivity across subtypes. Overall, these results show that a combinations of different universal influenza virus vaccine strategies can induce broad antibody and T-cell responses and can provide increased protection against influenza.

Adjuvant activity of multimolecular complexes based on Glycyrrhiza glabra saponins, lipids, and influenza virus glycoproteins.

Numerous studies have shown that immunostimulatory complexes containing Quil-A saponin and various antigens are effective in stimulating the immune response and can be used as vaccine preparations for animals and humans. However, Quil-A saponin possesses toxicity and haemolytic activity. In the present work, a saponin-containing preparation named "Glabilox" was isolated from the roots of a Glycyrrhiza glabra L. plant by high-performance liquid chromatography (HPLC). The results showed that Glabilox has no toxicity or haemolytic activity and can form stable immunostimulatory complexes. Subcutaneous immunization of mice with an immunostimulating complex containing Glabilox and H7N1 influenza virus antigens stimulated high levels of humoral and cellular immunity. Vaccination of chickens with the same immunostimulating complex protected 100% of the animals after experimental infection with a homologous virus. Comparative studies showed that the immunogenic and protective activity of immunostimulatory complexes containing Quil-A and immunostimulatory complexes containing Glabilox are comparable to each other. The results of these studies indicated that Glycyrrhiza glabra saponins show great promise as safe and effective adjuvants.

Influence of aging on germinal centre reaction and antibody response to inactivated influenza virus antigens in mice: sex-based differences.

The study examined sex-specificities in age-related changes in BALB/c mice IgG antibody responses to immunisation with trivalent inactivated split-virus influenza bulk. Aging diminished the total serum IgG antibody responses to H1N1 and H3N2 and B influenza virus antigens in mice of both sexes, but they remained greater in aged females. This sex difference in aged mice correlated with the greater post-immunisation increase in the frequency of spleen germinal centre (GC) B cells and more favourable T follicular regulatory (Tfr)/GC B cell ratio, as Tfr cells are suggested to control antibody production through suppression of glycolysis. The greater post-immunisation GC B cell response in aged females compared with males correlated with the greater proliferation of B cells and CD4+ cells in splenocyte cultures from aged females restimulated with inactivated split-virus influenza from the bulk. To support the greater post-immunisation increase in the frequency GC B cell in aged females was more favourable Tfr/T follicular helper (Tfh) cell ratio. Additionally, compared with aged males, in age-matched females the greater avidity of serum IgG antibodies was found. However, in aged females IgG2a/IgG1 antibody ratio, reflecting spleen Th1/Th2 cytokine balance, was shifted towards IgG1 when compared with age-matched male mice. This shift was ascribed to a more prominent decline in the titres of functionally important IgG2a antibodies in females with aging. The study suggest that biological sex should be considered as a variable in designing strategies to manipulate with immune outcome of immunisation in aged animals, and possibly, at very long distance, humans.

Etiological analysis of virus in children with respiratory tract infection during spring in Shenyang regions

Objective To investigate the distribution of respiratory viruses in the throat swabs from children with respiratory tract infection in Shenyang and provide reference for the diagnosis and treatment of clinical diseases. Methods A total of 756 children with respiratory tract infection who were admitted to Shengjing Hospital of China Medical University in Shenyang from February 2018 to May 2018 were enrolled in this study.The antigens of influenza virus A (INF-A), influenza virus B (INF-B), adenovirus (ADV), respiratory syncytial virus (RSV), and parainfluenza virus types I, Ⅱ, and Ⅲ (PIV-Ⅰ, PIV-Ⅱ and PIV-Ⅲ) in nasopharyngeal swabs were detected by direct immunofluorescence. Results Of all the 756 throat swabs from hospitalized children, 214 (28.31%) had positive virus detection results.RSV (11.38%) had the highest detection rate in single-positive cases, accounting for 47.78% of single positive cases.In mixed virus infection, RSV combined with other viral infections accounted for 73.53% of mixed virus infections, and RSV was the main pathogen found in the study population.Analysis of the detection rate of virus infection related with age and gender showed that the younger the age, the higher the detection rate(χ2=14.216, P=0.007). The detection rate of patients younger than 6 months was 39.73% and RSV was the mostly detected pathogen (75.86%). The detection rate of male patients was 30.07%, which was higher than that of female patients with 25.99%(χ2=3.982, P=0.046), and RSV infection and mixed infection were the major.There was no significant difference in virus detection rates between patients with different clinical diagnoses. Conclusion RSV is the main pathogen during spring from February 2018 to May 2018 in Shenyang.The virus detection rate of patients younger than 6 months is higher and RSV is the mostly detected pathogen. Key words: Respiratory virus; Antigen; Direct immunofluorescence; Children

Biodegradable Hyaluronic Acid Modified with Tetraglycine-l-octaarginine as a Safe Adjuvant for Mucosal Vaccination.

We have been investigating the potential use of polymers modified with cell-penetrating peptides as an adjuvant for mucosal vaccination and have already developed nondegradable poly( N-vinylacetamide- co-acrylic acid) (PNVA- co-AA) with which d-octaarginine, a typical cell-penetrating peptide, was grafted. Our previous murine infection experiments demonstrated that immunoglobulin G (IgG) and immunoglobulin A (IgA) were induced in systemic circulation and secreted on nasal mucosa, respectively, through 4-time nasal inoculations with a mixture of influenza viral antigens and d-octaarginine-linked PNVA- co-AA at 7-day intervals, and that immunized mice were perfectly protected from homologous virus infection. In the present study, we designed novel biodegradable polymers bearing cell-penetrating peptides from a perspective of clinical application. Hyaluronic acid whose glucuronic acid was modified with tetraglycine-l-octaarginine at a monosaccharide unit ratio of 30% was successfully developed. The hyaluronic acid derivative exhibited adjuvant activities identical to PNVA- co-AA bearing either d-octaarginine or tetraglycine-d-octaarginine under the above-mentioned inoculation schedule. We further found that there was no difference in humoral immunity between the 4-time inoculations at 7-day intervals and the 2-time inoculations at 28-day intervals. Intranasal IgA induced through the latter schedule with a smaller number of inoculations, which is clinically practical, exhibited cross-reactivity beyond the subtype of viral strains. In vitro toxicity studies demonstrated that the hyaluronic acid derivative was much less toxic than the corresponding PNVA- co-AA derivatives, and that both the polymers and their metabolites did not exhibit genotoxicity. Our results suggested that tetraglycine-l-octaarginine-linked hyaluronic acid would be a clinically valuable and safe adjuvant for mucosal vaccination.

Corn-derived alpha-D-glucan nanoparticles as adjuvant for intramuscular and intranasal immunization in pigs

Adjuvant potential of positively charged corn-derived nanoparticles (Nano-11) was earlier revealed in mice. We evaluated its adjuvant role to electrostatically adsorbed inactivated/killed swine influenza virus antigen (KAg) (Nano-11 + KAg) in pigs. Nano-11 facilitated the uptake of KAg by antigen presenting cells and induced secretion of proinflammatory cytokines. In pigs vaccinated by an intranasal mist containing Nano-11 + KAg, expression of T-helper 1 and T-helper 2 transcription factors and secretion of cross-reactive influenza antigen-specific mucosal IgA in the nasal cavity were observed. The enhanced frequencies of IFN-γ positive T-helper and cytotoxic T-cells in Nano-11 + KAg-vaccinates after heterologous virus challenge were also observed. Clinically, slightly reduced influenza signs and pneumonic lesions, with mild reduction in virus load in the respiratory tract of vaccinates were observed. In pigs immunized with Nano-11 adsorbed ovalbumin administered by intramuscular (IM) route, enhanced IgG1 and IgG2 antibodies were detected in serum. Thus, Nano-11 vaccine delivery system confers adjuvant effect in pigs.