Mucosal immune responses and protective efficacy elicited by oral administration AMP-ZnONPs-adjuvanted inactivated H9N2 virus in chickens.

The avian influenza virus is infected through the mucosal route, thus mucosal barrier defense is very important. While the inactivated H9N2 vaccine cannot achieve sufficient mucosal immunity, adjuvants are needed to induce mucosal and systemic immunity to prevent poultry from H9N2 influenza virus infection. Our previous study found that polysaccharide from Atractylodes macrocephala Koidz binding with zinc oxide nanoparticles (AMP-ZnONPs) had immune-enhancing effects in vitro. This study aimed to evaluate the mucosal immune responses of oral whole-inactivated H9N2 virus (WIV)+AMP-ZnONPs and its impact on the animal challenge protection, and the corresponding changes of pulmonary metabolomics after the second immunization. The results showed that compared to the WIV, the combined treatment of WIV and AMP-ZnONPs significantly enhanced the HI titer, IgG and specific sIgA levels, the number of goblet cells and intestinal epithelial lymphocytes (iIELs) as well as the expression of J-chain, polymeric immunoglobulin receptor (pIgR), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α) and transforming growth factor-ß (TGF-ß). In viral attack experiments, WIV combing with AMP-ZnONPs effectively reduced lung damage and viral titers in throat swabs. Interestingly, significant changes of both the IgA intestinal immune network and PPAR pathway could also be found in the WIV+AMP-ZnONPs group compared to the non-infected group. Taken together, these findings suggest that AMP-ZnONPs can serve as a potential mucosal vaccine adjuvant, thereby avoiding adverse stress and corresponding costs caused by vaccine injection.

Efficiency of natural oils as alternative adjuvants to mineral oils in inactivated avian influenza vaccine formulation.

1. The effectiveness of inactivated vaccines depends on selecting the suitable adjuvant for vaccine formulation. The potency of vaccines with low antigen content can be improved with the appropriate adjuvant. This could allow production of more doses and lower the production cost.2. This study evaluated the efficiency of vaccines prepared using oil extracted from natural sources including argan oil, almond oil, sesame seed oil, pumpkin oil, cactus oil and black seed oil as alternative adjuvants for improving the protection capacity of inactivated influenza virus vaccine as compared to commonly used mineral oils.3. Each vaccine formulation was evaluated for stability, safety and immunogenicity in chickens, as well as for reducing the viral shedding after challenge infection.4. The cactus, sesame and pumpkin seed oil-based vaccines were found to be potent and successfully induced the production of humoral immunity in vaccinated chickens.

Effect of Echinacea purpurea (L.) Moench and its extracts on the immunization outcome of avian influenza vaccine in broilers.

ETHNOPHARMACOLOGICAL RELEVANCE: Echinacea purpurea (L.) Moench (EP) is a perennial herbaceous flowering plant with immunomodulatory effects. However, the immunomodulatory effects of EP on broilers after vaccination are still unclear. AIM OF THE STUDY: The aim is to study the effect of EP and Echinacea purpurea (L.) Moench extracts(EE) on avian influenza virus (AIV) immunity, and further explore the potential mechanism of immune regulation. MATERIALS AND METHODS: Broilers were fed with feed additives containing 2% EP or 0.5% EE, and vaccinated against avian influenza. The samples were collected on the 7th, 21st, and 35th day after vaccination, and the feed conversion ratio (FCR) was calculated. Blood antibody titer, jejunal sIgA content, tight junction protein, gene and protein expression of TLR4-MAPK signaling pathway were also detected. RESULTS: The results showed that vaccination could cause immune stress, weight loss, increase sIgA content, and up-regulate the expression of tight junction proteins, including zonula occludens-1 (ZO-1), Occludin, and Claudin-1, as well as the genes of Toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), receptor-associated factor 6 (TRAF6), activator protein 1 (AP-1) protein gene expression on TLR4-mitogen-activated protein kinase (MAPK) signaling pathway, and the protein expression of MyD88, extracellular regulated protein kinases (ERK), and c-Jun N-terminal kinase (JNK). EP and EE could increase the body weight of broilers, further improve antibody titers, decrease FCR, increase sIgA levels, up-regulate the expression of tight junction proteins, including ZO-1, Occludin, and Claudin-1, as well as the genes of TLR4, MyD88, TRAF6, and AP-1 and the protein expression of MyD88, ERK, and JNK in the TLR4-MAPK signaling pathway. CONCLUSION: In conclusion, EP and EE can increase the broiler's production performance and improve vaccine immune effect through the TLR4-MAPK signaling pathway.

In ovo administration of retinoic acid enhances cell-mediated immune responses against an inactivated H9N2 avian influenza virus vaccine.

The H9N2 subtype avian influenza virus (AIV) is a low pathogenic AIV that infects avian species and lead to huge economical losses in the poultry industry. The unique immunomodulatory properties of Retinoic acid (RA), an active component of vitamin A, highlights its potential to enhance chicken's resistance to infectious diseases and perhaps vaccine-induced immunity. Therefore, the present study evaluated the effects of in ovo supplementation of RA on the immunogenicity and protective efficacy of an inactivated avian influenza virus vaccine. On embryonic day 18, eggs were inoculated with either 90 µmol RA/200 µL/egg or diluent into the amniotic sac. On days 7 and 21 post-hatch, birds were vaccinated with 15 µg of ß-propiolactone (BPL) inactivated H9N2 virus via the intramuscular route. One group received BPL in combination with an adjuvant, while the other group received saline solution and served as a non-vaccinated control group. Serum samples were collected on days 7, 14, 21, 28, 35, and 42 post-primary vaccination (ppv) for antibody analysis. On day 24 ppv, spleens were collected, and splenocytes were isolated to analyze cytokine expression, interferon gamma (IFN-γ) production, and cell population. On day 28 ppv, birds in all groups were infected with H9N2 virus and oral and cloacal swabs were collected for TCID50 (50 % Tissue Culture Infectious Dose) assay up to day 7 post-infection. The results demonstrated that in ovo administration of RA did not significantly enhance the AIV vaccine-induced antibody response against H9N2 virus compared to the group that received the vaccine alone. However, RA supplementation enhanced the frequency of macrophages (KUL01+), expression of inflammatory cytokines and production of IFN-γ by splenocytes. In addition, RA administration reduced oral shedding of AIV on day 5 post-infection. In conclusion, these findings suggest that RA can be supplemented in ovo to enhance AIV vaccine efficacy against LPAIV.

Orally administered BZL-sRNA-20 oligonucleotide targeting TLR4 effectively ameliorates acute lung injury in mice.

The global COVID-19 pandemic emerged at the end of December 2019. Acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are common lethal outcomes of bacterial lipopolysaccharide (LPS), avian influenza virus, and SARS-CoV-2. Toll-like receptor 4 (TLR4) is a key target in the pathological pathway of ARDS and ALI. Previous studies have reported that herbal small RNAs (sRNAs) are a functional medical component. BZL-sRNA-20 (Accession number: B59471456; Family ID: F2201.Q001979.B11) is a potent inhibitor of Toll-like receptor 4 (TLR4) and pro-inflammatory cytokines. Furthermore, BZL-sRNA-20 reduces intracellular levels of cytokines induced by lipoteichoic acid (LTA) and polyinosinic-polycytidylic acid (poly (I:C)). We found that BZL-sRNA-20 rescued the viability of cells infected with avian influenza H5N1, SARS-CoV-2, and several of its variants of concern (VOCs). Acute lung injury induced by LPS and SARS-CoV-2 in mice was significantly ameliorated by the oral medical decoctosome mimic (bencaosome; sphinganine (d22:0)+BZL-sRNA-20). Our findings suggest that BZL-sRNA-20 could be a pan-anti-ARDS ALI drug.

Antiviral activity of CAVAC-1901, a combination of 3 standardized medicinal plants, against highly pathogenic influenza A virus in chickens.

Three different medicinal plants that consisted of the formulated mixture (CAVAC-1901) have been traditionally used for distinct medicinal purposes in different areas. Angelica dahurica has been used as an important ingredient of a prescription, Gumiganghwal-tang, for the common cold and influenza. Curcuma longa has been utilized for the treatment of asthma, and jaundice. Pinus densiflora (Korean red pine) has been used to improve memory and brain function for the treatment of vascular. Industrial livestock, which are characterized by dense breeding, are vulnerable to influenza infection, causing severe economic loss and social problems. However, there are no viable alternatives due to the risk of the occurrence of variants. Therefore, the aim of this study was to discover anti-influenza combinations of different medicinal plants with the concept of a multicomponent and multitarget (MCMT) strategy in traditional Chinese medicine (TCM). As part of a continuous project, 3 medicinal plants whose inhibitory activity against influenza A was previously reported at the compound level, and the inhibition of cytopathic effects (CPEs) by these formulated mixtures was evaluated against influenza A virus H1N1. A selected combination with an optimal ratio exhibiting synergistic activity was assessed for its antiviral activity in chickens against the highly pathogenic avian influenza (HPAI) H5N6. The selected combination (CAVAC-1901) showed potent inhibitory effects on the expression of neuraminidase and nucleoprotein, by RT-qPCR, Western blot, and immunofluorescence assays. The antiviral activity was more evident in chickens infected with H5N6. The sample-treated group (50 mg/kg/d) decreased mortality and virus titers in various organs. Our results indirectly suggest synergistic inhibitory activity of the combination of 3 different medicinal plants with different modes of action. Taken together, an optimally formulated mixture (CAVAC-1901) could serve as an effective alternative to current measures to minimize damage caused by HPAIs.

Immunological, biochemical and pathological effects of vitamin C and Arabic gum co-administration on H9N2 avian influenza virus vaccinated and challenged laying Japanese quails.

AIM: This study evaluated the effect of co-administration of vitamin C and Arabic gum (AG) supplements on the response of vaccinated (VAC) and challenged laying Japanese quails with avian influenza virus (AIV) H9N2. MATERIALS AND METHODS: One hundred and fifty 49-day-old laying Japanese quails were divided into 5 groups (G1-G5): the G1 group was a negative control, G2 group was unvaccinated + H9N2 challenged (Ch), G3 group was unvaccinated + supplements + Ch, G4 group was VAC + Ch, and the G5 group was VAC + supplements + Ch. The supplements (vitamin C, 1 g/liter of drinking water and AG, 1% ration) were given for 5 weeks post-vaccination (PV). The birds were injected subcutaneously with an inactivated H9N2 vaccine at 49 days of age. The quails were then challenged intranasally with AIV H9N2 at the 3rd week PV. Blood, tracheal swab and tissue samples were collected at the 1st, 2nd, and 3rd weeks PV, and at different time points post-challenge (PC). RESULTS: Growth performance, egg production (%), egg and eggshell weights, HI antibody titers, clinical signs, lesions, mortality, virus shedding rates, leukogram, biochemical and immunological parameters and histopathological lesions PC showed significant differences (P < 0.05) between the vaccinated-unsupplemented (G4) group and the vaccinated-supplemented (G5) group. G5 showed the highest (P < 0.05) growth performance, egg production, HI antibody titers, and heterophil phagocytic activity and the lowest heterophil/lymphocyte (H/L) ratio, mortality, virus shedding rates, creatinine level and histopathological lesion scores in the lungs. CONCLUSION: The co-administration of vitamin C and AG for 5 weeks can improve growth performance, egg production and the immune response in vaccinated laying quails challenged with AIV H9N2.

Evaluation of Efficacy of Oil Adjuvanted H5N6 Inactivated Vaccine against Highly Pathogenic H5N6 and H5N1 Influenza Viruses Infected Chickens.

BACKGROUND: Over the last 20 years, circulating highly pathogenic (HP) Asian H5 subtype avian influenza viruses have caused global pandemics in poultry and sporadic infections in humans. Vaccines are a desirable solution to prevent viral infections in poultry and reduce transmission to humans. Herein, we investigated the efficacy of an oil-adjuvanted inactivated H5N6 vaccine against highly pathogenic H5N6 and H5N1 influenza virus infections in chickens. METHODS: The polybasic amino acid cleavage site depleted HA gene and NA gene of A/Waterfowl/Korea/S57/2016 (clade 2.3.4.4) (H5N6) was assembled with the rest of the A/PR/8/34 (H1N1) genes to construct the vaccine virus. The vaccine virus was propagated in fertilized eggs, partially purified using a tangential flow filtration (TFF) system, and inactivated using formalin. The chickens were intramuscularly immunized with 384 HA, 192HA, and 96HA units of oil-adjuvanted inactivated H5N6 vaccine. Antibody titer, survival rate, and lung pathology were evaluated against the homologous H5N6: A/waterfowl/Korea/S57/2016 (clade 2.3.4.4) and heterologous H5N1: A/Hong Kong/213/2003 (clade 1) viruses 12 and 4 weeks post-vaccination (p.v.), respectively. Data were statistically analyzed using the Mann-Whitney U test. RESULTS: The 384HA (n = 10) and 192HA (n = 5) antigen-immunized chickens showed 100% survival after lethal infections with homologous H5N6, and no virus shedding was observed from tracheal and cloacal routes. All chickens that received the 384HA vaccine survived the challenge of heterologous H5N1 after 4 weeks of immunization. The chickens that received the 384HA vaccine showed mean HI titers of 60 and 240 after 12 and 4 weeks of vaccination, respectively, against HP H5N6, whereas a mean HI titer of 80 was observed in sera collected 4 weeks after vaccination against HP H5N1. CONCLUSIONS: Our findings indicate that one dose of 384HA oil-adjuvanted inactivated H5N6 vaccine can induce a long-lasting immune response against both homologous H5N6 and heterologous H5N1 infections in chickens.

Glycine Nano-Selenium Enhances Immunoglobulin and Cytokine Production in Mice Immunized with H9N2 Avian Influenza Virus Vaccine.

This study was performed to investigate the immune enhancement effect of glycine nano-selenium, a microelement on H9N2 avian influenza virus vaccine (H9N2 AIV vaccine) in mice. Fifty (50) Specific Pathogen Free Kunming mice aged 4−6 weeks (18−20 g Body weight) were randomly divided into five groups: control normal group, which received no immunization + 0.5 mL 0.9% normal saline, positive control group, which received H9N2 AIV vaccine + 0.5 mL 0.9% normal saline, 0.25 mg/kg selenium group, which received H9N2 AIV vaccine + 0.5 mL 0.25 mg/kg selenium solution, 0.5 mg/kg selenium group, which received H9N2 AIV vaccine + 0.5 mL 0.5 mg/kg selenium solution, and 1 mg/kg selenium group, which received H9N2 AIV vaccine + 0.5 mL 1 mg/kg selenium solution. Hematoxylin and eosin staining, enzyme linked immunosorbent assay (ELISA), and quantitative real time polymerase chain reaction (qRT-PCR) methods were used to investigate the pathological changes, immunoglobulin levels, and cytokine gene expressions in this study. The results showed that all tested doses (0.25 mg/kg, 0.5 mg/kg and 1.00 mg/kg) of glycine nano-selenium did not lead to poisoning in mice. In addition, when compared to the positive control group, glycine nano-selenium increased the immunoglobin indexes (IgA, IgG, IgM and AIV-H9 IgG in serum) as well as the mRNA levels of IL-1ß, IL-6 and INF-γ in the liver, lungs, and spleen (p < 0.05). In summary, glycine nano-selenium could enhance the efficacy of avian influenza vaccine.

HA gene amino acid mutations contribute to antigenic variation and immune escape of H9N2 influenza virus.

Based on differences in the amino acid sequence of the protein haemagglutinin (HA), the H9N2 avian influenza virus (H9N2 virus) has been clustered into multiple lineages, and its rapidly ongoing evolution increases the difficulties faced by prevention and control programs. The HA protein, a major antigenic protein, and the amino acid mutations that alter viral antigenicity in particular have always been of interest. Likewise, it has been well documented that some amino acid mutations in HA alter viral antigenicity in the H9N2 virus, but little has been reported regarding how these antibody escape mutations affect antigenic variation. In this study, we were able to identify 15 HA mutations that were potentially relevant to viral antigenic drift, and we also found that a key amino acid mutation, A180V, at position 180 in HA (the numbering for mature H9 HA), the only site of the receptor binding sites that is not conserved, was directly responsible for viral antigenic variation. Moreover, the recombinant virus with alanine to valine substitution at position 180 in HA in the SH/F/98 backbone (rF/HAA180V virus) showed poor cross-reactivity to immune sera from animals immunized with the SH/F/98 (F/98, A180), SD/SS/94 (A180), JS/Y618/12 (T180), and rF/HAA180V (V180) viruses by microneutralization (MN) assay. The A180V substitution in the parent virus caused a significant decrease in cross-MN titres by enhancing the receptor binding activity, but it did not physically prevent antibody (Ab) binding. The strong receptor binding avidity prevented viral release from cells. Moreover, the A180V substitution promoted H9N2 virus escape from an in vitro pAb-neutralizing reaction, which also slightly affected the cross-protection in vivo. Our results suggest that the A180V mutation with a strong receptor binding avidity contributed to the low reactors in MN/HI assays and slightly affected vaccine efficacy but was not directly responsible for immune escape, which suggested that the A180V mutation might play a key role in the process of the adaptive evolution of H9N2 virus.

Immunogenicity and cross-protective efficacy of recombinant H5HA1 protein of clade 2.3.2.1a highly pathogenic H5N1 avian influenza virus expressed in E.coli.

The global spread of H5N1 highly pathogenic avian influenza virus (HPAIV) in poultry has caused great economic loss to the poultry farmers and industry with significant pandemic threat. The current study involved production of recombinant HA1 protein of clade 2.3.2.1a H5N1 HPAIV (rH5HA1) in E.coli and evaluation of its protective efficacy in chickens. Purification under denaturing conditions and refolding by dialysis against buffers containing decreasing concentrations of urea was found to preserve the biological activity of the expressed recombinant protein as assessed by hemagglutination assay, Western blot and ELISA. The Montanide ISA 71 VGA adjuvanted rH5HA1 protein was used for immunization of chickens. Humoral response was maintained at a minimum of 4log2 hemagglutination inhibition (HI) titre till 154 days post 2nd booster. We evaluated the protective efficacy of rH5HA1 protein in immunized chickens by challenging them with homologous (2.3.2.1a) and heterologous (2.3.2.1c) clades of H5N1 HPAIV. In both the groups, the HI titre significantly increased (P < 0.05) after challenge and the virus shedding significantly (P < 0.05) reduced between 3rd and 14th day post challenge. The virus shedding ratio in oro-pharyngeal swabs did not differ significantly between both the groups except on 7 days post challenge and during the entire experimental period in cloacal swabs. These results indicate that rH5HA1 was able to induce homologous and cross protective immune response in chickens and could be a potential vaccine candidate used for combating the global spread of H5N1 HPAIV threat. To our knowledge, this is the first study to report immunogenicity and protective efficacy of prokaryotic recombinant H5HA1 protein in chicken.

Safety and immunogenicity of monovalent H7N9 influenza vaccine with AS03 adjuvant given sequentially or simultaneously with a seasonal influenza vaccine: A randomized clinical trial.

BACKGROUND: Influenza A/H7N9 viruses have pandemic potential. METHODS: We conducted an open-label, randomized, controlled trial of AS03-adjuvanted 2017 inactivated influenza A/H7N9 vaccine (H7N9 IIV) in healthy adults. Group 1 received H7N9 IIV and seasonal quadrivalent influenza vaccine (IIV4) simultaneously, followed by H7N9 IIV three weeks later. Group 2 received IIV4 alone and then two doses of H7N9 IIV at three-week intervals. Group 3 received one dose of IIV4. We used hemagglutination inhibition (HAI) and microneutralization (MN) assays to measure geometric mean titers and seroprotection (≥1:40 titer) to vaccine strains and monitored for safety. RESULTS: Among 149 subjects, seroprotection by HAI three weeks after H7N9 IIV dose 2 was 51% (95 %CI 37%-65%) for Group 1 and 40% (95 %CI 25%-56%) for Group 2. Seroprotection by MN at the same timepoint was 84% (95 %CI 72%-93%) for Group 1 and 74% (95 %CI 60%-86%) for Group 2. By 180 days after H7N9 IIV dose 2, seroprotection by HAI or MN was low for Groups 1 and 2. Responses measured by HAI and MN against each IIV4 strain three weeks after IIV4 vaccination were similar in all groups. Solicited local and systemic reactions were similar after a single vaccination, while those receiving simultaneous H7N9 and IIV4 had slightly more reactogenicity. There were no serious adverse events or medically-attended adverse events related to study product receipt. CONCLUSIONS: Adjuvanted H7N9 IIV was modestly immunogenic whether administered simultaneously or sequentially with IIV4, though responses declined by 180 days. IIV4 was immunogenic regardless of schedule. CLINICAL TRIALS REGISTRATION: NCT03318315.

Chitosan-based formulation of hemagglutinin antigens for oculo-nasal booster vaccination of chickens against influenza viruses.

Avian influenza viruses (AIVs) and especially highly pathogenic (HP) AIVs of the H5 and H7 subtypes are of both veterinary and public health concern worldwide. In response to the demand for effective vaccines against H5N1 HPAIVs, we produced recombinant protein based on hemagglutinin (HA), a protective viral antigen. A fragment of the HA ectodomain, with a multibasic cleavage site deletion, was expressed in Escherichia coli, refolded, and chromatographically purified from inclusion bodies. Finally, the protein was formulated in Tris-HCl buffer of pH 8.0 or PBS of pH 7.4 to obtain antigens denoted rH5-1 and rH5-2, respectively. The systemic prime and boost immunizations proved that rH5-1 adsorbed to aluminum hydroxide induces anti-H5 HA neutralizing antibodies and protective immune responses against H5N1 HPAIVs in chickens. The present studies were aimed at stimulating immune responses via the mucosal routes using the systemic prime-mucosal boost strategy. Efficacy trials were performed in commercial layer chickens. For systemic and mucosal immunizations, H5 HA antigens were adjuvanted with aluminum hydroxide and chitosan glutamate, respectively. The first dose of rH5-2 was administered subcutaneously, while its second dose was administered subcutaneously, intraocularly, oculo-nasally, or intranasally. rH5-1 was delivered to the subcutaneously primed chickens by the intranasal route. Post-vaccination sera were analyzed for anti-H5 HA antibodies, using homologous ELISA and heterologous FluAC H5 and hemagglutination inhibition tests. Intraocularly and oculo-nasally delivered rH5-2 mixed with chitosan glutamate was capable of stimulating anti-H5 HA IgY antibody responses in the subcutaneously primed chickens; however, it was ineffective when administered by the intranasal route. Efficient intranasal boosting was achieved using rH5-1. The enhanced production of antigen-specific antibodies was reflected in the development of H5-subtype specific and hemagglutination inhibiting antibodies. Conclusively, the subcutaneous prime and oculo-nasal boost vaccination is proposed as the target strategy for future optimization.

Effects of four herbs as a dietary on properties of egg and immune response against Newcastle and avian influenza vaccine in Japanese laying quail.

This study shows the effectiveness of diet containing Trachyspermum copticum (TC), Majorana hortensis Minch (MH), Stachys lavandulifolia Vahl (SL), and Zingiber officinale (ZO) on the growth performance, biochemical factors, and qualitative agents of eggs of Japanese quail (Coturnix japonica) and their immune responses against Newcastle and Avian Influenza vaccine. For this prepose, 675 quails were divided into 9 groups with three replicates and fed with different treatment diets (basic diet with no supplements (control treatment diet) and diets supplemented with one of two levels (0.5 and 2%) of each plant powders). Data showed that the use of TC 2% increased the Haugh unit significantly (P < 0.05) compared with the control (P < 0.05). At the end of the experiment, shell weight (g) and shell thickness were also remarkably enhanced in treated groups compared with the control group. Moreover, the findings of this study showed the thiobarbituric acid and yolk cholesterol level reduced remarkably (P < 0.05) in the MH and SL groups without significant adverse effect on albumen protein (%) and total protein (%) level. In this study, TC-2%, ZO-2%, and SL-2% all increased the antibody titers against avian influenza. The use of a diet containing MH-2% increased Newcastle disease in Japanese quail in comparison to both controls and different levels of other medicinal herb powders. Based on these results, using these four herbal plant powders in Japanese quail, diets could positively affect their egg qualitative and biochemical factors.

Carboxymethyl chitosan bounded iron oxide nanoparticles and gamma-irradiated avian influenza subtype H9N2 vaccine to development of immunity on mouse and chicken.

BACKGROUND: Avian influenza virus (AIV) subtype H9N2 is a low pathogenic avian influenza virus (LPAIV). OBJECTIVE: This study aims to evaluate the humoral and cellular immunity in vaccinated mice and broiler chicken by irradiated AIV antigen plus carboxymethyl chitosan bounded iron oxide nanoparticles (CMC-IO NPs) as an adjuvant. METHODS: AIV subtype H9N2 with 108.5 EID50 /ml and haemagglutinin antigen assay about 10 log2 was irradiated by 30 kGy gamma radiation dose. Then, the gamma-irradiated AIV was used as an inactivated vaccine and conjugated with CMC-IO NPs to improve immune responses on mice. IO NPs must be applied in all activated tests using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide sodium salt (sulfo-NHS), and then functionalized by CMC as IO-CMC. Fourier transform infrared (FTIR) spectra on functionalized IO-CMC showed a peak of 638 cm-1 which is a band between metal and O (Fe-O). RESULTS: Based on the comparison between the two X-ray diffraction (XRD) patterns on Fe2 O3 -NPs and IO-CMC, the characteristics of IO-NPs did not change after carboxymethylation. A CHN Analyzer was applied to measure the molecular weight of IO-CMC that was calculated as 1045 g. IO-CMC, irradiated AIV-IO-CMC and formalin AIV-IO-CMC were injected into 42 BALB/c mice in six groups. The fourth group was the negative control, and the fifth and sixth groups were inoculated by irradiated AIV-ISA70 and formalin AIV-ISA70 vaccines. An increase in haemagglutination inhibition (HI) antibody titration was observed in the irradiated AIV-IO-CMC and formalin AIV-IO-CMC groups (p < 0.05). In addition, increases in the lymphoproliferative activity of re-stimulated splenic lymphocytes, interfron-γ (IFN-γ) and interleukin-2 (IL-2) concentration in the irradiated AIV-IO-CMC group demonstrated the activation of Type 1 helper cells. The concentration of IL-4 was without any significant increases in non-group. CONCLUSIONS: Accordingly, Th2 activation represented no increase. Finally, the finding showed that AIV-IO-CMC was effective on enhancing immunogenicity as irradiated AIV antigen administered with a clinically acceptable adjuvant (i.e. IO-CMC).

Development and application of a visual microarray for synchronously detecting H5N1, H7N9 and H9N2 avian influenza virus RNA.

The aim of this study was to develop a microarray assay for the simultaneous detection of the H5, H7, H9, N1, N9 and N2 genes of the avian influenza virus (AIV) using a Nanogold-streptavidin and silver-stain-enhanced nucleic acid dot-blot hybridisation system. The conserved sequences of H5 genes from H5N1, H7 genes from H7N9, H9 genes from H9N2, N9 genes from H7N9 and N2 genes from H9N2 AIV were cloned, together with that of N1 obtained commercially, and were used as templates for generating the probes using biotin-labeled primers, which targeted the conserved regions of H5, H7, H9, N1, N9 and N2 genes, respectively. The oligonucleotide probes were diluted using the spotting buffer and ddH2O, and each probe was then spotted to each specific position on the microarray. The PCR products including biotin-labeled lambda, NP, H5, H7, H9, N1, N9 and N2 were mixed, 200 µL of which was then added to the microarray chamber after denaturing. Following a hybridization incubation at 45℃ for 120 min, the microarray was then incubated with nanogold-streptavidin about 4 µg/mL for 30 min. After the supplementary of 200 µL of silver buffer A and silver buffer B in the chamber, the hybridization results were assessed by direct visualization in the dark at room temperature. The microarray assay was optimized and its specificity, sensitivity and stability were evaluated. The optimal conditions comprised a probe concentration of 50 µmol/L, a hybridization temperature of 45℃ and a hybridization time of 2 h. The optimal concentration of nanogold-streptavidin was 4 µg/mL and the optimal staining time was 7 min. The results of specificity evaluation showed that no cross-binding of the probes with each other and no cross-hybridization with Newcastle disease virus, infectious bronchitis virus and infectious laryngotracheitis virus was observed. The optimized microarray assay was significantly more sensitivity than the reverse-transcription PCR assay. The microarray was available after storing at less 90 d at 4 ℃. The optimized microarray assay was validated on clinical specimens and the results showed that it had over 95.6 % correlation with reverse-transcription PCR method. Therefore, the microarray assay could be used for the high throughput detection of AIV infections due to H5N1, H7N9 and H9N2.

Comparative Study on the Efficacy of MF 59, ISA70 VG, and Nano-Aluminum Hydroxide Adjuvants, Alone and with Nano-Selenium on Humoral Immunity Induced by a Bivalent Newcastle+Avian Influenza Vaccine in Chickens.

Newcastle disease (ND) and Avian influenza (AI) are the major problems and the most economically important viral diseases in the poultry industry; therefore, vaccination against these diseases is considered one of the most effective ways of prevention. Extensive studies have been conducted to improve the performance of vaccines, and one of the major achievements of these studies is the preparation of adjuvants as stimulants of the immune system and one of the most important compounds in killed vaccines. An immunogenicity comparison of three adjuvants including, ISA70VG, Nano-Aluminum Hydroxide (Nano-Alum), and MF59 alone or with Nano-Selenium (Nano-Se), was performed using bivalent Newcastle plus Avian Influenza (ND+AI) killed vaccine. In this study, 105 specific-pathogen-free chicks (Ross-308) were divided into 7 treatments, including T1 (control group), T2 (ISA70VG), T3 (ISA70VG plus Nano-Se), T4 (Nano-Alum Hydroxide), T5 (Nano-Alum+Nano-Se), T6 (MF59), and T7 (MF59+Nano-Se). The vaccine was injected subcutaneously on day 21 in the back of the neck area. The blood samples were taken on days 14, 21, 28, 35, 42, and 49 post-vaccination. Serums of the samples were titrated by the haemagglutination inhibition (HI) test against Newcastle and Avian influenza. Based on the results, the highest HI test titers were observed for the T2 and T3 treatments, while the T6 and T7 treatments had the lowest titers. Moreover, regardless of the type of the adjuvants, adding Nano-Se increased the antibody titer in the vaccinated groups. In conclusion, a combination of the ISA70VG adjuvant and Nano-Se induced excellent antibody titers using bivalent ND+AI killed vaccine.

Effect of Baicalin on Bacterial Secondary Infection and Inflammation Caused by H9N2 AIV Infection in Chickens.

H9N2 subtype avian influenza virus (H9N2 AIV) is a low pathogenic virus that is widely prevalent all over the world. H9N2 AIV causes immunosuppression in the host and often leads to high rates of mortality due to secondary infection with Escherichia. Due to the drug resistance of bacteria, many antibiotics are not effective in the treatment of secondary bacterial infection. Therefore, the purpose of this study is to find effective nonantibiotic drugs for the treatment of H9N2 AIV infection-induced secondary bacterial infection and inflammation. This study proves, for the first time, that baicalin, a Chinese herbal medicine, can regulate Lactobacillus to replace Escherichia induced by H9N2 AIV, so as to resolve the intestinal flora disorder. In addition, baicalin can effectively prevent intestinal bacterial translocation of SPF chickens' post-H9N2 AIV infection, thus inhibiting secondary bacterial infection. Furthermore, baicalin can effectively treat H9N2 AIV-induced inflammation by inhibiting intestinal structural damage, inhibiting damage to ileal mucus layer construction and tight junctions, improving antioxidant capacity, affecting blood biochemical indexes, and inhibiting the production of inflammatory cytokines. Taken together, these results provide a new theoretical basis for clinical prevention and control of H9N2 AIV infection-induced secondary bacterial infection and inflammation.

Quick and improved immune responses to inactivated H9N2 avian influenza vaccine by purified active fraction of Albizia julibrissin saponins.

BACKGROUND: H9N2 Low pathogenic avian influenza virus (LPAIV) raises public health concerns and its eradication in poultry becomes even more important in preventing influenza. AJSAF is a purified active saponin fraction from the stem bark of Albizzia julibrissin. In this study, AJSAF was evaluated for the adjuvant potentials on immune responses to inactivated H9N2 avian influenza virus vaccine (IH9V) in mice and chicken in comparison with commercially oil-adjuvant. RESULTS: AJSAF significantly induced faster and higher H9 subtype avian influenza virus antigen (H9-Ag)-specific IgG, IgG1, IgG2a and IgG2b antibody titers in mice and haemagglutination inhibition (HI) and IgY antibody levels in chicken immunized with IH9V. AJSAF also markedly promoted Con A-, LPS- and H9-Ag-stimulated splenocyte proliferation and natural killer cell activity. Furthermore, AJSAF significantly induced the production of both Th1 (IL-2 and IFN-γ) and Th2 (IL-10) cytokines, and up-regulated the mRNA expression levels of Th1 and Th2 cytokines and transcription factors in splenocytes from the IH9V-immunized mice. Although oil-formulated inactivated H9N2 avian influenza vaccine (CH9V) also elicited higher H9-Ag-specific IgG and IgG1 in mice and HI antibody titer in chicken, this robust humoral response was later produced. Moreover, serum IgG2a and IgG2b antibody titers in CH9V-immunized mice were significantly lower than those of IH9V alone group. CONCLUSIONS: AJSAF could improve antigen-specific humoral and cellular immune responses, and simultaneously trigger a Th1/Th2 response to IH9V. AJSAF might be a safe and efficacious adjuvant candidate for H9N2 avian influenza vaccine.

Selection and antigenic characterization of immune-escape mutants of H7N2 low pathogenic avian influenza virus using homologous polyclonal sera.

Understanding the dynamics of the selection of influenza A immune escape variants by serum antibody is critical for designing effective vaccination programs for animals, especially poultry where large populations have a short generation time and may be vaccinated with high frequency. In this report, immune-escape mutants of A/turkey/New York/4450/1994 H7N2 low pathogenic avian influenza virus, were selected by serially passaging the virus in the presence of continuously increasing concentrations of homologous chicken polyclonal sera. Amino acid mutations were identified by sequencing the parental hemagglutinin (HA) gene and every 10 passages by both Sanger and deep sequencing, and the antigenic distance of the mutants to the parent strain was determined. Progressively, a total of five amino acid mutations were observed over the course of 30 passages. Based on their absence from the parental virus with deep sequencing, the mutations appear to have developed de novo. The antigenic distance between the selected mutants and the parent strain increased as the number of amino acid mutations accumulated and the concentration of antibodies had to be periodically increased to maintain the same reduction in virus titer during selection. This selection system demonstrates how H7 avian influenza viruses behave under selection with homologous sera, and provides a glimpse of their evolutionary dynamics, which can be applied to developing vaccination programs that maximize the effectiveness of a vaccine over time.