Inactivated H5N1 Research Articles

Immunisation of chickens with inactivated and/or infectious H9N2 avian influenza virus leads to differential immune B-cell repertoire development.

Avian influenza viruses (AIVs) are a major economic burden to the poultry industry and pose serious zoonotic risks, with human infections being reported every year. To date, the vaccination of birds remains the most important method for the prevention and control of AIV outbreaks. Most national vaccination strategies against AIV infection use whole virus-inactivated vaccines, which predominantly trigger a systemic antibody-mediated immune response. There are currently no studies that have examined the antibody repertoire of birds that were infected with and/or vaccinated against AIV. To this end, we evaluate the changes in the H9N2-specific IgM and IgY repertoires in chickens subjected to vaccination(s) and/or infectious challenge. We show that a large proportion of the IgM and IgY clones were shared across multiple individuals, and these public clonal responses are dependent on both the immunisation status of the birds and the specific tissue that was examined. Furthermore, the analysis revealed specific clonal expansions that are restricted to particular H9N2 immunisation regimes. These results indicate that both the nature and number of immunisations are important drivers of the antibody responses and repertoire profiles in chickens following H9N2 antigenic stimulation. We discuss how the repertoire biology of avian B-cell responses may affect the success of AIV vaccination in chickens, in particular the implications of public versus private clonal selection.

Recombinant avian-derived antiviral proteins cIFITM1, cIFITM3, and cViperin as effective adjuvants in inactivated H9N2 subtype avian influenza vaccines

Vaccine adjuvants, serving as non-specific immune enhancers, play a pivotal role in the immunoprevention and control of animal diseases. This study utilized prokaryotic expression systems to express and purify chicken-derived cIFITM1, cIFITM3, and cViperin, which were then formulated as adjuvants with H9N2 avian influenza virus antigens to create inactivated vaccines. These vaccines were administered to SPF chickens to investigate their immunopotentiating functions. Additionally, the proteins were assessed for their ability to act as standalone immune enhancers. The results demonstrated that cIFITM1, cIFITM3, and cViperin significantly elevated serum hemagglutination inhibition (HI) antibody titers. Notably, when used individually, these proteins markedly enhanced the antiviral capabilities of challenged chickens, leading to alleviated clinical symptoms, reduced tracheal virus replication, diminished virus shedding, and lessened histopathological damage, with cIFITM1 exhibiting the most pronounced effect. Furthermore, the protective efficacy of two H9N2 recombinant virus inactivated vaccines supplemented with cIFITM1 adjuvant was validated, achieving a 100 % vaccine protection efficiency. In conclusion, cIFITM1, cIFITM3, and cViperin as adjuvants for influenza vaccines effectively inhibit virus replication and shedding, highlighting their significant potential in influenza prevention and control.

H3N2 canine influenza virus-like particle vaccine with great protection in beagle dogs.

A new antigenically and genetically distinct canine influenza virus (CIV) H3N2 clade possessing mutations associated with mammalian adaptation emerged in 2016 and substituted previously circulating strains. This clade poses a risk for zoonotic infection. In our study, intramuscular injection of the H3N2 virus-like particle (VLP) vaccine and inactivated H3N2 CIV confer completely sterilizing protection against homologous H3N2 canine influenza virus challenge. Our results provide further support for the possibility of developing VLP vaccines that can reliably induce immunity in animal species.

A synthetic TLR4 agonist significantly increases humoral immune responses and the protective ability of an MDCK-cell-derived inactivated H7N9 vaccine in mice.

Antigenically divergent H7N9 viruses pose a potential threat to public health, with the poor immunogenicity of candidate H7N9 vaccines demonstrated in clinical trials underscoring the urgent need for more-effective H7N9 vaccines. In the present study, mice were immunized with various doses of a suspended-MDCK-cell-derived inactivated H7N9 vaccine, which was based on a low-pathogenic H7N9 virus, to assess cross-reactive immunity and cross-protection against antigenically divergent H7N9 viruses. We found that the CRX-527 adjuvant, a synthetic TLR4 agonist, significantly enhanced the humoral immune responses of the suspended-MDCK-cell-derived H7N9 vaccine, with significant antigen-sparing and immune-enhancing effects, including robust virus-specific IgG, hemagglutination-inhibiting (HI), neuraminidase-inhibiting (NI), and virus-neutralizing (VN) antibody responses, which are crucial for protection against influenza virus infection. Moreover, the CRX-527-adjuvanted H7N9 vaccine also elicited cross-protective immunity and cross-protection against a highly pathogenic H7N9 virus with a single vaccination. Notably, NI and VN antibodies might play an important role in cross-protection against lethal influenza virus infections. This study showed that a synthetic TLR4 agonist adjuvant has a potent immunopotentiating effect, which might be considered worth further development as a means of increasing vaccine effectiveness.

Sustained release of inactivated H1N1 virus from degradable microparticles for extended vaccination

Influenza vaccines administered as intramuscularly injected inactivated viruses or intranasally administered live-attenuated viruses usually provide short-term protection against influenza infections. Biodegradable particles that provide sustained release of the antigen has been studied as an approach to extend vaccine protection. Here, we investigate sustained release of ultraviolet killed influenza A virus (A/PR/8/34(H1N1)) (kPR8) loaded into poly(D,L-lactic-co-glycolic acid) (PLGA) microparticles. Particles were prepared using the double emulsion method, and polymer molecular weight (MW), polymer hydrophobicity, polymer concentration in the organic phase, and the amount of killed virus were varied to obtain a range of particles. Formulations included PLGA 50:50 (2–6, 7–17 kDa), PLGA 75:25 (4–15 kDa), and 50/50 PLGA 75:25 (4–15 kDa)/PCL (14 kDa). Additionally, NaOH was co-encapsulated in some cases to enhance particle degradation. The structure of the particles was explored by size measurements and electron microscopy. The kPR8 release profiles were measured using hemagglutinin ELISA. The concentration of the polymer (PLGA) in the organic phase and polymer MW significantly influenced virus loading, while polymer MW and co-encapsulation of NaOH modulated the release profiles. Mice receiving a single intramuscular injection of NaOH microparticle-encapsulated kPR8 were partially protected against a lethal influenza challenge 32 weeks post immunization. Microparticle (MP) vaccination induced a gradual increase in PR8-specific IgGs dominated by IgG1 in contrast to the rapid IgG2a-biased response elicited by soluble kPR8 immunization. Our results indicate that vaccine-NaOH co-loaded PLGA particles show potential as a single dose vaccination strategy for extended protection against influenza virus infection.

THE IMMUNITY DURATION AND INTENSITY IN INDUSTRIAL LAYING HENS FOLLOWING VACCINATION WITH INACTIVATED H5N1 AVIAN INFLUENZA VACCINE

After the severe AIV H5 outbreak in Kazakhstan in 2020 the extensive use of AIV H5 vaccines started in the industrial poultry farms to limit the H5N1 influenza spread. Traditional methods, such as stamping out are no longer a viable option in countries where Highly Pathogenic Avian Influenza (HPAI) has become endemic. However, available vaccines and vaccination protocols have been widely researched in laboratory conditions, and limited testing has been conducted on commercial layers for the immunity persistence in field conditions. Immunity persistence after AIV vaccination can be quite different between laboratory and field conditions. Our basic goal was to assess the intensity and duration of immunity in commercial layers following 1, 2 and 3 vaccinations. H5N1 hemagglutination inhibition (HI) antibodies were observed 370 days after vaccination of chickens using three schemes of vaccination. However, at 370 days post vaccination 1 experimental group obtaining one vaccination had comparatively low HI titers (mean titer 6,5±1,2 log2), the other two groups having two and three vaccinations showed 8,8±2,4 mean log2 and 9,4±2,2 mean log2 respectively. The described results showed that inactivated H5N1 vaccine can produce lengthy, intense and homogenous immunity under field conditions following 2 and 3 vaccinations.

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-β (TGF-β). 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.

Efficacy of an inactivated influenza vaccine adjuvanted with Toll-like receptor ligands against transmission of H9N2 avian influenza virus in chickens

Avian influenza viruses (AIV), including the H9N2 subtype, pose a major threat to the poultry industry as well as to human health. Although vaccination provides a protective control measure, its effect on transmission remains uncertain in chickens. The objective of the present study was to investigate the efficacy of beta-propiolactone (BPL) whole inactivated H9N2 virus (WIV) vaccine either alone or in combination with CpG ODN 2007 (CpG), poly(I:C) or AddaVax™ (ADD) to prevent H9N2 AIV transmission in chickens. The seeder chickens (trial 1) and recipient chickens (trial 2) were vaccinated twice with different vaccine formulations. Ten days after secondary vaccination, seeder chickens were infected with H9N2 AIV (trial 1) and co-housed with healthy recipient chickens. In trial 2, the recipient chickens were vaccinated and then exposed to H9N2 AIV-infected seeder chickens. Our results demonstrated that BPL+ CpG and BPL+ poly(I:C) treated chickens exhibited reduced oral and cloacal shedding in both trials post-exposure (PE). The number of H9N2 AIV+ recipient chickens in the BPL+ CpG group (trial 1) was lower than in other vaccinated groups, and the reduction was higher in BPL+ CpG recipient chickens in trial 2. BPL+ CpG vaccinated chickens demonstrated enhanced systemic antibody responses with high IgM and IgY titers with higher rates of seroprotection by day 21 post-primary vaccination (ppv). Additionally, the induction of IFN-γ expression and production was higher in the BPL+ CpG treated chickens. Interleukin (IL)− 2 expression was upregulated in both BPL+ CpG and BPL+ poly(I:C) groups at 12 and 24 hr post-stimulation.

Effect of Vaccination on Distribution and Immune Response of Avian Influenza Virus H9N2 in Coturnix coturnix.

Influenza viruses can multiply in quails and be transmitted to other animal species. As vaccination reduces virus shedding in chickens, the effect of the killed H9N2 avian influenza virus (AIV) on tissue distribution and virus shedding was evaluated in quails. One hundred 20-day-old quails were divided into six equal groups, kept in separate pens, and fed ad libitum. Before vaccination, blood samples were randomly collected from the wing veins. Four groups were vaccinated with the inactivated H9N2 Razi Institute vaccine at 21 days subcutaneously at the back of neck. Three weeks later, two groups were re-vaccinated. Two weeks later, at the age of 56 days, three groups were challenged with 100 μL of allantoic fluid containing 105 EID50 H9N2 through the oculonasal route. Blood samples were collected from quails at 42, 56, 63, and 70 days from each group to determine AIV antibodies by the hemagglutination inhibition test. Three quails were randomly selected and euthanized from each group on days 1, 3, and 6 post-inoculation (PI). Tissue samples were collected, and the RT-PCR test was performed. No clinical signs or gross lesions existed in any of the groups during the experiment. However, the virus was detected in different tissues on the first, third, and sixth days after the challenge in unvaccinated challenged birds. Virus detection was significantly more frequent in the quails vaccinated once and challenged than in the twice-vaccinated challenged group (P≤0.05). On the third day of PI, the virus was detected in some organs of the challenged groups. On the sixth day of PI, the virus was detected only in the lungs of two unvaccinated and once-vaccinated challenged birds. It was concluded that the vaccination of quails against AIV H9 is necessary to protect them from clinical signs, as well as respiratory tract and intestine replication. Two-time vaccination significantly protects the respiratory and intestine tracts, compared to one-time vaccination (P≤0.05).

Wearable MXene‐Graphene Sensing of Influenza and SARS‐CoV‐2 Virus in Air and Breath: From Lab to Clinic

AbstractThe rapidly expanding severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and its variants demand a continuous monitoring method through portable and wearable devices. Utilizing the rich surface chemistry and high chemical‐to‐electrical signal conversion of 2D MXene‐graphene heterostructure thin films, a field‐effect‐transistor (FET) sensor, which has a flexible substrate to be assembled onto the mask and combines with a Bluetooth system for wireless transmission is developed, to detect the influenza and SARS‐CoV‐2 viruses in air and breath. At first, the developed sensors are examined in the laboratory through direct contact with sensing targets in solution form. The results show a low limit of detection (LOD) of 1 fg mL−1 for recombinant SARS‐CoV‐2 spike protein and 125 copies mL−1 for inactivated influenza A (H1N1) virus with high specificity in differing recombinant SARS‐CoV‐2 spike protein and inactivated H1N1 virus. Then the sensors are tested under various simulated human breathing modes through controlled exposure to atomizer‐generated aerosols in an enclosed chamber and mask coverage. The results show the high sensitivity of the developed sensors under varying distances to the source, viral load, flow rate, and enclosed conditions. At last, clinical tests are carried out to demonstrate the robustness and potential field applications of the sensors.

Influenza virus immune imprinting dictates the clinical outcomes in ferrets challenged with highly pathogenic avian influenza virus H5N1.

Zoonotic transmission of H5N1 highly pathogenic avian influenza virus (HPAIV) into the human population is an increasing global threat. The recent 2022 HPAIV outbreak significantly highlighted this possibility, increasing concern in the general population. The clinical outcomes of H5N1 influenza virus exposure can be determined by an individual's primary influenza virus infection (imprinting) or vaccination status. Immunological imprinting with Group 1 - (H1N1, H2N2, and H2N3) increases survival rates following H5N1 viral infection compared to Group 2 - (H3N2) imprinted individuals. Vaccination against H5N1 influenza viruses can offer protection to at-risk populations; however, stockpiled inactivated H5N1 influenza vaccines are not readily available to the public. We hypothesize that the immunological response to vaccination and subsequent clinical outcome following H5N1 influenza virus infection is correlated with the immunological imprinting status of an individual. To test this hypothesis, our lab established a ferret pre-immune model of disease. Naïve ferrets were intranasally inoculated with seasonal influenza viruses and allowed to recover for 84 days prior to H5N1 virus infection. Ferrets imprinted following H1N1 and H2N3 virus infections were completely protected against lethal H5N1 influenza virus challenge (100% survival), with few to no clinical symptoms. In comparison, H3N2 influenza virus-imprinted ferrets had severe clinical symptoms, delayed disease progression, and a sublethal phenotype (40% mortality). Consecutive infections with H1N1 influenza viruses followed by an H3N2 influenza virus infection did not abrogate the immune protection induced by the original H1N1 influenza virus infection. In addition, ferrets consecutively infected with H1N1 and H2N3 viruses had no clinical symptoms or weight loss. H3N2 pre-immune ferrets were vaccinated with a broadly reactive H5 HA-based or H1 NA-based vaccine (Hu-CO 2). These ferrets were protected against H5N1 influenza virus challenge, whereas ferrets vaccinated with the H1N1 wild-type CA/09 rHA vaccine had similar phenotypes as non-vaccinated H3N2-imprinted ferrets with 40% survival. Overall, Group 2 imprinted ferrets, which were vaccinated with heterologous Group 1 HA vaccines, had redirected immune responses to Group 1 influenza viral antigens and rescued a sublethal phenotype to complete protection.

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 β-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.

Pilot-scale production of inactivated monoglycosylated split H1N1 influenza virus vaccine provides cross-strain protection against influenza viruses

Influenza epidemics and pandemics caused by newly emerging virus strains highlight an urgent need to develop a universal vaccine against viruses. Previously, a monoglycosylated X-181mg vaccine demonstrated that the HA possessing a single N-acetylglucosamine at each N-glycosylation site is superior to confer broader protection in mice than conventional vaccines. However, the greatest challenge in conducting clinical trials is the need to develop robust manufacturing processes capable of producing vaccines at the pilot scale with the desired stability, potency, and efficacy. Whether the monoglycosylated virus vaccine platform can be applied to the new vaccine strain in a timely manner and whether the mass-produced vaccine has the proper immunogenicity to induce cross-protective immunity remains unclear. Here, we show that a pilot-scale manufacturing process produced a monoglycosylated A/Brisbane/02/2018(H1N1) virus vaccine (IVR-190mg) with a single glycan at each glycosylation site of HA and NA. Compared with the fully glycosylated virus vaccine (IVR-190fg), the IVR-190mg provided broader cross-protection in mice against a wide range of H1N1 variants. The enhanced antibody responses induced by IVR-190mg immunization include higher hemagglutination-inhibition titers, higher neutralization activity, more anti-HA head domain, more anti-HA stem antibodies, higher neuraminidase activity inhibition titers, and notably, higher antibody-dependent cellular cytotoxicity. Additionally, the IVR-190mg also induced a more balanced Th1/Th2 response and elicited broader splenic CD4+ and CD8+ T-cell responses than IVR-190fg. This study demonstrated that IVR-190mg produced using a pilot-scale manufacturing process elicits comprehensive cross-strain immune responses that have great potential to substantially mitigate the need for yearly reformulation of strain-specific inactivated vaccines.

The efficacy of an inactivated avian influenza H5N1 vaccine against an African strain of HPAI H5N8 (clade 2.3.4.4 B)

ABSTRACT Highly pathogenic avian influenza (HPAI) viruses from the Goose/Guangdong/96-lineage emerged in Southeast Asia and subsequently spread to the Middle East, Africa and Europe, infecting a range of birds and mammals (including humans). This lineage of H5 viruses can efficiently establish itself in wild birds after circulating among gallinaceous poultry, facilitating reassortment with low pathogenic avian influenza (LPAI) virus strains, enhancing dispersal over long distances and contributing to endemicity. The detection of HPAI H5N8 virus (clade 2.3.4.4B) in 2017 in the Mpumalanga Province of South Africa marked the beginning of an epidemic that devastated the South African poultry industry. Vaccines were tested to assess protection against the circulating field strain. This article describes the performance of a reverse genetics inactivated H5N1 vaccine from Zoetis (RG-H5N1), with 96.1% identity to the circulating HPAI H5N8 virus. Two locally formulated benchmarks, one containing an H5N8 antigen homologous to the field strain (Benchmark-H5N8), the other containing a heterologous (87.6% identity to field virus) LPAI H5N1 antigen (Benchmark-H5N1), were included for comparison. Efficacy was assessed in specific pathogen-free (SPF) chickens using a prime-boost approach (injections at days 21 and 45), followed by a challenge with a South African HPAI H5N8 isolate (70 days of age). The Zoetis RG-H5N1 vaccine and Benchmark-H5N8 outperformed the Benchmark-H5N1 in terms of humoral response against the H5N8 antigen and reduction of shedding. The Zoetis RG-H5N1 vaccine protected 100% of the chickens against clinical disease and death. This study confirmed that antigenically matched inactivated vaccines could induce robust protection and markedly reduce viral shedding. RESEARCH HIGHLIGHTS Conditionally licensed vaccine protected against HPAI H5N8 (clade 2.3.4.4B). Complete protection against clinical disease and mortality. Drastic reduction of viral shedding after challenge.

Development of an Inactivated Avian Influenza Virus Vaccine against Circulating H9N2 in Chickens and Ducks.

Avian influenza virus (AIV) subtype H9N2 is the most widespread AIV in poultry worldwide, causing great economic losses in the global poultry industry. Chickens and ducks are the major hosts and play essential roles in the transmission and evolution of H9N2 AIV. Vaccines are considered an effective strategy for fighting H9N2 infection. However, due to the differences in immune responses to infection, vaccines against H9N2 AIV suitable for use in both chickens and ducks have not been well studied. This study developed an inactivated H9N2 vaccine based on a duck-origin H9N2 AIV and assessed its effectiveness in the laboratory. The results showed that the inactivated H9N2 vaccine elicited significant haemagglutination inhibition (HI) antibodies in both chickens and ducks. Virus challenge experiments revealed that immunization with this vaccine significantly blocked virus shedding after infection by both homogenous and heterologous H9N2 viruses. The vaccine was efficacious in chicken and duck flocks under normal field conditions. We also found that egg-yolk antibodies were produced by laying birds immunized with the inactivated vaccine, and high levels of maternal antibodies were detected in the serum of the offspring. Taken together, our study showed that this inactivated H9N2 vaccine could be extremely favourable for the prevention of H9N2 in both chickens and ducks.

Protective Efficacy of Inactivated H9N2 Vaccine in Turkey Poults under Both Experimental and Field Conditions.

Low pathogenic avian influenza (LPAI) H9N2 virus is one of the major poultry pathogens associated with severe economic losses in the poultry industry (broiler, layers, breeders, and grandparents' flocks), especially in endemic regions including the Middle East, North Africa, and Asian countries. This work is an attempt to evaluate the efficacy of whole inactivated H9N2 vaccine (MEFLUVACTM H9) in turkey poults kept under laboratory and commercial farm conditions. Here, 10,000 white turkey poults (1-day old) free from maternally derived immunity against H9N2 virus were divided into four groups; G1 involved 10 vaccinated birds kept under biosafety level-3 (BLS-3) as a laboratory vaccinated and challenged group, while G2 had 9970 vaccinated turkeys raised on a commercial farm. Ten of those birds were moved to BLS-3 for daily cloacal and tracheal swabbing to check for the absence of any life-threating disease, before conducting analyses. G3 (10 birds) served as a non-vaccinated challenged control under BSL-3 conditions, while G4 (10 birds) was used as a non-vaccinated and non-challenged control under BSL-3 conditions. Sera were collected on days 7-, 14-, 21-, and 28-post-vaccinations to monitor the humoral immune response using a hemagglutination-inhibition (HI) test. At these same intervals, cloacal and tracheal swabs were also checked for any viral infection. The challenge was conducted 28 days post-vaccination (PV) using AI-H9N2 in BSL-3 by intranasal inoculation of 6-log10 embryo infective dose50 (EID50). At 3-, 6-, and 10-days post-challenge, oropharyngeal swabs were taken from challenged birds to quantify viral shedding by quantitative polymerase chain reaction (qRT-PCR). The results of this study showed that vaccinated groups (G1/2) developed HI titers of 1.38, 4.38, 5.88, and 7.25 log2 in G1 vs. 1.2, 3.8, 4.9 and 6.2 log2 in G2 when measured at 7-, 14-, 21- and 28-days PV, respectively, while undetectable levels were recorded in non-vaccinated groups (G3/4). Birds in G3 showed 90% clinical sickness vs. 10% and 20% in G1/2, respectively, over a 10-day monitoring period following challenge. Vaccinated birds showed a significant reduction in virus shedding in terms of the number of shedders, amount of shed virus and shedding interval over the non-vaccinated challenged birds. Regarding mortality, all groups did not show any mortality, which confirms that the circulating H9N2 virus still has low pathogenicity and cannot cause mortality. However, the virus may cause up to 90% clinical sickness in non-vaccinated birds vs. 10% and 20% in laboratory- and farm-vaccinated birds, respectively, highlighting the role of the vaccine in limiting clinical sickness cases. In conclusion, under the current trial circumstances, MEFLUVACTM-H9 provided protective seroconversion titers, significant clinical sickness protection and significant reduction in virus shedding either in laboratory- or farm-vaccinated groups after a single vaccine dose.

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

AimThis 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 methodsOne 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).ResultsGrowth 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.ConclusionThe 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.

Anamnestic broadly reactive antibodies induced by H7N9 virus more efficiently bind to seasonal H3N2 strains

ABSTRACT The very first influenza virus exposure in a human during infancy is known to imprint the host immune system. However, it is unclear how the memory B cells that first target virus epitopes affect antibody response to the stalk of hemagglutinin (HA) domain of influenza virus. Our study is designed to measure the cross-reactivity of antibodies induced by inactivated H7N9 virus using isolated human peripheral blood B cells. Most of the participants displayed higher levels of plasma IgG against the seasonal strains A/Vic11 and A/Cali09 than those binding to historical outbreak A/HK68 and A/PR8. H3 stalk-binding antibodies were detected in plasma at a 1:5000 dilution in 12 of 13 donors, H1 stalk-binding antibodies in all donors, indicating the existence of H3 and H1 stalk-reactive memory B cells. A moderate to high level of broadly cross-reactive antibodies was induced in memory B cells from all donors after in vitro stimulation of B cells with H7N9 virus. H3 stalk-binding antibodies were also detected in most subjects, with cross-reactivity to H1 and H7 stalk domains. The stalk-reactive antibodies bound to five H3 strains spanning 45 years and different H1, H2, H3, H5, H6, H7, H9 and B strains. Interestingly, H1- and H3-reactive IgG were much higher than H7-binding antibodies after 6 days of H7N9 stimulation. Our results demonstrate that HA stalk-reactive antibodies induced by H7N9 viruses more efficiently bound to yearly circulating both H3N2 and H1N1 strains than the boosting strain, indicating that HA stalk immunological imprint can be extended across currently circulating strains or vaccines.

Impact of sodium butyrate on stimulating of some host defense peptides and body performance in broiler vaccinated with different avian influenza (H9N2) vaccines

Our study is designated to determine the impact of SB in the induction HDPs, including AvBD-10 and CATH-B1, accompanied by two different inactivated H9N2 vaccines and their effect on body performance. One hundred fifty, day-old chicks were separated into five groups (30 chicks for each, three replicates): groups A and C were vaccinated with classical avian influenza H9N2 and developed H9N2P inactivated vaccines, respectively, but groups B and D were treated with sodium butyrate (SB) by a dosage of 1gm/liter of drinking water daily till the end of the trail, and these groups (B and D) received the same type of vaccines as they given to group A and C respectively, while group E is a control group. The results illustrated that SB improved the AvBD-10 level significantly in the treated group (B and D) at 14 days in comparison with groups A and E, but without significant with group C. Whereas at 35 days, this improvement occurred distinctly in treated groups B and D. The same improvement revealed with CATH-B1 at 35 days of experiments. Moreover, the supplementation of SB improved FCR in groups B and D at 35 days of the experiment, respectively, but no influence on WG between all groups at the same age. Thus, we concluded that supplemented SB enhanced innate immunity by stimulating the induction of AvBD-10 and CATH-B1. Also, these supplementations improved FCR but did not influence WG.

Comparative Study of the Efficiency of the Inactivated H9n2 Vaccine against Avian Influenza in Broiler Chickens

Aim: Our work is devoted to a serological study of avian influenza disease in broiler chickens, after five different vaccine protocols. Methods: a total of 500 broiler chicks (day-old) were equitably divided into 5 lots. in each batch a specific vaccination protocol was followed. The relative level of serum antibodies against H9N2 virus present in the samples is determined at days: 1, 10, 24, 33, and 51 of age. Results: for batches 1 and 2: the kinetics of the antibodies present a stable curve not associated with significant modifications (p&gt; 0.05) during the entire experimental period, whereas for batches 3, 4 and 5: a peak is observed at D 33 for the first two batches and at D51 for the last Conclusion: the development of active immunity conferred by vaccinations has not been proven because in groups 3, 4 and 5 the titers of antibodies reach high values only in the case of response to the provoked infectious condition and not following immunization by vaccines. Keywords: antibodies, ELISA method, immunity, serotype H9N2, vaccine.