Hemagglutination Inhibition Research Articles

An oleanic acid decorated gold nanorod for highly efficient inhibition of hemagglutinin and visible rapid detection of the influenza virus

Accurate diagnosis and effective antiviral treatments are urgently needed for the prevention and control of flu caused by influenza viruses. In this study, a novel oleanic acid (OA) functionalized gold nanorod OA-AuNP was prepared through a convenient ligand-exchange reaction. As hemagglutinin (HA) on the viral surface binds strongly to the multiple OA molecules on the surface of the nanoparticle, the prepared OA-AuNP was found to exhibit potent antiviral activity against a wide range of influenza A virus strains. Furthermore, the change in color resulting from the specific binding between HA and OA and the resultant aggregation of the OA-AuNP can be visually observed or measured by UV–vis spectra with a detection limit of 2 and 0.18 hemagglutination units (HAU), respectively, which is comparable to the commercially available influenza colloid gold rapid diagnostic kits. These findings demonstrate the potential of the OA-AuNP for the development of novel multivalent antiviral conjugates and the diagnosis of influenza virus.

Characterization of Amino Acid Mutations of Newcastle Disease Virus (NDV) In Swan Geese (Anser cygnoides) In East Java, Indonesia

Background: Newcastle disease (ND) is a highly contagious viral disease affecting the poultry industry. The NDV is classified into three strains based on their relative virulence, namely velogenic or highly virulent, mesogenic or moderately virulent, and lentogenic or lowly virulent. The clinical manifestations of the disease vary depending on many factors, such as host susceptibility and the virulence of the NDV strain. Objective: This study aims to analyze the amino acid mutations of the NDV in unvaccinated swan goose (Anser cygnoides) from various locations in Java. Methods: Samples were collected through cloacal swabs and isolated by inoculation in Specific Pathogen-Free (SPF) embryonated eggs that were nine days old. Hemagglutination and hemagglutination inhibition tests were conducted to confirm that the isolated virus was NDV. The isolated virus was processed using reverse transcription-polymerase chain reaction (RT-PCR) with primers that amplified partial sequences of the fusion (F) gene, which was analyzed to determine the pathotype. Results: The results indicated the presence of mutations in several regions. The amino acid changes occurred in 17 variable sites (7.2%) between RefSeq/JF950510 and ND/SW1/2018, 12 variable sites (5.1%) between RefSeq/JF950510 and ND/SW2/2018, 13 variable sites (5.5%) between RefSeq/JF950510 and ND/SW3/2018, and 19 variable sites (8.1%) between RefSeq/JF950510 and ND/SW4/2018. The amino acid sequences of the cleavage site of the fusion (F) protein revealed that all isolates had low virulence. Conclusion: The results indicated that mutations in the region outside the cleavage site not were incapable of altering the virulence of the virus.

Efficiency of Immunoblot While Determination of Antibodies to Treponema Pallidum in Cerebrospinal Fluid

BACKGROUND: A decrease in the incidence of syphilis has been observed in the world and in Ukraine in recent years. At the same time, an increase in cases of neurosyphilis is recorded. Diagnosis of neurosyphilis is quite difficult and based on the correct interpretation of the complex of various diagnostic tests. OBJECTIVES: The paper is aimed to determine diagnostic potential of treponemal tests (TTs) and evaluate effectiveness of Treponema pallidum immunoblot (TPI) while research on cerebrospinal fluid (CSF) in differential diagnosis of neurosyphilis. MATERIALS AND METHODS: The research object was CSF of patients with late forms of syphilis. The regulated serological and immunological methods in accordance with current guidelines and orders of the Ministry of Healthcare of Ukraine were used for laboratory diagnosis of neurosyphilis: Enzyme immunoassays (EIA), fluorescent treponemal antibody (FTA), T. pallidum hemagglutination assay (TPHA), and TPI. RESULTS: Effectiveness of TTs in the diagnosis of neurosyphilis while research on 23 samples of CSF was carried out by the following methods: EIA (Immunoglobulin [Ig]M + IgG), FTA, andTPHA. The above-mentioned TTs used in serological diagnosis of CSF do not always meet the problem of confirming neurosyphilis diagnosis. According to these investigations, both positive and false positive results were obtained. In order to verify the diagnosis, a study on positive and false positive samples of CSF by TPI method was carried out. Positive results were obtained in 13 samples with the established duration of the disease. CONCLUSIONS: TPI is an optimal treponemal immunological method of examination of CSF to diagnose neurosyphilis with a high degree of reliability. The use of TPI while research on CSF makes it possible to verify the diagnosis of neurosyphilis by differentiated detection of antibodies to the most immunogenic antigens of T. pallidum eliminating the subjective factor of the reaction and simplifies diagnostic procedure.

Level of influenza virus strain antibodies in healthy vaccinated people at the end of the COVID-19 pandemic

Introduction. Abnormally low global influenza detection rates were observed at the peak of ongoing COVID-19 infection. In the 2021–2022 season, influenza resumed circulation, and in December 2022 its incidence rate returned to the 2015–2016 season levels, which exceeded the highest value recorded in 2018 worldwide. There is a need to assess changes in pattern of population immunity after influenza vaccination during the global COVID-19 pandemic. The study was aimed at analyzing level of vaccine influenza virus strain antibodies in healthy people formed during the COVID-19 pandemic. Materials and methods. A total of 123 healthy volunteers immunized with influenza quadrivalent inactivated subunit adjuvanted vaccine and 47 unimmunized volunteers were enrolled in the study. Influenza virus strains antibodies were assessed by performing a hemagglutination inhibition (HI) test one month after vaccination. Results. A significantly increased seroprotection rate (SPR) (p 0.05) was show in main group that reached 78.9% against the A/Victoria/2570/2019(H1N1)pdm09 strain, 94.3% — against A/H3N2/Darwin strain /9/2021, 69.1% — against B/Phuket/3073/13 strain and 48.8% — against B/Austria/1359417/2021 strain one month after vaccination. The GMT ratio to influenza A(H1N1), B (Victoria) and B (Yamagata) viruses exceeded the required minimum value (p 0.05) and comprised 4.1 (CI 3.68–4.54), 3.80 (CI 3.33–4.33) and 4.76 (CI 3.87–5.47), respectively. The post-vaccination GMT ratio to influenza A(H3N2) virus was 2.45, which is lower than the required level of 2.50 (p 0.05). The seroconversion rate (SCR) for strains A/Victoria/2570/2019(H1N1)pdm09, B/Phuket/3073/2013 and B/Austria/1359417/2021 was 56.1%, 50.4% and 51.2%, respectively. The SCR of the influenza A(H3N2) virus like GMT ratio was minimal among the four strains (p 0.05) and comprised 38.2%. Conclusion. The vaccine-related immunogenic activity met the requirements for inactivated seasonal influenza vaccines by at least one criterion.

Potential Involvement of the South American Lungfish Intelectin-2 in Innate-Associated Immune Modulation.

Intelectins belong to a family of lectins with specific and transitory carbohydrate interaction capabilities. These interactions are related to the activity of agglutinating pathogens, as intelectins play a significant role in immunity. Despite the prominent immune defense function of intelectins, limited information about its structural characteristics and carbohydrate interaction properties is available. This study investigated an intelectin transcript identified in RNA-seq data obtained from the South American lungfish (Lepidosiren paradoxa), namely LpITLN2-B. The structural analyses predicted LpITLN2-B to be a homo-trimeric globular protein with the fibrinogen-like functional domain (FReD), exhibiting a molecular mass of 57 kDa. The quaternary structure is subdivided into three monomers, A, B, and C, and each domain comprises 11 β-sheets: an anti-parallel β-sheet, a β-hairpin, and a disordered β-sheet structure. Molecular docking demonstrates a significant interaction with disaccharides rather than monosaccharides. The preferential interaction with disaccharides highlights the potential interaction with pathogen molecules, such as LPS and Poly(I:C). The hemagglutination assay inhibited lectins activity, especially maltose and sucrose, highlighting lectin activity in L. paradoxa samples. Overall, our results show the potential relevance of LpITLN2-B in L. paradoxa immune defense against pathogens.

Evolutionary dynamics and comparative pathogenicity of clade 2.3.4.4b H5 subtype avian influenza viruses, China, 2021–2022

The recent concurrent emergence of H5N1, H5N6, and H5N8 avian influenza viruses (AIVs) has led to significant avian mortality globally. Since 2020, frequent human-animal interactions have been documented. To gain insight into the novel H5 subtype AIVs (i.e., H5N1, H5N6 and H5N8), we collected 6102 samples from various regions of China between January 2021 and September 2022, and identified 41 H5Nx strains. Comparative analyses on the evolution and biological properties of these isolates were conducted. Phylogenetic analysis revealed that the 41 H5Nx strains belonged to clade 2.3.4.4b, with 13 related to H5N1, 19 to H5N6, and 9 to H5N8. Analysis based on global 2.3.4.4b viruses showed that all the viruses described in this study were likely originated from H5N8, exhibiting a heterogeneous evolutionary history between H5N1 and H5N6 during 2015–2022 worldwide. H5N1 showed a higher rate of evolution in 2021–2022 and more sites under positive selection pressure in 2015–2022. The antigenic profiles of the novel H5N1 and H5N6 exhibited notable variations. Further hemagglutination inhibition assay suggested that some A(H5N1) viruses may be antigenically distinct from the circulating H5N6 and H5N8 strains. Mammalian challenge assays demonstrated that the H5N8 virus (21GD001_H5N8) displayed the highest pathogenicity in mice, followed by the H5N1 virus (B1557_H5N1) and then the H5N6 virus (220086_H5N6), suggesting a heterogeneous virulence profile of H5 AIVs in the mammalian hosts. Based on the above results, we speculate that A(H5N1) viruses have a higher risk of emergence in the future. Collectively, these findings unveil a new landscape of different evolutionary history and biological characteristics of novel H5 AIVs in clade 2.3.4.4b, contributing to a better understanding of designing more effective strategies for the prevention and control of novel H5 AIVs.

Molecular And Serological Detection Of Newcastle Disease Virus In Live-Bird Markets, Jos, Plateau State In Nigeria

Newcastle disease (ND) is a highly infectious viral disease of birds caused by the Newcastle disease virus (NDV) and it has been reported in domestic birds in Nigeria. Waterfowls and village poultry in live bird markets (LBM) acts as reservoirs, potentially reintroducing the virus to commercial poultry. This study aims at molecular and serological detection of NDV at live bird markets in Plateau State, Nigeria. A cross sectional analysis involved 309 pooled cloacal and tracheal swabs over three months were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR), RT-qPCR, virus isolation and haemagglutination and haemagglutinationinhibition test (HA &HI). Virus isolation was attempted in 9 to 11 days old specific antibody negative (SAN) embryonated chicken eggs and fifteen samples showed haemagglutination. Subsequent tests confimed nine of these as NDV positive through haemagglutination and haemagglutination inhibition (HI) tests. Conventional RT-PCR and RT-qPCR further validated five of the nine NDV positive isolates. The phylogenetic analysis of partial F gene nucleotide sequences revealed that all three isolates belonged to class II genotype XIV.2_XIVb. This finding underscores the persistent threat of NDV to local poultry, necessitating comprehensive virological surveillance to understand, isolate and characterize the virus in Nigeria. Therefore, monitoring for emerging lineages and sub-lineages in Nigeria birds is crucial for safeguarding commercial poultry production.

Advax-SM™-Adjuvanted COBRA (H1/H3) Hemagglutinin Influenza Vaccines.

Adjuvants enhance immune responses stimulated by vaccines. To date, many seasonal influenza vaccines are not formulated with an adjuvant. In the present study, the adjuvant Advax-SM™ was combined with next generation, broadly reactive influenza hemagglutinin (HA) vaccines that were designed using a computationally optimized broadly reactive antigen (COBRA) methodology. Advax-SM™ is a novel adjuvant comprising inulin polysaccharide and CpG55.2, a TLR9 agonist. COBRA HA vaccines were combined with Advax-SM™ or a comparator squalene emulsion (SE) adjuvant and administered to mice intramuscularly. Mice vaccinated with Advax-SM™ adjuvanted COBRA HA vaccines had increased serum levels of anti-influenza IgG and IgA, high hemagglutination inhibition activity against a panel of H1N1 and H3N2 influenza viruses, and increased anti-influenza antibody secreting cells isolated from spleens. COBRA HA plus Advax-SM™ immunized mice were protected against both morbidity and mortality following viral challenge and, at postmortem, had no detectable lung viral titers or lung inflammation. Overall, the Advax-SM™-adjuvanted COBRA HA formulation provided effective protection against drifted H1N1 and H3N2 influenza viruses.

Sex Differences in the Immunogenicity and Efficacy of Seasonal Influenza Vaccines: A Meta-analysis of Randomized Controlled Trials.

Sex impacts individuals' response to vaccination. However, most vaccine studies do not report these differences disaggregated by sex. The aim of this study was to assess sex differences in the immunogenicity and efficacy of influenza vaccine. We performed a meta-analysis using phase 3 randomized controlled trial data conducted between 2010 and 2018. Using hemagglutination inhibition antibody titers for each strain, differences in geometric mean ratios (GMRs) were calculated by sex. Risk ratios (RRs) comparing seroconversion proportions were pooled for females and males using random-effects models. Vaccine efficacy (VE) was assessed. Data were analyzed by age group (18-64 vs ≥65 years). A total of 33 092 healthy adults from 19 studies were included for immunogenicity analysis, and 6740 from 1 study for VE. Whereas no sex differences in immunogenicity were found in adults <65 years old, older females had a significantly greater chance to seroconvert compared to older males for all strains: RRH1N1 = 1.17 [95% confidence interval {CI}, 1.12-1.23]; RRH3N2 = 1.09 [95% CI, 1.05-1.14]; RRVictoria = 1.23 [95% CI, 1.14-1.31]; RRYamagata = 1.22 [95% CI, 1.14-1.30]. GMRs were also higher in older females for all strains compared to older males. VE in preventing laboratory-confirmed influenza was higher in older females compared to older males with VEs of 27.32% (95% CI, 1.15%-46.56%) and 6.06% (95% CI, -37.68% to 35.90%), respectively. Our results suggest a higher immunogenicity and VE in females compared to males in older adults. These differences in immunogenicity and VE support the disaggregation of vaccine data by sex in clinical trials and observational studies. CRD42018112260.

Antimicrobial and Antiplasmodial Activities of Endophytic Fungi Associated with Psidium guajava

Infections due to antimicrobial-resistant microorganisms have become widespread in recent years. Thus, searching for novel antimicrobial agents to combat such pathogens has become crucial. The current study aimed to evaluate the antimicrobial, antiplasmodial, and immunomodulatory activities of the extracts of endophytic fungi isolated from Psidium guajava. Isolation, identification, fermentation, and extraction of the secondary metabolites of the fungal endophytes were carried out following standard procedures. The extracts were subjected to High-Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD) analysis to detect their bioactive components. The Antimicrobial activity and Minimum Inhibitory Concentration of the fungal extracts were evaluated against pure cultures of Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Salmonella typhi, and Klebsiella pneumonia using Agar well diffusion and Agar dilution method respectively. The acute toxicity study (LD50) was carried out using Lorke’s method. The extracts were tested in vivo in mice for antiplasmodial activity against Plasmodium berghei and in vitro against Plasmodium falciparum using Peter and Reyley's curative test method and WHO standardized micro-test system with modification respectively. The immunomodulatory activity of the extracts was evaluated by cyclophosphamide-induced myelosuppression (hematological parameters). Active extracts were further subjected to Delayed-Type Hypersensitivity Response (DTHR) and Haemagglutination Inhibition Assay using Sheep Red Blood Cells as antigens. The result showed Alternaria sp. (PGL1, PGL2, PGL3), from P. guajava. The HPLC-DAD analysis revealed the presence of bioactive compounds previously reported to have antimicrobial, antiplasmodial, and immunomodulatory properties. The fungal extracts exhibited varying degrees of antimicrobial activity. The LD50 of the fungi extracts was&gt;5000 mg/kg in mice. The extracts at 100 and 200 mg/kg body weight in mice showed varying degrees of antiplasmodial activity. Growth of P. berghei was significantly (p&lt;0.001) inhibited, curative effect ranges from 59.09 – 100%. Schizont maturation of P. falciparum isolates was inhibited and the highest level of inhibition was observed at 1 mg/ml (p&lt;0.05). The fungal extracts reversed the effect of cyclophosphamide-induced reduction in total white blood cell counts and % neutrophil. This study showed that the tested plant harbors species of endophytic fungi that contain numerous secondary metabolites. The endophytic fungi showed prophylactic, immunostimulatory, and antiplasmodial activities, which can be exploited to develop antimicrobial, antiplasmodial, and immunomodulatory agents.

MRNA vaccines encoding influenza virus hemagglutinin (HA) elicits immunity in mice from influenza A virus challenge.

Influenza viruses cause epidemics and can cause pandemics with substantial morbidity with some mortality every year. Seasonal influenza vaccines have incomplete effectiveness and elicit a narrow antibody response that often does not protect against mutations occurring in influenza viruses. Thus, various vaccine approaches have been investigated to improve safety and efficacy. Here, we evaluate an mRNA influenza vaccine encoding hemagglutinin (HA) proteins in a BALB/c mouse model. The results show that mRNA vaccination elicits neutralizing and serum antibodies to each influenza virus strain contained in the current quadrivalent vaccine that is designed to protect against four different influenza viruses including two influenza A viruses (IAV) and two influenza B (IBV), as well as several antigenically distinct influenza virus strains in both hemagglutination inhibition assay (HAI) and virus neutralization assays. The quadrivalent mRNA vaccines had antibody titers comparable to the antibodies elicited by the monovalent vaccines to each tested virus regardless of dosage following an mRNA booster vaccine. Mice vaccinated with mRNA encoding an H1 HA had decreased weight loss and decreased lung viral titers compared to mice not vaccinated with an mRNA encoding an H1 HA. Overall, this study demonstrates the efficacy of mRNA-based seasonal influenza vaccines are their potential to replace both the currently available split-inactivated, and live-attenuated seasonal influenza vaccines.

Trivalent mRNA vaccine-candidate against seasonal flu with cross-specific humoral immune response.

Seasonal influenza remains a serious global health problem, leading to high mortality rates among the elderly and individuals with comorbidities. Vaccination is generally accepted as the most effective strategy for influenza prevention. While current influenza vaccines are effective, they still have limitations, including narrow specificity for certain serological variants, which may result in a mismatch between vaccine antigens and circulating strains. Additionally, the rapid variability of the virus poses challenges in providing extended protection beyond a single season. Therefore, mRNA technology is particularly promising for influenza prevention, as it enables the rapid development of multivalent vaccines and allows for quick updates of their antigenic composition. mRNA vaccines have already proven successful in preventing COVID-19 by eliciting rapid cellular and humoral immune responses. In this study, we present the development of a trivalent mRNA vaccine candidate, evaluate its immunogenicity using the hemagglutination inhibition assay, ELISA, and assess its efficacy in animals. We demonstrate the higher immunogenicity of the mRNA vaccine candidate compared to the inactivated split influenza vaccine and its enhanced ability to generate a cross-specific humoral immune response. These findings highlight the potential mRNA technology in overcoming current limitations of influenza vaccines and hold promise for ensuring greater efficacy in preventing seasonal influenza outbreaks.

The immunodominance of antigenic site Sb on the H1 influenza virus hemagglutinin increases with high immunoglobulin titers of the cohorts and with young age, but not sex

The head domain of the hemagglutinin of influenza viruses plays a dominant role in the antibody response due to the presence of immunodominant antigenic sites that are the main targets of host neutralizing antibodies. For the H1 hemagglutinin, five major antigenic sites defined as Sa, Sb, Ca1, Ca2, and Cb have been described. Although previous studies have focused on defining the hierarchy of the antigenic sites of the hemagglutinin in different human cohorts, it is still unclear if the immunodominance profile of the antigenic sites might change with the antibody levels of individuals or if other demographic factors (such as exposure history, sex, or age) could also influence the importance of the antigenic sites. The major antigenic sites of influenza viruses hemagglutinins are responsible for eliciting most of the hemagglutination inhibition antibodies in the host. To determine the antibody prevalence towards each major antigenic site, we evaluated the hemagglutination inhibition against a panel of mutant H1 viruses, each one lacking one of the “classic” antigenic sites. Our results showed that the individuals from the Stop Flu NYU cohort had an immunodominant response towards the sites Sb and Ca2 of H1 hemagglutinin. A simple logistic regression analysis of the immunodominance profiles and the hemagglutination inhibition titers displayed by each donor revealed that individuals with high hemagglutination inhibition titers against the wild-type influenza virus exhibited higher probabilities of displaying an immunodominance profile dominated by Sb, followed by Ca2 (Sb > Ca2 profile), while individuals with low hemagglutination inhibition titers presented a higher chance of displaying an immunodominance profile in which Sb and Ca2 presented the same level of immunodominance (Sb = Ca2 profile). Finally, while age exhibited an influence on the immunodominance of the antigenic sites, biological sex was not related to displaying a specific immunodominance profile.

Characterization of anti-soybean agglutinin (SBA) IgY antibodies: a new strategy for neutralization of the detrimental biological activity of SBA.

Anti-soybean agglutinin (SBA) IgY was produced, and its potential to neutralize the haemagglutinating activity of SBA in vitro was tested. Thirty-five-week-old hens [treatment (n = 5) and control (n = 5)] were immunized with SBA or injected with saline 4 times every 15 days. Eggs were collected after the last immunization, and IgY was extracted using the polyethylene glycol (PEG) method. Serum anti-SBA IgY titres in immunized hens increased after the first immunization and reached a plateau between days 45 and 60. In contrast, specific IgY titres in the control group remained at basal levels throughout the evaluation. Average IgY titres were significantly higher in the treatment group on days 15, 30, 45, and 60. Total IgY content in the egg yolk extract was 38.7 ± 1.6 and 37.7 ± 1.5 mg/ml for the treatment and control groups, respectively. The specific anti-SBA IgY titer detected in the egg yolk extract was significantly higher (p < 0.001) for hens in the treatment group compared to the control group, with OD450nm values of 0.98 ± 0.05 and 0.058 ± 0.02, respectively. The specificity of anti-SBA IgY was confirmed by the Western blotting, and the inhibition of SBA-induced haemagglutination in vitro was compared with D-galactose, a known molecule that binds to SBA and blocks its binding to erythrocytes. The inhibition of SBA-induced haemagglutination by the anti-SBA IgY reached 512 units of haemagglutination inhibition (UHI), compared to 8 or 256 UHI, respectively, when IgY from control chickens or D-galactose was used. Thus, anti-SBA IgY antibodies were efficiently produced in large quantities and effectively inhibited SBA-induced haemagglutination in vitro.

Immune profiling of responses to influenza vaccination in patients with myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs) are associated with immune dysregulation and increased susceptibility to infection, emphasizing the importance of vaccination for patients. This pilot study evaluated immune responses to influenza vaccination in MPN patients compared with healthy donors using mass cytometry and serology. We observed diminished CXCR5+ B-cell, CXCR3+ T-cell, activated CD127+ memory T-cell subsets, and a trend toward lower hemagglutinin inhibition titer in MPN patients. These results indicate that patients with MPN exhibit distinct responses to influenza vaccination suggestive of impaired migration to lymphoid organs and T-cell maturation which may impact the development of protective immunity.

Duration of maternally derived antibodies of porcine parvovirus in growing pigs and presence of antibodies in gilts and sows vaccinated with three different parvovirus vaccines

While gilts and sows are regularly vaccinated against the porcine parvovirus (PPV), little is known on the presence of antibodies in vaccinated sows nor the decline of maternally derived antibodies (MDA) in their offspring. On twelve farms serum samples were taken from 180 gilts and sows vaccinated at least twice with one of three different commercial PPV vaccines. On nine farms, additional 270 serum samples were collected from growing pigs of three different age categories. All 450 samples were examined for PPV antibodies (Abs) by ELISA and haemagglutination inhibition (HI) assay. In total, 65% of all gilts vaccinated twice with either vaccine 1 or vaccine 3 were seronegative by HI assay. In each farm, there were at least three animals with high Ab titres (≥ 1:1280) indicating the presence of PPV in all twelve study farms. However, PPV DNA could not be detected in collected faecal samples. While low to moderately high Ab titres (1:10–1:640) were measured in 98% of twelve-weeks-old pigs, ELISA was only positive in 30% of the same pigs. Though, the statement on the duration of MDA may depend on the applied test, we could confirm an exponential decay of MDA. In addition, we could demonstrate that applied serological tools are insufficient for the confirmation of successful vaccination.

Mapping Genetic Markers Associated with Antigenicity and Host Range in H9N2 Influenza A Viruses Infecting Poultry in Pakistan.

The aim of the current study was to map the genetic diversity in the haemagglutinin (HA) glycoprotein of influenza A viruses (IAVs) of the H9N2 subtype. Twenty-five H9N2 IAVs were isolated from broiler chickens from March to July 2019. The HA gene was amplified, and phylogenetic analysis was performed to determine the evolutionary relationship. Important antigenic amino acid residues of HA attributed to immune escape and zoonotic potential were compared among H9N2 IAVs. Phylogenetic analysis revealed that sublineage B2 under the G1 lineage in Pakistan was found to be diversified, and newly sequenced H9N2 isolates were nested into two clades (A and B). Mutations linked to the antigenic variation and potential immune escape were observed as G72E (1/25, 4%), A180T (3/25, 12%), and A180V (1/25, 4%). A twofold significant reduction (P < 0.01) in log2 hemagglutination inhibition titers was observed with H9N2 IAV naturally harboring amino acid V180 instead of A180 in HA protein. Moreover, in the last 20 years, complete substitution at residues (T127D, D135N, and L150N) and partial substitution at residues (72, 74, 131, 148, 180, 183, 188, 216, 217, and 249, mature H9 HA numbering) associated with changes in antigenicity were observed. The presence of L216 in all H9N2 IAV isolates and T/V180 in four isolates in the receptor-binding site reveals the potential of these viruses to cross the species barrier to infect human or mammals. The current study observed the circulation of antigenically diverse H9N2 IAV variants that possess potential mutations that can escape the host immune system.

Generation and characterization of a nanobody against the avian influenza virus H7 subtype

Avian influenza virus (AIV) H7N9 diseases have been recently reported, raising concerns about a potential pandemic. Thus, there is an urgent need for effective therapeutics for AIV H7N9 infections. Herein, camelid immunization and yeast two-hybrid techniques were used to identify potent neutralizing nanobodies (Nbs) targeting the H7 subtype hemagglutinin. First, we evaluated the binding specificity and hemagglutination inhibition activity of the screened Nbs against the H7 subtype hemagglutinin. Nb-Z77, with high hemagglutination inhibition activity was selected from the screened Nbs to optimize the yeast expression conditions and construct oligomeric forms of Nb-Z77 using various ligation methods. The oligomers Nb-Z77-DiGS, Nb-Z77-TriGS, Nb-Z77-Fc and Nb-Z77-Foldon were successfully constructed and expressed. Nb-Z77-DiGS and Nb-Z77-Foldon exhibited considerably greater activity than did Nb-Z77 against H7 subtype hemagglutinin, with median effective concentrations of 384.7 and 27.33 pM and binding affinity values of 213 and 5.21 pM, respectively. Nb-Z77-DiGS and Nb-Z77-Foldon completely inhibited the hemagglutination activity of the inactivated virus H7-Re1 at the lowest concentration of 0.938 μg/mL. This study screened a strain of Nb with high hemagglutination inhibition activity and enhanced its antiviral activity through oligomerization, which may have great potential for developing effective agents for the prevention, diagnosis, and treatment of AIV H7 subtype infection.

Effects of astragalus extract, levamisole and ascorbic acid on humoral immunity in chickens vaccinated with newcastle disease vaccines

This study was designed to assess the effects of Astragalus extract, levamisole and ascorbic acid on the humoral immune response of chickens vaccinated with live and inactivated Newcastle Disease (ND) vaccines. Sixty day-old Cobb500 broiler chicks were used for the study. At day old, the maternally derived antibodies (MDA) to ND virus was determined and the chicks were then housed in a bio-secured pen with water and feed given ad-lib. On the sixth day, the MDA decay was determined thereafter, the chicks were shared into 4 treatment groups. Group A (Astragalus extract); Group B (levamisole); Group C (ascorbic acid) and Group D (Control) of 15 chicks each. Response to ND vaccinations was determined through bleeding to obtain sera for haemagglutination inhibition test at 7 and 14 days except for the second booster with inactivated Komarov vaccine where it was done at 7, 14 and 21 days post vaccination respectively. Antibody titres in the chicks 7 days post first dose of vaccination with La Sota was high in all the treatment groups above the control with 100 % of the chicks having protective antibody titre of ≥4 log2 until day 30 when the antibody titres in all the groups dropped drastically following the second dose of live La Sota vaccination as the first booster vaccine. However, following the second booster with inactivated Komarov the antibody titres increased in all the treatment groups in comparison to the control especially in groups B and C with GMTs of 5.8 ± 0.19 log2 and 6.1 ± 0.27 log2 respectively. We observed that ascorbic acid and Levamisole may have humoral immuno-stimulating effects on vaccinated chickens through yet to be fully explored mechanism. It is recommended that ascorbic acid or levamisole could be used during vaccinations as immuno-stimulating agents to enhance humoral antibody response in vaccinated flocks.

Assessment of Different Newcastle Disease Virus Antigens and Inactivators of Binary Ethylene Amine and Formalin for the Hemagglutination Inhibition Assay.

Newcastle disease is a severe viral threat to the global poultry industry due to its high prevalence and rapid transmission. Evaluating vaccination timing and effectiveness is crucial, often accomplished through the hemagglutination inhibition (HI) assay. This test relies on the virus's agglutination ability in certain animals, utilizing various inactivated antigens. Our study aimed to assess multiple Newcastle viral antigens ( LaSota, clone, thermo-resistant strain, B1, and V4 ) inactivated by binary ethylene amine (BEA) and formalin, seeking the best antigen and inactivator for the HI assay. We prepared the different ND antigens include; LaSota, Clone, thermo resistant, B1, V4 and the mixture of the antigens then inactivated them using BEA and formalin. The hemagglutination (HA) assay determined mean titers, comparing BEA and formalin inactivation. These antigens were also subjected to the HI test using 112 serum samples from different commercial poultry flocks to assess their performance. BEA-inactivated antigens exhibited significantly higher mean titers in the HA assay than formalin-inactivated antigens. In the evaluation of different antigens in the HI test, the mean titer of antigen B1 followed by clone and LaSota displayed a higher mean titer than others. In conclusion, this study recommends using Hitchner pathotype antigens, specifically the B1 vaccine, for Newcastle disease HI testing. BEA is the preferred inactivator, preserving antigen structure particularly the structure of hemagglutinin antigen while minimizing risks. These findings can enhance serological testing accuracy, contributing to more effective disease control and prevention in the poultry industry.