Anti-hemagglutinin Antibodies Research Articles

Targeted Elimination of Influenza Virus and Infected Cells with Near-Infrared Antiviral Photoimmunotherapy (NIR-AVPIT)

Background: Seasonal influenza causes significant morbidity and mortality each year. Since viruses can easily acquire drug-resistant mutations, it is necessary to develop new antiviral strategies with different targets. Near-infrared photoimmunotherapy (NIR-PIT) is a type of anti-cancer therapy that has recently attracted considerable attention, with favorable outcomes reported for several cancers. In this study, we investigated whether this approach could be used as a novel anti-influenza therapy to destroy influenza virus and infected cells. Methods: To evaluate the efficacy of near-infrared antiviral photoimmunotherapy (NIR-AVPIT), we prepared an anti-hemagglutinin (HA) monoclonal antibody without neutralizing activity against influenza A virus (FluV) labeled with IR-700 and reacted it with FluV and infected cells, as well as HA-expressing HEK293 cells. Results: NIR-AVPIT strongly inactivated FluV virions, suppressed cytopathic effects, and achieved more than a 4-log reduction in viral RNA amplification. Treatment of FluV-infected cells with the antibody-IR700 complex and NIR in the early stages of infection significantly inhibited viral propagation, and double treatment with time apart exerted a greater inhibitory effect. NIR-AVPIT rapidly induced morphological changes in HA-expressing HEK293 cells and inhibited the proliferation of these cells. Conclusions: These results suggest that NIR-AVPIT targeting HA antigens could inactivate FluV and eliminate infected cells in vitro. This strategy is a promising approach to treat various viral infections, including influenza.

Triton X-100-treated virus-based ELLA demonstrates discordant antigenic evolution of influenza B virus hemagglutinin and neuraminidase.

Neuraminidase (NA)-specific antibodies have been associated with protection against influenza and thus NA is considered a promising target for next-generation vaccines against influenza A (IAV) and B viruses (IBV). NA inhibition (NI) by antibodies is typically assessed using an enzyme-linked lectin assay (ELLA). However, ELLA can be confounded by anti-hemagglutinin (anti-HA) antibodies that block NA by steric hindrance (termed HA interference). Although strategies have been employed to overcome HA interference for IAV, similar approaches have not been assessed for IBV. We found that HA interference is common in ELLA using IBV, rendering the technique unreliable. Anti-HA antibodies were not completely depleted from sera by HA-expressing cell lines, and this approach was of limited utility. In contrast, we find that treatment of virions with Triton X-100, but not Tween-20 or ether, efficiently separates the HA and NA components and overcomes interference caused by anti-HA antibodies. We also characterize a panel of recombinant IBV NA proteins that further validated the results from Triton X-100-treated virus-based ELLA. Using these reagents and assays, we demonstrate discordant antigenic evolution between IBV NA and HA over the last 80 years. This optimized ELLA protocol will facilitate further in-depth serological surveys of IBV immunity as well as antigenic characterization of the IBV NA on a larger scale.IMPORTANCEInfluenza B viruses (IBVs) contribute to annual epidemics and may cause severe disease, especially in children. Consequently, several approaches are being explored to improve vaccine efficacy, including the addition of neuraminidase (NA). Antigen selection and assessment of serological responses will require a reliable serological assay to specifically quantify NA inhibition (NI). Although such assays have been assessed for influenza A viruses (IAVs), this has not been done of influenza B viruses. Our study identifies a readily applicable strategy to measure the inhibitory activity of neuraminidase-specific antibodies against influenza B virus without interference from anti-hemagglutinin (anti-HA) antibodies. This will aid broader serological assessment of influenza B virus-specific antibodies and antigenic characterization of the influenza B virus neuraminidase.

Influenza Hemagglutinin Antibody Levels in the Elderly: Impact of Sex, Age, and Influenza/COVID-19 Vaccination Status During the 2021/2022 Epidemic Season in Warsaw, Poland.

BACKGROUND The purpose of the study was to determine the level of antihemagglutinin antibodies in the serum of patients in the geriatric population in Doctor's Surgery NZOZ Nucleus Warsaw, Poland, during the epidemic season 2021/2022 using the hemagglutination inhibition assay (HAI), according to anti-influenza and anti-COVID-19 vaccination, age, and sex. MATERIAL AND METHODS Serum samples taken from 256 patients aged 65 to 99 years were examined for anti-hemagglutinin antibodies and protective levels of antibodies against antigens: A/Victoria/2570/2019 (H1N1)pdm09, A/Cambodia/e0826360/2020(H3N2), B/Washington/02/2019 (B/Victoria lineage), and B/Phuket/3073/2013 (B/Yamagata lineage) of the quadrivalent influenza vaccine for epidemic season 2021/2022. RESULTS The highest protective level, ie, the percentage of people with antibody titers ≥40 was 87.5% and was recorded for subtype A/Cambodia/e0826360/2020(H3N2), the dominant type causing infections in the epidemic season 2021/2022 confirmed by molecular biology methods. Geometric mean titer (GMT) values and protective levels for B/Washington/02/2019 (B/Victoria lineage) antigen were higher for men than women (respectively 38.4 vs 67.6; P<0.001 and 58.0% vs 74.6%; P<0.001). The protective levels of antibodies among patients vaccinated vs unvaccinated against COVID-19 were higher for B/Washington/02/2019 (B/Victoria lineage) and B/Phuket/3073/2013 (B/Yamagata lineage) antigens (64.2% vs 44.4%; P=0.023 and 78.6% vs 55.6%; P=0.004). GMT values for vaccinated against COVID-19 were also higher. There were no significant differences between younger (65-79 years) and older (≥80 years) seniors. CONCLUSIONS The analysis shows differences in the level of individual antibodies, GMT and the protective level depending on subtypes of influenza A or B virus, B/Victoria or B/Yamagata lineage, sex, and previous vaccination history against influenza and COVID-19.

Influenza virus antibodies inhibit antigen-specific de novo B cell responses in mice.

Antibody responses to influenza vaccines tend to be focused on epitopes encountered during prior influenza exposures, with little production of de novo responses to novel epitopes. To examine the contribution of circulating antibodies to this phenomenon, we passively transferred a hemagglutinin (HA)-specific monoclonal antibody (mAb) into mice before immunizing with whole inactivated virions. The HA mAb inhibited de novo HA-specific antibodies, plasmablasts, germinal center B cells, and memory B cells, while responses to a second antigen in the vaccine, neuraminidase (NA), were uninhibited. The HA mAb potently inhibited de novo antibody responses against epitopes near the HA mAb binding site. The HA mAb also promoted IgG1 class switching, an effect that, unlike the inhibition of HA responses, relied on signaling through Fc-gamma receptors. These studies suggest that circulating antibodies inhibit de novo B cell responses in an antigen-specific manner, which likely contributes to differences in antibody specificities elicited during primary and secondary influenza virus exposures.IMPORTANCEMost humans are exposed to influenza viruses in childhood and then subsequently exposed to antigenically drifted influenza variants later in life. It is unclear if antibodies elicited by earlier influenza virus exposures impact immunity against new influenza virus strains. Here, we used a mouse model to investigate how an anti-hemagglutinin (HA) monoclonal antibody (mAb) affects de novo B cell and antibody responses to the protein targeted by the monoclonal antibody (HA) and a second protein not targeted by the monoclonal antibody [neuraminidase (NA)]. Collectively, our studies suggest that circulating anti-influenza virus antibodies can potently modulate the magnitude and specificity of antibody responses elicited by secondary influenza virus exposures.

Occurrence of Circulating Antibodies against the Hemagglutinins of Influenza Viruses in the 2022/2023 Epidemic Season in Poland.

The aim of this study was to determine the level of anti-hemagglutinin antibodies in blood sera collected from patients during the 2022/2023 epidemic season in Poland. A total of 700 sera samples from patients across the country were tested. The samples were divided into seven groups according to the age of the patients, with 100 samples from each age group. The hemagglutination inhibition test (OZHA) was used to determine the level of anti-hemagglutinin antibodies. The test results have confirmed the presence of anti-hemagglutinin antibodies for antigens A/Victoria/2570/2019 (H1N1)pdm09, A/Darwin/9/2021 (H3N2), B/Austria/1359417/2021 (B/Yamagata lineage) and B/ Phuket/3073/2013 (B/Victoria lineage) present in the influenza vaccine recommended by the World Health Organization (WHO) for the 2022/2023 epidemic season. The highest geometric mean antibody titres (GMT) and protection rate values (%) were recorded for hemagglutinin A/H3N2. In Poland, in the 2022/2023 epidemic season, the percentage of the population vaccinated against influenza was 5.7%. Therefore, the test results can be interpreted as the response of the immune system in patients who have been previously infected with an influenza virus.

Prevalence of circulating antibodies against hemagglutinin of influenza viruses in epidemic season 2021/2022 in Poland.

The aim of the study was to determine the level of anti-hemagglutinin antibodies in the serum of patients during the 2021/2022 epidemic season in Poland. A total of 700 sera samples were tested, divided according to the age of the patients into 7 age groups: 0-4years of age, 5-9years of age, 10-14years of age, 15-25years of age, 26-44years of age, 45-64years of age and ≥65years of age, 100 samples were collected from each age group. Anti-hemagglutinin antibody levels was determined using the haemagglutination inhibition assay (OZHA). The results obtained confirm the presence of anti-hemagglutinin antibodies for the antigens A/Victoria/2570/2019 (H1N1) pdm09, A/Cambodia/e0826360/2020 (H3N2), B/Washington/02/2019 and B/Phuket/3073/2013 recommended by World Health Organization (WHO) for the 2021/2022 epidemic season. The analysis of the results shows differences in the levels of individual anti-hemagglutinin antibodies in the considered age groups. In view of very low percentage of the vaccinated population in Poland, which was 6.90% in the 2021/2022 epidemic season, the results obtained in the study would have to be interpreted as the immune system response in patients after a previous influenza virus infection.

Longitudinal Analysis of Neuraminidase and Hemagglutinin Antibodies to Influenza A Viruses after Immunization with Seasonal Inactivated Influenza Vaccines.

Neuraminidase (NA)-based immunity could reduce the harmful impact of novel antigenic variants of influenza viruses. The detection of neuraminidase-inhibiting (NI) antibodies in parallel with anti-hemagglutinin (HA) antibodies may enhance research on the immunogenicity and duration of antibody responses to influenza vaccines. To assess anti-NA antibodies after vaccination with seasonal inactivated influenza vaccines, we used the enzyme-linked lectin assay, and anti-HA antibodies were detected in the hemagglutination inhibition assay. The dynamics of the anti-NA antibody response differed depending on the virus subtype: antibodies to A/H3N2 virus neuraminidase increased later than antibodies to A/H1N1pdm09 subtype neuraminidase and persisted longer. In contrast to HA antibodies, the fold increase in antibody titers to NA after vaccination poorly depended on the preexisting level. At the same time, NA antibody levels after vaccination directly correlated with titers before vaccination. A difference was found in response to NA antigen between split and subunit-adjuvanted vaccines and in NA functional activity in the vaccine formulations.

Imprinted Anti-Hemagglutinin and Anti-Neuraminidase Antibody Responses after Childhood Infections of A(H1N1) and A(H1N1)pdm09 Influenza Viruses.

Immune imprinting is a driver known to shape the anti-hemagglutinin (HA) antibody landscape of individuals born within the same birth cohort. With the HA and neuraminidase (NA) proteins evolving at different rates under immune selection pressures, anti-HA and anti-NA antibody responses since childhood influenza virus infections have not been evaluated in parallel at the individual level. This is partly due to the limited knowledge of changes in NA antigenicity, as seasonal influenza vaccines have focused on generating neutralizing anti-HA antibodies against HA antigenic variants. Here, we systematically characterized the NA antigenic variants of seasonal A(H1N1) viruses from 1977 to 1991 and completed the antigenic profile of N1 NAs from 1977 to 2015. We identified that NA proteins of A/USSR/90/77, A/Singapore/06/86, and A/Texas/36/91 were antigenically distinct and mapped N386K as a key determinant of the NA antigenic change from A/USSR/90/77 to A/Singapore/06/86. With comprehensive panels of HA and NA antigenic variants of A(H1N1) and A(H1N1)pdm09 viruses, we determined hemagglutinin inhibition (HI) and neuraminidase inhibition (NI) antibodies from 130 subjects born between 1950 and 2015. Age-dependent imprinting was observed for both anti-HA and anti-NA antibodies, with the peak HI and NI titers predominantly detected from subjects at 4 to 12 years old during the year of initial virus isolation, except the age-independent anti-HA antibody response against A(H1N1)pdm09 viruses. More participants possessed antibodies that reacted to multiple antigenically distinct NA proteins than those with antibodies that reacted to multiple antigenically distinct HA proteins. Our results support the need to include NA proteins in seasonal influenza vaccine preparations. IMPORTANCE Seasonal influenza vaccines have aimed to generate neutralizing anti-HA antibodies for protection since licensure. More recently, anti-NA antibodies have been established as an additional correlate of protection. While HA and NA antigenic changes occurred discordantly, the anti-HA and anti-NA antibody profiles have rarely been analyzed in parallel at the individual level, due to the limited knowledge on NA antigenic changes. By characterizing NA antigenic changes of A(H1N1) viruses, we determined the anti-HA and anti-NA antibody landscape against antigenically distinct A(H1N1) and A(H1N1)pdm09 viruses using sera of 130 subjects born between 1950 and 2015. We observed age-dependent imprinting of both anti-HA and anti-NA antibodies against strains circulated during the first decade of life. A total of 67.7% (88/130) and 90% (117/130) of participants developed cross-reactive antibodies to multiple HA and NA antigens at titers ≥1:40. With slower NA antigenic changes and cross-reactive anti-NA antibody responses, including NA protein in influenza vaccine preparation may enhance vaccine efficacy.

Hemagglutinin Antibodies in the Polish Population during the 2019/2020 Epidemic Season.

The aim of the study was to determine the level of antibodies against hemagglutinin of influenza viruses in the serum of subjects belonging to seven different age groups in the 2019/2020 epidemic season. The level of anti-hemagglutinin antibodies was tested using the hemagglutination inhibition (HAI) test. The tests included 700 sera from all over Poland. Their results confirmed the presence of antibodies against the following influenza virus antigens: A/Brisbane/02/2018 (H1N1)pdm09 (48% of samples), A/Kansas/14/2017/ (H3N2) (74% of samples), B/Colorado/06/ 2017 Victoria line (26% of samples), and B/Phuket/3073/2013 Yamagata line (63% of samples). The level of antibodies against hemagglutinin varied between the age groups. The highest average (geometric mean) antibody titer (68.0) and the highest response rate (62%) were found for the strain A/Kansas/14/2017/ (H3N2). During the epidemic season in Poland, only 4.4% of the population was vaccinated.

Electro-Nano Diagnostic Platform Based on Antibody–Antigen Interaction: An Electrochemical Immunosensor for Influenza A Virus Detection

H1N1 is a kind of influenza A virus that causes serious health issues throughout the world. Its symptoms are more serious than seasonal flu and can sometimes be lethal. For this reason, rapid, accurate, and effective diagnostic tests are needed. In this study, an electrochemical immunosensor for the sensitive, selective, and practical detection of the H1N1 virus was developed. The sensor platform included multi-walled carbon nanotube gold-platinum (MWCNT-Au-Pt) hybrid nanomaterial and anti-hemagglutinin (anti-H1) monoclonal antibody. For the construction of this biosensor, a gold screen-printed electrode (AuSPE) was used as a transducer. Firstly, AuSPE was modified with MWCNT-Au-Pt hybrid nanomaterial via drop casting. Anti-H1 antibody was immobilized onto the electrode surface after the modification process with cysteamine was applied. Then, the effect of the interaction time with cysteamine for surface modification was investigated. Following that, the experimental parameters, such as the amount of hybrid nanomaterial and the concentration of anti-H1 were optimized. Under the optimized conditions, the analytical characteristics of the developed electrochemical immunosensor were investigated for the H1N1 virus by using electrochemical impedance spectroscopy. As a result, a linear range was obtained between 2.5-25.0 µg/mL with a limit of the detection value of 3.54 µg/mL. The relative standard deviation value for 20 µg/mL of the H1N1 virus was also calculated and found as 0.45% (n = 3). In order to determine the selectivity of the developed anti-H1-based electrochemical influenza A immunosensor, the response of this system towards the H3N2 virus was investigated. The matrix effect was also investigated by using synthetic saliva supplemented with H1N1 virus.

EVALUATION OF THE EFFICIENCY OF THE SEROLOGICAL HAI METHOD FOR THE DETECTION OF ANTIBODIES TO THE EGG DROP SYNDROME VIRUS

The article describes the features of the course of the disease of laying hens with Egg drop syndrome (EDS). This is a contagious viral disease of commercial and breeding laying hens, which is characterized by damage to the reproductive organs, a sharp decrease in laying capacity, changes in the pigmentation and shape of laid eggs, the appearance of effusions, thinning, deformations, and in some places the complete absence of a shell, a decrease in the percentage of hatching of chickens from hatching eggs and a deterioration in their viability. Morbidity is 10–70 %, mortality is 1–10 %. The disease in chickens occurs during the egg-laying period, until then no clinical signs of infection are observed. Three forms of EDS have been described: classic, endemic, and sporadic. The diagnosis of EDS is established on the basis of the results of laboratory tests, taking into account epizootological data, clinical signs and pathological anatomical changes. At the same time, special attention is paid to the decrease in egg production and the appearance of deformed and shelles eggs in clinically healthy chickens, the death of embryos and day-old chicks. Laboratory diagnostics is based on express, virological and retrospective methods. One of the most common methods of diagnosing EDS is retrospective diagnosis, which consists in detecting a diagnostic (minimum 4-fold) increase in the antibody titer in paired blood sera of chickens by the methods of HAI, IHA or ELISA. HAI is recommended for mass serological examinations of poultry farms. The detection of Anti-Hemagglutinin Antibody (Anti-HA) in the studied blood sera at a titer of 1:16 (4 log2) and higher in farms where specific prevention of EDS is not carried out indicates the circulation of the field strain of EDS virus among poultry. It should be noted that chickens that do not yet lay deformed eggs can be infected with the virus, but do not give a humoral immune response to latent infection. Thus, in flocks of chickens that were infected transovarially, antibodies are not synthesized during the breeding period. A negative serological test in chickens under the age of 20 weeks does not guarantee the absence of EDS infection. Differential diagnosis requires the exclusion of infectious bronchitis, infectious laryngotracheitis, Newcastle disease and respiratory mycoplasmosis. An analysis of 20 inactivated vaccines from various manufacturing companies registered in Ukraine for the specific prevention of EDS was carried out, including monovalent – 3 and associated, namely 3-component – 10 and 4-component – 7. During serological monitoring, it was established, that the average titers in the sera of laying hens to the EDS virus from batches of different ages due to different terms after vaccination with inactivated vaccines ranged from 7.0 log2 to 13.2 log2. The average titers to the EDS virus above the basic norm were 16 % among birds of 4-8 batches (280, 310 and 460 days). It was established that the percentage of protective antibodies to the EDS virus in 25 batches of laying hens ranged from 78 to 100. The use of inactivated vaccines against EDS stimulates the formation of an active immune response in laying hens in 84 % of the studied batches. Carrying out constant monitoring of the effectiveness of inactivated vaccines against by means of serological monitoring of blood sera from birds in the HAI will allow, depending on the epizootic situation, to timely adjust the vaccination scheme and ensure stable economic indicators in commercial and breeding flocks of laying hens.

Importance of Influenza Anti-Hemagglutinin Antibodies During the SARS-CoV-2 Pandemic in the 2019/2020 Epidemic Season in Poland.

BackgroundThe aim of this study was to determine the level of anti-hemagglutinin antibodies in the serum of recovered patients during the SARS-CoV-2 pandemic in the 2019/2020 epidemic season in Poland, and the course of COVID-19.Material/MethodsThe material for the study consisted of the sera of COVID-19 convalescents obtained from the following 9 Regional Blood Donation and Blood Supply Centers located in 8 voivodeships. The hemagglutination inhibition reaction assay (HAI) using 8 viral hemagglutination units was used to determine antibody levels, in accordance with WHO recommendations.ResultsThis research confirms that a patient’s declared severity of the course of SARS-CoV-2 infection is influenced by the patient’s age and concomitant diseases. There was no statistically significant correlation between the level of anti-hemagglutinin antibodies and the severity of the course of a SARS-CoV-2 infection. Based on the serological tests conducted, it can be unequivocally concluded that both vaccinated and influenza-infected patients had a response rate in line with the requirements of the European Commission and the Committee for Medicinal Products for Human Use hemagglutinin antibodies for 4 influenza virus antigens tested.ConclusionsPatients who confirmed their antibody levels with the Commission of the European Communities and the Committee for Propriety Medicinal Products (CPMP) requirements had a mild COVID-19 course. The results of our research emphasize the role of anti-hemagglutinin antibodies in the course of SARS-CoV-2 infection. COVID-19 convalescents have a higher response rate against all 4 types of anti-hemagglutinin antibodies analyzed.

Measles and Antibody-Dependent Enhancement (ADE): History and Mechanisms

Antibody-dependent enhancement (ADE) is an inadequate response to reinfection or vaccination. ADE was described for influenza and dengue fever: patients already exposed are likely to develop a more severe infection when exposed to a virus of another type than the first. Vaccine antibodies also appear to be responsible for an increased risk of severe disease in a naive person. In COVID-19, ADE is likely with antibodies acquired following infection or vaccination. The aggravation of the disease by measles vaccination has been shown for the inactivated virus vaccines. Atypical measles was also described after the live attenuated vaccine (LAV). ADE mechanisms are the penetration into cells of virus-antibody complexes promoted by FcγR or complement receptors and by an imbalance between neutralizing and facilitating antibodies. The role of maternal antibodies in ADE has been suggested after influenza vaccination in piglets. Facilitation of virus entry into the cell by complement fixation and an imbalance between anti-hemagglutinin and anti-fusion protein antibody levels have been suggested as a mechanism for atypical measles after the inactivated vaccine. Antibodies induced by the current LAV can induce ADE in vitro by binding to FcγR and the same imbalance. A recent vaccination campaign during an outbreak and the comparative history of measles before and during the vaccine era may alert to a possible ADE by the current LAV: it could be caused in infants by maternal antibodies and in adults by waning vaccine immunity. Improvement of current LAV or the development of a new type of vaccine could eliminate this phenomenon.

Development of PDA Nanoparticles for H9N2 Avian Influenza BPP-V/BP-IV Epitope Peptide Vaccines: Immunogenicity and Delivery Efficiency Improvement.

The protection of current influenza vaccines is limited due to the viral antigenic shifts and antigenic drifts. The universal influenza vaccine is a new hotspot in vaccine research that aims to overcome these problems. Polydopamine (PDA), a versatile biomaterial, has the advantages of an excellent biocompatibility, controllable particle size, and distinctive drug loading approach in drug delivery systems. To enhance the immunogenicities and delivery efficiencies of H9N2 avian influenza virus (AIV) epitope peptide vaccines, PDA nanoparticles conjugated with the BPP-V and BP-IV epitope peptides were used to prepare the nano BPP-V and BP-IV epitope peptide vaccines, respectively. The characteristics of the newly developed epitope peptide vaccines were then evaluated, revealing particle sizes ranging from approximately 240 to 290 nm (PDI<0.3), indicating that the synthesized nanoparticles were stable. Simultaneously, the immunoprotective effects of nano BPP-V and BP-IV epitope peptide vaccines were assessed. The nano BPP-V and BP-IV epitope vaccines, especially nano BP-IV epitope vaccine, quickly induced anti-hemagglutinin (HA) antibody production and a sustained immune response, significantly promoted humoral and cellular immune responses, reduced viral lung damage and provided effective protection against AIV viral infection. Together, these results reveal that PDA, as a delivery carrier, can improve the immunogenicities and delivery efficiencies of H9N2 AIV nano epitope vaccines, thereby providing a theoretical basis for the design and development of PDA as a carrier of new universal influenza vaccines.

Genetic and Antigenic Characterization of Avian Avulavirus Type 6 (AAvV-6) Circulating in Canadian Wild Birds (2005–2017)

We describe for the first time the genetic and antigenic characterization of 18 avian avulavirus type-6 viruses (AAvV-6) that were isolated from wild waterfowl in the Americas over the span of 12 years. Only one of the AAvV-6 viruses isolated failed to hemagglutinate chicken red blood cells. We were able to obtain full genome sequences of 16 and 2 fusion gene sequences from the remaining 2 isolates. This is more than double the number of full genome sequences available at the NCBI database. These AAvV-6 viruses phylogenetically grouped into the 2 existing AAvV-6 genotype subgroups indicating the existence of an intercontinental epidemiological link with other AAvV-6 viruses isolated from migratory waterfowl from different Eurasian countries. Antigenic maps made using HI assay data for these isolates showed that the two genetic groups were also antigenically distinct. An isolate representing each genotype was inoculated in specific pathogen free (SPF) chickens, however, no clinical symptoms were observed. A duplex fusion gene based real-time assay for the detection and genotyping of AAvV-6 to genotype 1 and 2 was developed. Using the developed assay, the viral shedding pattern in the infected chickens was examined. The chickens infected with both genotypes were able to shed the virus orally for about a week, however, no significant cloacal shedding was detected in chickens of both groups. Chickens in both groups developed detectable levels of anti-hemagglutinin antibodies 7 days after infection.

An influenza HA stalk reactive polymeric IgA antibody exhibits anti-viral function regulated by binary interaction between HA and the antibody.

IgA antibodies, which are secreted onto the mucosal surface as secretory IgA antibodies (SIgAs), play an important role in preventing influenza virus infection. A recent study reported that anti-hemagglutinin (HA) head-targeting antibodies increase anti-viral functions such as hemagglutination inhibition (HI) and virus neutralization (NT), in addition to HA binding activity (reactivity) via IgA polymerization. However, the functional properties of anti-viral IgA antibodies with mechanisms of action distinct from those of anti-HA head-targeting antibodies remain elusive. Here, we characterized the functional properties of IgG, monomeric IgA, and polymeric IgA anti-HA stalk-binding clones F11 and FI6, and B12 (a low affinity anti-HA stalk clone), as well as Fab-deficient (ΔFab) IgA antibodies. We found that IgA polymerization impacts the functional properties of anti-HA stalk antibodies. Unlike anti-HA head antibodies, the anti-viral functions of anti-HA stalk antibodies were not simply enhanced by IgA polymerization. The data suggest that two modes of binding (Fab paratope-mediated binding to the HA stalk, and IgA Fc glycan-mediated binding to the HA receptor binding site (RBS)) occur during interaction between anti-stalk HA IgA antibodies and HA. In situations where Fab paratope-mediated binding to the HA stalk exceeded IgA Fc glycan-mediated binding to HA RBS, IgA polymerization increased anti-viral functions. By contrast, when IgA Fc glycan-mediated binding to the HA RBS was dominant, anti-viral activity will fall upon IgA polymerization. In summary, the results suggest that coordination between these two independent binding modules determines whether IgA polymerization has a negative or positive effect on the anti-viral functions of anti-HA stalk IgA antibodies.

Cross-neutralizing Anti-hemagglutinin Antibodies Isolated from Patients Infected with Avian Influenza A (H5N1) Virus

ObjectiveTo recover broad-neutralizing monoclonal antibodies (BnAbs) from avian influenza A (H5N1) virus infection cases and investigate their genetic and functional features. MethodsWe screened the Abs repertoires of expanded B cells circulating in the peripheral blood of H5N1 patients. The genetic basis, biological functions, and epitopes of the obtained BnAbs were assessed and modeled. ResultsTwo BnAbs, 2-12D5, and 3-37G7.1, were respectively obtained from two human H5N1 cases on days 12 and 21 after disease onset. Both Abs demonstrated cross-neutralizing and Ab-dependent cellular cytotoxicity (ADCC) activity. Albeit derived from distinct Ab lineages, i.e., VH1-69-D2-15-JH4 (2-12D5) and VH1-2-D3-9-JH5 (3-32G7.1), the BnAbs were directed toward CR6261-like epitopes in the HA stem, and HA2 I45 in the hydrophobic pocket was the critical residue for their binding. Signature motifs for binding with the HA stem, namely, IFY in VH1-69-encoded Abs and LXYFXW in D3-9-encoded Abs, were also observed in 2-12D5 and 3-32G7.1, respectively. ConclusionsCross-reactive B cells of different germline origins could be activated and re-circulated by avian influenza virus. The HA stem epitopes targeted by the BnAbs, and the two Ab-encoding genes usage implied the VH1-69 and D3-9 are the ideal candidates triggered by influenza virus for vaccine development.

Response to a DNA vaccine against the H5N1 virus depending on the chicken line and number of doses

BackgroundAvian influenza virus infections cause significant economic losses on poultry farms and pose the threat of a possible pandemic outbreak. Routine vaccination of poultry against avian influenza is not recommended in Europe, however it has been ordered in some other countries, and more countries are considering use of the avian influenza vaccine as a component of their control strategy. Although a variety of such vaccines have been tested, most research has concentrated on specific antibodies and challenge experiments.MethodsWe monitored the transcriptomic response to a DNA vaccine encoding hemagglutinin from the highly pathogenic H5N1 avian influenza virus in the spleens of broiler and layer chickens. Moreover, in layer chickens the response to one and two doses of the vaccine was compared.ResultsAll groups of birds immunized with two doses of the vaccine responded at the humoral level by producing specific anti-hemagglutinin antibodies. A response to the vaccine was also detected in the spleen transcriptomes. Differential expression of many genes encoding noncoding RNA and proteins functionally connected to the neuroendocrine-immune system was observed in different immunized groups.ConclusionBroiler chickens showed a higher number and wider range of fold-changes in the transcriptional response than laying hens.

Hemagglutination inhibition antibody titers as a correlate of protection against influenza disease in the 2018/2019 epidemic season in Poland.

The aim of this study was to determine the level of antibodies against hemagglutinin of influenza viruses in the sera of people in the seven age groups in the epidemic season 2018/2019 in Poland. The level of anti-hemagglutinin antibodies was determined by hemagglutination inhibition test (HAI). 1050 clinical samples from all over the country were tested. The level of antibodies against influenza viruses was highest in the 10-14 age group for A/Singapore/INFIMH-16-0019/2016 (H3N2) and B/Phuket/3073/2013 Yamagata lineage antigens. These results confirm the circulation of four antigenically different influenza virus strains, two subtypes of influenza A virus - A/Michigan/45/2015 (H1N1)pdm09 and A/Singapore/INFIMH-16-0019/2016 (H3N2) and two lineages of influenza B virus - B/Colorado/06/2017 - Victoria lineage and B/Phuket/3073/2013 Yamagata lineage.

Influenza Anti-Stalk Antibodies: Development of a New Method for the Evaluation of the Immune Responses to Universal Vaccine.

Growing interest in universal influenza vaccines and novel administration routes has led to the development of alternative serological assays that are able to detect antibodies against conserved epitopes. We present a competitive ELISA method that is able to accurately determine the ratio of serum immunoglobulin G directed against the different domains of the hemagglutinin, the head and the stalk. Human serum samples were treated with two variants of the hemagglutinin protein from the A/California/7/2009 influenza virus. The signals detected were assigned to different groups of antibodies and presented as a ratio between head and stalk domains. A subset of selected sera was also tested by hemagglutination inhibition, single radial hemolysis, microneutralization, and enzyme-linked lectin assays. Pre-vaccination samples from adults showed a quite high presence of anti-stalk antibodies, and the results were substantially in line with those of the classical serological assays. By contrast, pre-vaccination samples from children did not present anti-stalk antibodies, and the majority of the anti-hemagglutinin antibodies that were detected after vaccination were directed against the head domain. The presented approach, when supported by further assays, can be used to assess the presence of specific anti-stalk antibodies and the potential boost of broadly protective antibodies, especially in the case of novel universal influenza vaccine approaches.