Higher Hemagglutination Inhibition Antibody Research Articles

Evaluation of the Newcastle disease virus genotype VII–mismatched vaccines in SPF chickens: A challenge efficacy study

Newcastle disease virus (NDV) strains, while falling under a single serotype, are classified into distinct genotypes. Genotype VII virulent NDVs pose a significant threat to poultry due to their association with high mortality rates and economic losses. This study aimed to evaluate the efficacy of three commercial live vaccines based on genotype II against genotype VII virulent NDV (vNDV) in specific pathogen-free (SPF) chickens. Forty one-day-old chickens were randomly divided into four groups (n = 10) and inoculated with one dose of each ND pneumotropic vaccine—B1, Clone.12IR, and La Sota—or received phosphate-buffered saline (PBS) as a control at eight days of age via eye drop. At 28 days of age (20th post-vaccination days), chickens were intramuscularly challenged with genotype VII virulent NDV (≥ 105 LD50). Serum samples were collected at 28 days of age (challenge day), 7 and 14 post-challenge days to measure NDV antibodies via the hemagglutination inhibition (HI) test. Cloacal and oropharyngeal swabs were taken on the 3rd, 5th, 7th, and 10th post-challenge days to evaluate virus shedding. Vaccinated groups exhibited significantly higher antibody titers and greater protection levels compared to the control group (P≤ 0.001). While HI antibody titer was not different at 28 and 35 days of age between vaccinated chickens, the Clone.12IR groups showed higher HI antibody titer compared to B1 at day 42 of age (9.43 vs. 7.42; P≤ 0.002). La Sota and Clone.12IR vaccines demonstrated superior protection against mortality compared to the B1 vaccine (90 %, 80% vs. 60 %, respectively) with 6.0 and 2.67 odds ratio of survivability. All three mismatched vaccines effectively curbed the shedding of virulent genotype VII NDV, with 0 % to 11 % positive cloacal samples up to the 3rd post-challenge day. These findings demonstrate that in the experimental setting, the administration of mismatched ND vaccines, particularly La Sota and Clone.12IR, confer protection against genotype VII virulent NDV and control viral shedding, which can help to develop effective vaccination strategies to mitigate the impact of vNDV outbreaks in the poultry farms.

Study of Cellular and Humoral Immunity and Histopathology of Target Tissues Following Newcastle Clone12IR Vaccine Administration in SPF Chickens.

Newcastle disease (ND) is a highly contagious viral infection affecting poultry production in many countries. Strict biosecurity and the administration of live attenuated vaccines against the ND virus (NDV) are the main implements of controlling programs. This study evaluated the efficacy and potency of the Razi Clone12IR Newcastle vaccine in specific pathogen-free (SPF) chickens. Chickens were vaccinated with either the Razi Clone12IR vaccine (group A1, n=20) or an imported Clone vaccine (B1, n=20) in the first week of life and boosted in the second week via eye drop, while negative control chickens received PBS (C1, n=20). Half of the birds in each group were challenged with the virulent NDV strain in the third post-vaccination week (A2, B2, and C2 groups). Specific antibody responses were determined in the collected sera by the hemagglutination inhibition (HI) assay for up to eight weeks. Cell-mediated immunity (CMI) was determined by the lymphocyte proliferation assay three and six weeks after the second vaccination. Sections of the tissues and organs, including the trachea, lungs, cecal tonsils, spleen, the bursa of Fabricius, liver, and small intestine, were subjected to histopathology. The immunized groups A1 and B1 showed significantly higher HI antibody titers before the challenge than the control group. In addition, lymphocyte proliferation responses significantly increased in the peripheral blood of the vaccinated groups. After the challenge, the A2 and B2 groups conferred good protection and drastically reduced virus shedding. No main lesions were noted in the tissues or organs of the vaccinated group in histopathology. In a few cases, mild microscopic lesions were observed, including the infiltration of inflammatory cells, which was related to the effect of the vaccine virus. These results indicate that the Razi Clone12IR vaccine is safe and can be an efficient tool for NDV infections by inducing protective humoral and CMI responses.

Immunological profile of mice immunized with a polyvalent virosome-based influenza vaccine

BackgroundInfluenza A virus (IAV) causes respiratory disease in pigs and is a major concern for public health. Vaccination of pigs is the most successful measure to mitigate the impact of the disease in the herds. Influenza-based virosome is an effective immunomodulating carrier that replicates the natural antigen presentation pathway and has tolerability profile due to their purity and biocompatibility.MethodsThis study aimed to develop a polyvalent virosome influenza vaccine containing the hemagglutinin and neuraminidase proteins derived from the swine IAVs (swIAVs) H1N1, H1N2 and H3N2 subtypes, and to investigate its effectiveness in mice as a potential vaccine for swine. Mice were immunized with two vaccine doses (1 and 15 days), intramuscularly and intranasally. At 21 days and eight months later after the second vaccine dose, mice were euthanized. The humoral and cellular immune responses in mice vaccinated intranasally or intramuscularly with a polyvalent influenza virosomal vaccine were investigated.ResultsOnly intramuscular vaccination induced high hemagglutination inhibition (HI) titers. Seroconversion and seroprotection (> 4-fold rise in HI antibody titers, reaching a titer of ≥ 1:40) were achieved in 80% of mice (intramuscularly vaccinated group) at 21 days after booster immunization. Virus-neutralizing antibody titers against IAV were detected at 8 months after vaccination, indicating long-lasting immunity. Overall, mice immunized with the virosome displayed greater ability for B, effector-T and memory-T cells from the spleen to respond to H1N1, H1N2 and H3N2 antigens.ConclusionsAll findings showed an efficient immune response against IAVs in mice vaccinated with a polyvalent virosome-based influenza vaccine.

Study of Cellular and Humoral Immunity and Histopathology of Target Tissues Following Newcastle Clone12IR Vaccine Administration in SPF Chickens

Newcastle disease (ND) is a highly contagious viral infection affecting poultry production in many countries. Strict biosecurity and the administration of live attenuated vaccines against the ND virus (NDV) are the main implements of controlling programs. This study evaluated the efficacy and potency of the Razi Clone12IR Newcastle vaccine in specific pathogen-free (SPF) chickens. Chickens were vaccinated with either the Razi Clone12IR vaccine (group A1, n=20) or an imported Clone vaccine (B1, n=20) in the first week of life and boosted in the second week via eye drop, while negative control chickens received PBS (C1, n=20). Half of the birds in each group were challenged with the virulent NDV strain in the third post-vaccination week (A2, B2, and C2 groups). Specific antibody responses were determined in the collected sera by the hemagglutination inhibition (HI) assay for up to eight weeks. Cell-mediated immunity (CMI) was determined by the lymphocyte proliferation assay three and six weeks after the second vaccination. Sections of the tissues and organs, including the trachea, lungs, cecal tonsils, spleen, the bursa of Fabricius, liver, and small intestine, were subjected to histopathology. The immunized groups A1 and B1 showed significantly higher HI antibody titers before the challenge than the control group. In addition, lymphocyte proliferation responses significantly increased in the peripheral blood of the vaccinated groups. After the challenge, the A2 and B2 groups conferred good protection and drastically reduced virus shedding. No main lesions were noted in the tissues or organs of the vaccinated group in histopathology. In a few cases, mild microscopic lesions were observed, including the infiltration of inflammatory cells, which was related to the effect of the vaccine virus. These results indicate that the Razi Clone12IR vaccine is safe and can be an efficient tool for NDV infections by inducing protective humoral and CMI responses.

Low quality antibody responses in critically ill patients hospitalized with pandemic influenza A(H1N1)pdm09 virus infection

Although some adults infected with influenza 2009 A(H1N1)pdm09 viruses mounted high hemagglutination inhibition (HAI) antibody response, they still suffered from severe disease, or even death. Here, we analyzed antibody profiles in patients (n = 31, 17–65 years) admitted to intensive care units (ICUs) with lung failure and invasive mechanical ventilation use due to infection with A(H1N1)pdm09 viruses during 2009–2011. We performed a comprehensive analysis of the quality and quantity of antibody responses using HAI, virus neutralization, biolayer interferometry, enzyme-linked-lectin and enzyme-linked immunosorbent assays. At time of the ICU admission, 45% (14/31) of the patients had HAI antibody titers ≥ 80 in the first serum (S1), most (13/14) exhibited narrowly-focused HAI and/or anti-HA-head binding antibodies targeting single epitopes in or around the receptor binding site. In contrast, 42% (13/31) of the patients with HAI titers ≤ 10 in S1 had non-neutralizing anti-HA-stem antibodies against A(H1N1)pdm09 viruses. Only 19% (6/31) of the patients showed HA-specific IgG1-dominant antibody responses. Three of 5 fatal patients possessed highly focused cross-type HAI antibodies targeting the (K130 + Q223)-epitopes with extremely low avidity. Our findings suggest that narrowly-focused low-quality antibody responses targeting specific HA-epitopes may have contributed to severe infection of the lower respiratory tract.

Thermoresistant Newcastle disease vaccine effectively protects SPF, native, and commercial chickens in challenge with virulent virus

BackgroundDue to the more stability and a better homogenecity in immune response, the use of thermoresistant vaccines in different chicken types has been increased.ObjectiveThis study aimed to evaluate the efficacy of a newly developed Newcastle disease vaccine (ND.TR.IR) originating from I‐2 strain in specific pathogen‐free (SPF) and native and broiler chickens.MethodsFollowing determination of pathogenicity indices on the candidate seed, three efficacy examinations were conducted. In the first experiment, 120 1‐day‐old SPF chickens were randomly allocated to six groups and either vaccinated with ND.TR.IR via eye drop at 1, 7, and 21 days of age (V1, V7, and V21), or considered as non‐vaccinated control groups (C1, C7, and C21). At 20th post‐vaccination day, sera hemagglutination inhibition (HI) antibody titres against ND virus (NDV) were measured and then the chickens were challenged by virulent NDV (vNDV). In the second and third experiments, the efficacy of ND.TR.IR vaccine was compared to routine vaccination program (B1 and LaSota) in native and broiler chickens that were vaccinated at 10 and 20 days of age, respectively. The HI antibody titres were measured on 10, 20, 30, and 40 days of age, and also challenge efficacy test with vNDV was conducted on 30 days of age.ResultsThe studied virus, as a vaccinal seed, complied with the pathogenicity indices of avirulent NDV and molecular identity of I‐2 strain. In the efficacy evaluation trials, the vaccinated chickens had higher HI antibody titres against NDV compared with their corresponding control chickens (p < 0.05). Results of the challenge tests indicated 95% and 100% protection against vNDV in native, SPF, and broiler‐vaccinated chickens, respectively.ConclusionsThe present findings indicated that administration of ND.TR.IR induced appropriate HI antibody titres against NDV in SPF, native, and broiler chickens associated with good protection in efficacy test.

Comparison of Chicken Immune Responses to Immunization with Vaccine La Sota or ZG1999HDS Strain of Newcastle Disease Virus.

Newcastle disease (ND) is a highly contagious avian disease. Global control of ND is mainly based on vaccination of poultry; however, reported outbreaks of ND in vaccinated flocks indicate a constant need to re-evaluate the existing vaccines and a development of the new ones. In this study, 4-week-old male chickens of the layer commercial hybrid were immunized oculonasally with a commercial NDV live La Sota vaccine (LS group), a suspension of lyophilized NDV strain ZG1999HDS (ZG group), or saline (Control (K) group). Antibody response was determined by haemagglutination inhibition (HI) assay. Cell-mediated immunity (CMI) was characterized by immunophenotyping of leukocyte’s and T-lymphocyte’s subpopulations (flow cytometry). Applied NDV strains did not cause any adverse reaction in treated chickens. Both strains induced the significantly higher HI antibody response in comparison to the control group, and overall antibody titer was higher in ZG group than in LS group. CMI, manifested as a higher proliferation of B- and T-helper cells, yielded better results in the ZG groups than in the LS group. Based on the obtained results, we conclude that the strain ZG1999HDS is immunogenic and is a suitable candidate for further research and development of poultry vaccines.

Inflammasome Activity in Response to Influenza Vaccination Is Maintained in Monocyte-Derived Peripheral Blood Macrophages in Older Adults.

Introduction: Each year, a disproportionate number of the total seasonal influenza-related hospitalizations (90%) and deaths (70%) occur among adults who are >65 years old. Inflammasome activation has been shown to be important for protection against influenza infection in animal models but has not yet been demonstrated in humans. We hypothesized that age-related dysfunction (immunosenescence) of the inflammasome may be associated with poor influenza-vaccine response among older adults. Methods: A cohort of younger (18–40 years of age) and older (≥65 years of age) adults was recruited prior to the 2014–2015 influenza season. We measured hemagglutination inhibition (HAI) titers in serum before and 28 days after receipt of the seasonal inactivated influenza vaccine. Inflammasome-related gene expression and protein secretion were quantified in monocyte-derived macrophages following stimulation with influenza A/H1N1 virus. Results: Younger adults exhibited higher HAI titers compared to older adults following vaccination, although inflammasome-related protein secretion in response to influenza stimulation was similar between the age groups. Expression of P2RX7 following influenza stimulation was lower among older adults. Interestingly, CFLAR expression was significantly higher among females (p = 2.42 × 10−5) following influenza stimulation and this gene may play an important role in the development of higher HAI antibody titers among older females. Conclusion: Inflammasome activation in response to influenza vaccination appears to be maintained in monocyte-derived macrophages from older adults and does not explain the poor influenza vaccine responses generally observed among this age group.

Protective efficacy of inactivated Newcastle disease virus vaccines prepared in two different oil-based adjuvants

Despite the availability of Newcastle disease (ND) vaccines for more than six decades, disease outbreaks continue to occur with huge economic consequences to the global poultry industry. The aim of this study is to develop a safe and effective inactivated vaccine based on a recently isolated Newcastle disease virus (NDV) strain IBS025/13 and evaluate its protective efficacy in chicken following challenge with a highly virulent genotype VII isolate. Firstly, high titre of IBS025/13 was exposed to various concentrations of binary ethylenimine (BEI) to determine the optimal conditions for complete inactivation of the virus. The inactivated virus was then prepared in form of a stable water-in-oil emulsion of black seed oil (BSO) or Freund’s incomplete adjuvant (FIA) and used as vaccines in specific pathogen-free chicken. Efficacy of various vaccine preparations was also evaluated based on the ability of the vaccine to protect against clinical disease, mortality and virus shedding following challenge with highly virulent genotype\\VII NDV isolate. The results indicate that exposure of NDV IBS025/13 to 10 mM of BEI for 21 h at 37 °C could completely inactivate the virus without tempering with the structural integrity of the viral hemagglutin-neuraminidase protein. More so, the inactivated vaccines adjuvanted with either BSO- or FIA-induced high hemagglutination inhibition antibody titre that protected the vaccinated birds against clinical disease and in some cases virus shedding, especially when used together with live attenuated vaccines. Thus, genotype VII-based NDV-inactivated vaccines formulated in BSO could substantially improve poultry disease control particularly when combined with live attenuated vaccines.

Efficacy of Heterologous Prime-Boost Vaccination with H3N2 Influenza Viruses in Pre-Immune Individuals: Studies in the Pig Model.

In a previous study in influenza-naïve pigs, heterologous prime-boost vaccination with monovalent, adjuvanted whole inactivated vaccines (WIV) based on the European swine influenza A virus (SwIAV) strain, A/swine/Gent/172/2008 (G08), followed by the US SwIAV strain, A/swine/Pennsylvania/A01076777/2010 (PA10), was shown to induce broadly cross-reactive hemagglutination inhibition (HI) antibodies against 12 out of 15 antigenically distinct H3N2 influenza strains. Here, we used the pig model to examine the efficacy of that particular heterologous prime-boost vaccination regimen, in individuals with pre-existing infection-immunity. Pigs were first inoculated intranasally with the human H3N2 strain, A/Nanchang/933/1995. Seven weeks later, they were vaccinated intramuscularly with G08 followed by PA10 or vice versa. We examined serum antibody responses against the hemagglutinin and neuraminidase, and antibody-secreting cell (ASC) responses in peripheral blood, draining lymph nodes, and nasal mucosa (NMC), in ELISPOT assays. Vaccination induced up to 10-fold higher HI antibody titers than in naïve pigs, with broader cross-reactivity, and protection against challenge with an antigenically distant H3N2 strain. It also boosted ASC responses in lymph nodes and NMC. Our results show that intramuscular administration of WIV can lead to enhanced antibody responses and cross-reactivity in pre-immune subjects, and recall of ASC responses in lymph nodes and NMC.

Immunization of turkeys with a DNA vaccine expressing the haemagglutinin gene of low pathogenic avian influenza virus subtype H9N2

Low pathogenic avian influenza H9N2 is still circulating in the Middle East causing respiratory manifestations and severe economic losses in poultry. In the present study, an H9 plasmid-based DNA vaccine targeting the HA gene of H9N2 A/CK/Egypt/SCU8/2014 was developed and evaluated in turkeys. The full length of HA was cloned into vector plasmids under the control of a cytomegalovirus promoter. The in-vitro expression of the recombinant HA was demonstrated in HeLa cells transfected with the plasmids pVAX1-H9 or pCR-H9 using western blot and Immunofluorescent assay (IFA). The efficacy of pVAX-H9 and pCR- H9, naked or saponin-adjuvanted, was evaluated in turkey poults at 3 weeks and challenged with A/CK/Egypt/SCU8/2014 (106 EID50/bird at 3 weeks post-vaccination. The efficacy was assesses based on virus shedding, oropharyngeal and cloacal, as well as seroconversion using haemagglutination inhibition (HI) test. All immunized birds showed high HI antibody titers (7–8 log2) at 3 weeks post-vaccination. None of the birds vaccinated with naked or saponin-adjuvanted pVAX-H9 or pCR-H9 showed any clinical signs. The pVAX-H9 and pCR-H9 alone did not prevent cloacal and oropharyngeal virus shedding, however, saponin-adjuvanted pVAX1-H9 and pCR-H9 prevented cloacal and oropharyngeal virus shedding at 3 and 5 days post challenge, respectively. In conclusion, DNA vaccination with pVAX1-H9 and pCR-H9 could protect turkey from the H9N2 virus, but vaccination regimes need to be improved.

The immunogenicity of the virus-like particles derived from the VP2 protein of porcine parvovirus

Porcine parvovirus (PPV) is a major cause of the syndrome of sow reproductive failure that can cause economic losses. In this study, we developed a subunit vaccine against porcine parvovirus (PPV), composed of virus-like particles (VLPs) derived from a prokaryotic system, and evaluated its potential against PPV infection. The soluble recombinant VP2 protein was expressed in E. coli Transetta(DE3) cells using a pCold II prokaryotic expression vector at a low temperature of 15 °C. After expression and purification, the recombinant VP2 protein was successfully assembled into VLPs with a similar shape of PPV viron and also hemagglutination activity. PPV VLPs formulated in a water-in-oil-in-water adjuvant evoked high hemagglutination inhibition antibody and neutralization antibody titres in both guinea pigs, used as reference model, and target species, pigs. Immunization with VLPs vaccine stimulated high hemagglutination inhibition antibody and neutralization antibody responses in guinea pigs, used as reference, and target species, weaned pigs, and primiparous gilts. PPV VLPs from E. coli yielded complete fetal protection against PPV infection in primiparous gilts immunized with a single-dose vaccine. PPV VLPs inhibited the replication and spread of PPV in primiparous gilts, which was confirmed by the detection of PPV DNA and infectious PPV in nasal and rectal swabs of challenged sows. These results suggest that VLPs-based PPV vaccine is a promising PPV vaccine candidate.

Nano-emulsion as a vaccine adjuvant can enhance the humoral immunity against influenza in aged and young mice

Objective To evaluate the immunogenicity of split influenza H1N1 vaccine formulated with an oil-in-water nano-emulsion adjuvant in aged mice and young mice. Methods A nano-emulsion adjuvant formulated split influenza H1N1 vaccine was used to immunize aged and young mice through intramuscular injection. Each mouse was immunized with 0.012 μg of hemagglutinin (HA) twice with an interval of 28 d. Hemagglutination inhibition (HI) titers in serum were measured 27 d after first immunization. Serum HI, IgG1 and IgG2a titers were detected 14 d after the last immunization. No adjuvant-formulated vaccine and normal saline (NS) were used to set up control groups. Virus challenge test was carried out using 10 times the median lethal dose (LD50) of A/Puerto Rico/8/34 (H1N1) strain two weeks after the last immunization and the protective effects were assessed through measuring the dynamic changes in body weight and survival rate. Results Higher levels of serum HI, IgG1 and IgG2a antibodies and higher HI antibody conversion rates were induced in the adjuvant groups, especially in the aged mice group, than in the control groups. Nano-emulsion adjuvant improved the immunogenicity of HA and mouse immunity to A/Puerto Rico/8/34 (H1N1). Conclusions Nano-emulsion adjuvant could enhance the immunogenicity of influenza antigens, especially in aged mice. Key words: Adjuvant; Influenza; Nano-emulsion adjuvant; Aged mice; Immunogenicity

Characterization of antibody and memory T-cell response in H7N9 survivors: a cross-sectional analysis

ObjectivesDespite the importance of immunological memory for protective immunity against viral infection, whether H7N9-specific antibodies and memory T-cell responses remain detectable years after the original infection is unknown. MethodsA cross-sectional study was conducted to investigate the immune memory responses of H7N9 patients who contracted the disease and survived during the 2013–2016 epidemics in China. Sustainability of antibodies and T-cell memory to H7N9 virus were examined. Healthy individuals receiving routine medical examinations in a physical examination centre were recruited as control. ResultsA total of 75 survivors were enrolled and classified into four groups based on the time elapsed from illness onset to specimen collection: 3 months (n = 14), 14 months (n = 14), 26 months (n = 28) and 36 months (n = 19). Approximately 36 months after infection, the geometric mean titres of virus-specific antibodies were significantly lower than titres in patients 3 months after infection, but 16 of 19 (84.2%) survivors in the 36-month interval had microneutralization (MN) titres ≥40. Despite the overall declining trend, the percentages of virus-specific cytokine-secreting memory CD4+ and CD8+ T cells remained higher in survivors at nearly all time-points in comparison with control individuals. Linear regression analysis showed that severe disease (mean titre ratio 2.77, 95% CI 1.17–6.49) was associated with higher haemagglutination inhibition (HI) titre and female sex for both HI (1.92, 1.02–3.57) and MN (3.33, 1.26–9.09) antibody, whereas female sex (mean percentage ratio 1.69, 95% CI 1.08–2.63), underlying medical conditions (1.94, 95% CI 1.09–3.46) and lack of antiviral therapy (2.08, 95% CI 1.04–4.17) were predictors for higher T-cell responses. ConclusionsSurvivors of H7N9 virus infection produced long-term antibodies and memory T-cell responses. Our findings warrant further serological investigation in general and high-risk populations and have important implications for vaccine design and development.

Recombinant baculovirus vaccine expressing hemagglutinin of H7N9 avian influenza virus confers full protection against lethal highly pathogenic H7N9 virus infection in chickens.

The emergent highly pathogenic avian influenza A (H7N9) (HPAI) virus is a major public concern in China. Therefore, it is crucially important to develop an effective vaccine against this virus. In this study, we constructed a baculovirus vaccine expressing the hemagglutinin (HA) of H7N9 strain A/Chicken/Jiaxing/148/2014 (JX148). The recombinant baculovirus (rBac-JX148HA) generated in this study showed good growth in insect cells and good safety, and it stably expressed the HA protein. We compared the immunogenicity and efficacy of the inactivated whole-virus vaccine JX148 and rBac-JX148HA. One chicken in the JX148-treated group died on day 4 post-challenge, and three chickens had typical clinical symptoms (survival rate, 90%; morbidity, 40%). However, no chickens immunized with rBac-JX148HA showed clinical signs during the 14-day observation period. An analysis of viral shedding and viral replication demonstrated that rBac-JX148HA more efficiently inhibited viral shedding and viral replication than the inactivated whole-virus vaccine. Taken together, these results indicate that the inactivated recombinant baculovirus vaccine induces a high hemagglutination inhibition antibody titer, provides complete protection against challenge with the highly pathogenic H7N9 virus, and effectively inhibits viral shedding. Therefore, the candidate vaccine has potential utility in the prevention and control of H7N9 avian influenza and is also appropriate for veterinary vaccines using cell suspension culture technology.

Antibody Responses toward the Major Antigenic Sites of Influenza B Virus Hemagglutinin in Mice, Ferrets, and Humans.

The influenza B virus hemagglutinin contains four major antigenic sites (the 120 loop, the 150 loop, the 160 loop, and the 190 helix) within the head domain. These immunodominant antigenic sites are the main targets of neutralizing antibodies and are subject to antigenic drift. Yet little is known about the specific antibody responses toward each site in terms of antibody prevalence and hemagglutination inhibition activity. In this study, we used modified hemagglutinins of influenza B virus which display only one or none of the major antigenic sites to measure antibody responses toward the classical as well as the noncanonical epitopes in mice, ferrets, and humans. With our novel reagents, we found that both hemagglutination inhibition antibodies and total IgGs were mostly induced by the major antigenic sites. However, in human adults, we observed high hemagglutination inhibition antibody responses toward the noncanonical epitopes. By stratifying the human samples into age groups, we found that the noncanonical antibody responses appeared to increase with age.IMPORTANCE This study dissected the specific antibody responses toward the major antigenic sites and the noncanonical epitopes of influenza B virus hemagglutinin in animals and humans using novel reagents. These findings will guide the design of the next generation of influenza virus vaccines.

Gut microbiota modulates type I interferon and antibody-mediated immune responses in chickens infected with influenza virus subtype H9N2.

Commensal gut microbes play a critical role in shaping host defences against pathogens, including influenza viruses. The current study was conducted to assess the role and mechanisms of action of commensal gut microbiota on the innate and antibody-mediated responses of layer chickens against influenza virus subtype H9N2. A total of 104 one-day-old specific pathogen free chickens were assigned to either of the four treatments, which included two levels of antibiotics treatment (ABX- and ABX+) and two levels of H9N2 virus infection (H9N2- and H9N2+). At day 17 of age, chickens in the H9N2+ group were infected via the oral-nasal route with 400 μl of 107 TCID50/ml (200 μl/each route). Oropharyngeal and cloacal swabs at days 1, 3, 5, 7 and 9 post-infection (p.i.) for virus shedding, tissue samples at 12 h, 24 h and 36 h p.i. for mRNA measurement, and serum samples at days 7 and 14 p.i. for hemagglutination inhibition (HI) assay and IgG antibodies were collected. Virus shedding analysis showed that antibiotic treated (depleted)-H9N2 virus infected chickens showed a significantly higher oropharyngeal virus shedding at all time points, and cloacal shedding at days 3 and 5 p.i. compared to control treated (undepleted)-H9N2 infected chickens. Analysis of mRNA expression showed that infection of depleted chickens with H9N2 virus resulted in significantly down-regulated type I interferon responses both in the respiratory and gastrointestinal tracts compared to undepleted-H9N2 infected chickens. However, antibody-mediated immune response analysis showed a significantly higher HI antibody titre and IgG levels in the serum of chickens depleted with antibiotics and infected with H9N2 virus compared to undepleted-H9N2 infected chickens. In conclusion, findings from the current study suggest that the gut microbiota of chickens plays an important role in the initiation of innate responses against influenza virus infection, while the antibody-mediated immune response remains unaffected.

Antigenic variation of LaSota and genotype VII Newcastle disease virus (NDV) and their efficacy against challenge with velogenic NDV

Continued monitoring and evaluation of vaccine efficacy against prevalent or newly isolated strains has great importance in advising Newcastle disease (ND) immunization strategy. In this study, we systematically analysed the antigenic variation between genotype VII NDV aSG10 and the commercial vaccine strain LaSota, and assessed their efficacy against challenge with velogenic NDV by serological analysis and animal testing. We show that these two viruses are antigenically distinguishable; anti-NDV aSG10 hyper-immune sera demonstrated higher haemagglutination inhibition (HI) titres (11.13±0.30log2) against the aSG10 virus, compared with titres against LaSota (9.53±0.50log2). Conversely, the hyper-immune sera from LaSota showed higher HI titres against LaSota virus (9.73±0.36log2), but 2-fold lower HI titre against aSG10 (8.87±0.38log2). Each serum neutralised heterologous virus, but neutralisation titres were always 3- to 6-fold higher against its homologous strain than heterologous virus. The cross-reactivity R value between aSG10 and LaSota was 0.23, indicating that they are loosely related with major antigenic differences within a single serotype. The results of animal tests revealed that the aSG10 vaccine had a significantly higher protection rate than the LaSota vaccine against genotype VII NDV, regardless of intramuscular (IM) or eye drop/intranasal (ED/IN) route of SG10 challenge. Compared with IM administration, chicken flocks needed higher HI antibody levels to obtain sufficient protection when challenged by the natural ED/IN route. These results are highly informative for better control of ND in the poultry industry.

Safety, immunogenicity and shedding of LAIV4 in HIV-infected and uninfected children

ObjectivesHIV-infected children have poor responses to inactivated influenza vaccines. Live vaccines (LAIVs) are highly efficacious in children, but they are not used in HIV-infected children du e to limited information. We investigated the safety, immunogenicity and viral shedding of LAIV4 in HIV-infected compared with uninfected children. DesignForty-six HIV-infected and 56 uninfected children 2 to 25 years old, who had been previously vaccinated against influenza, consented to receive a single dose of LAIV4. All grade adverse events (AEs) were recorded in the first month post-vaccination and serious AEs (SAEs) throughout the influenza season. Nasopharyngeal swabs for influenza PCR and IgA ELISA and blood for hemagglutination inhibition antibody (HAI) measurements were collected at entry, 2–5, 7–10 and 21–28 days post-vaccination. ResultsThe HIV-infected subjects had median CD4+ cells of 649 cells/μL and plasma HIV RNA of 20 copies/mL. AEs were similar in the two groups. There were no vaccine-related SAEs. Shedding of ≥1 vaccine virus was detected in 67% HIV-infected and 50% uninfected participants (p=0.14). HAI titers did not appreciably change, but mucosal IgA antibodies significantly increased post-vaccination in both groups. High baseline HAI and IgA antibody concentrations were associated with decreased viral shedding in controls, but not in HIV-infected subjects. Similar proportions of HIV-infected vaccinees and controls reported influenza-like illnesses (12% and 6%) throughout the season. ConclusionsLAIV4 was equally safe and immunogenic and caused similar viral shedding in HIV-infected and uninfected children. A correlate of protection against vaccine viral shedding was not identified in HIV-infected participants, although both circulating and mucosal antibodies correlated with protection in controls.

AF03-adjuvanted and non-adjuvanted pandemic influenza A (H1N1) 2009 vaccines induce strong antibody responses in seasonal influenza vaccine-primed and unprimed mice

Pandemic influenza vaccines have been manufactured using the A/California/07/2009 (H1N1) strain as recommended by the World Health Organization. We evaluated in mice the immunogenicity of pandemic (H1N1) 2009 vaccine and the impact of prior vaccination against seasonal trivalent influenza vaccines (TIV) on antibody responses against pandemic (H1N1) 2009. In naïve mice, a single dose of unadjuvanted H1N1 vaccine (3μg of HA) was shown to elicit hemagglutination inhibition (HI) antibody titers >40, a titer associated with protection in humans against seasonal influenza. A second vaccine dose of pandemic (H1N1) 2009 vaccine strongly increased these titers, which were consistently higher in mice previously primed with TIV than in naïve mice. At a low immunization dose (0.3μg of HA), the AF03-adjuvanted vaccine elicited higher HI antibody titers than the corresponding unadjuvanted vaccines in both naïve and TIV-primed animals, suggesting a potential for antigen dose-sparing. These results are in accordance with the use in humans of a split-virion inactivated pandemic (H1N1) 2009 vaccine formulated with or without AF03 adjuvant to protect children and young adults against influenza A (H1N1) 2009 infection.