Higher Hemagglutination Inhibition Titers Research Articles

Induction of Local Secretory IgA and Multifunctional CD4⁺ T-helper Cells Following Intranasal Immunization with a H5N1 Whole Inactivated Influenza Virus Vaccine in BALB/c Mice.

Avian influenza subunit vaccines have been shown to be poorly immunogenic, leading to the re-evaluation of the immunogenic and dose-sparing potential of whole virus vaccines. In this study, we investigated the immune responses after one or two doses of intramuscular or intranasal whole inactivated influenza H5N1 virus vaccine in BALB/c mice. Serum samples and nasal washings were collected weekly post-vaccination and analysed using enzyme-linked immunosorbent assay (ELISA). Sera were also analysed by the haemagglutination inhibition (HI) assay. Antibody-secreting cells were measured in lymphocytes from spleen and bone marrow via enzyme-linked immunospot (ELISPOT). Splenocytes were stimulated in vitro, and T-helper profiles were measured through multiplex bead assay in the supernatants, or intracellularly by multiparametric flow cytometry. Both vaccine routes induced high HI titres following the second immunization (intramuscular = 370, intranasal = 230). Moreover, the intramuscular group showed significantly higher levels of serum IgG (P < 0.01), IgG1 (P < 0.01) and IgG2a (P < 0.01) following the second vaccine dose, while the intranasal group exhibited significantly higher levels of serum IgA (P < 0.05) and local IgA (P < 0.01) in the nasal washings. Also, IgA antibody-secreting cells were found in significantly higher numbers in the intranasal group in both the spleen (P < 0.01) and the bone marrow (P < 0.01). Moreover, Th1 (TNF-α, IL-2, IFN-γ) and Th2 (IL-4, IL-5, IL-10) cytokines were expressed by both groups, yet only the intranasal group expressed the Th17 marker IL-17. As the intranasal vaccines induce local IgA and are easily administered, we suggest the intranasally administered whole virus vaccine as a promising candidate for a pandemic H5N1 vaccine.

SM-07 * A PILOT STUDY OF HIGH-DOSE INFLUENZA VACCINE IMMUNOGENICITY IN PATIENTS WITH PRIMARY CENTRAL NERVOUS SYSTEM MALIGNANCY

BACKGROUND: In a previous investigation, we demonstrated reduced immunogenicity to the standard trivalent inactivated influenza vaccine in patients with primary central nervous system (CNS) malignancy (Neuro-Oncol 2014, PMID 24714522). Other populations with similar reduced responses have shown improved immunogenicity to a high-dose vaccine. OBJECTIVE: A pilot prospective cohort study was designed to evaluate the immunogenicity of the Fluzone® High-Dose influenza vaccine in patients with CNS malignancy. METHODS: Baseline data on diagnosis, chemotherapy, radiation, glucocorticoids, and timing of treatment were collected. All patients underwent vaccination with of the Fluzone® High-Dose influenza vaccine. Serum samples are actively being collected at baseline, day 28, and 3 months following vaccine administration. Hemagglutination inhibition (HAI) titers will be measured to determine seroconversion (4-fold rise) and seroprotection (titer 1:40). RESULTS: A total of 27 patients were enrolled. Mean age at baseline was 52.7 years (+/-12.6); 41% male; 93% white and 7% black. Diagnoses included high-grade glioma (85%), CNS lymphoma (11%), and meningioma (4%). At enrollment, 8 patients (30%) were taking glucocorticoids, 4 (15%) were undergoing active radiation, 14 (52%) were on chemotherapy, and 5 (19%) began chemotherapy following vaccine administration. Prior radiation (n = 19, 70%) and chemotherapy (n = 14, 52%) were common. Seven patients (26%) reported post-vaccination symptoms potentially attributable to vaccination; 6 experienced only local soreness or redness at the injection site and 1 suffered headache, nausea, vomiting. All symptoms were transient and resolved completely. At baseline, 8 patients (30%) had absolute CD4 < 300. Serum samples for HAI assay are being collected. Seroconversion and seroprotection rates are forthcoming. CONCLUSION: Studies have shown that populations known to respond poorly to standard yearly influenza vaccination (e.g. the elderly) can mount an effective immunologic response to the high-dose vaccine. This prospective cohort study will provide preliminary data regarding the immunologic response to high-dose vaccination in patients with primary CNS malignancy. Funding: The Fluzone® High Dose vaccine and HAI titer measurements were provided by Sanofi Pasteur. Acknowledgement: This research was supported by the Clinical Research Unit of the Wake Forest Translational Science Institute.

Cocirculation of two distinct genetic and antigenic lineages of proposed influenza D virus in cattle.

Viruses with approximately 50% homology to human influenza C virus (ICV) have recently been isolated from swine and cattle. The overall low homology to ICV, lack of antibody cross-reactivity to ICV in hemagglutination inhibition (HI) and agar gel immunodiffusion assays, and inability to productively reassort with ICV led to the proposal that these viruses represented a new genus of influenza virus, influenzavirus D (IDV). To further our understanding of the epidemiology of IDV, real-time reverse transcription-PCR was performed on a set of 208 samples from bovines with respiratory disease. Ten samples (4.8%) were positive and six viruses were successfully isolated in vitro. Phylogenetic analysis of full-genome sequences of these six new viruses and four previously reported viruses revealed two distinct cocirculating lineages represented by D/swine/Oklahoma/1334/2011 (D/OK) and D/bovine/Oklahoma/660/2013 (D/660), which frequently reassorted with one another. Antigenic analysis using the HI assay and lineage-representative D/OK and D/660 antiserum found up to an approximate 10-fold loss in cross-reactivity against heterologous clade antiserum. One isolate, D/bovine/Texas/3-13/2011 (D/3-13), clustered with the D/660 lineage, but also had high HI titers to heterologous (D/OK) clade antiserum. Molecular modeling of the hemagglutinin esterase fusion protein of D/3-13 identified a mutation at position 212 as a possible antigenic determinant responsible for the discrepant HI results. These results suggest that IDV is common in bovines with respiratory disease and that at least two genetic and antigenically distinct clades cocirculate. A novel bovine influenza virus was recently identified. Detailed genetic and antigenic studies led to the proposal that this virus represents a new genus of influenza, influenzavirus D (IDV). Here, we show that IDV is common in clinical samples of bovine respiratory disease complex (BRDC), with a prevalence similar to that of other established BRDC etiological agents. These results are in good agreement with the near-ubiquitous seroprevalence of IDV previously found. Phylogenetic analysis of complete genome sequences found evidence for two distinct cocirculating lineages of IDV which freely reassort. Significant antigenic differences, which generally agreed with the surface glycoprotein hemagglutinin esterase phylogeny, were observed between the two lineages. Based on these results, and on the ability of IDV to infect and transmit in multiple mammalian species, additional studies to determine the pathogenic potential of IDV are warranted.

Influenza Vaccination of Pregnant Women and Protection of Their Infants

BackgroundThere are limited data on the efficacy of vaccination against confirmed influenza in pregnant women with and those without human immunodeficiency virus (HIV) infection and protection of their infants.MethodsWe conducted two double-blind, randomized, placebo-controlled trials of trivalent inactivated influenza vaccine (IIV3) in South Africa during 2011 in pregnant women infected with HIV and during 2011 and 2012 in pregnant women who were not infected. The immunogenicity, safety, and efficacy of IIV3 in pregnant women and their infants were evaluated until 24 weeks after birth. Immune responses were measured with a hemagglutination inhibition (HAI) assay, and influenza was diagnosed by means of reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assays of respiratory samples.ResultsThe study cohorts included 2116 pregnant women who were not infected with HIV and 194 pregnant women who were infected with HIV. At 1 month after vaccination, seroconversion rates and the proportion of participants with HAI titers of 1:40 or more were higher among IIV3 recipients than among placebo recipients in both cohorts. Newborns of IIV3 recipients also had higher HAI titers than newborns of placebo recipients. The attack rate for RT-PCR–confirmed influenza among both HIV-uninfected placebo recipients and their infants was 3.6%. The attack rates among HIV-uninfected IIV3 recipients and their infants were 1.8% and 1.9%, respectively, and the respective vaccine-efficacy rates were 50.4% (95% confidence interval [CI], 14.5 to 71.2) and 48.8% (95% CI, 11.6 to 70.4). Among HIV-infected women, the attack rate for placebo recipients was 17.0% and the rate for IIV3 recipients was 7.0%; the vaccine-efficacy rate for these IIV3 recipients was 57.7% (95% CI, 0.2 to 82.1).ConclusionsInfluenza vaccine was immunogenic in HIV-uninfected and HIV-infected pregnant women and provided partial protection against confirmed influenza in both groups of women and in infants who were not exposed to HIV. (Funded by the Bill and Melinda Gates Foundation and others; ClinicalTrials.gov numbers, NCT01306669 and NCT01306682.)

Serological Responses Following Influenza A(H7N9) Virus Infection

To the Editor—We commend Yang et al [1] for their important work using serology to draw new epidemiological conclusions about the recent epidemic of the novel A(H7N9) influenza virus. By determining the levels of antibody titers against this virus among poultry workers and the general population of Zhejiang province, in which a relatively high number of confirmed infections of A(H7N9) virus occurred, they found that the former have higher hemagglutination inhibition (HI) titers against the A(H7N9) influenza virus, on average, compared to the latter. This is important evidence that subclinical human infections of A(H7N9) virus have occurred among poultry workers, and further supports the role of poultry in the A(H7N9) epidemic in China. Furthermore, finding low antibody titers among the general population confirms the absence of a major “silent” A(H7N9) epidemic. The authors also reported that postinfection antibody titers appeared to be lower among the hospitalized A(H7N9) patients who died compared to those who survived, and speculated that therefore “the presence of antibodies may improve clinical outcome in infected patients.” However, using the data kindly provided by the authors in their Supplementary Table 1B, we were not able to find evidence that survival was associated with higher antibody titers among the cohort of patients in this study, after adjustment for the time from onset of symptoms to collection of sera. We fitted a nonlinear model using the nls function of R version 3.0.1, with the logarithm of the HI titer as the outcome variable and survival status and a sigmoidal function of time since symptom onset as the explanatory variables. The fitted model is shown against the raw data in Figure ​Figure1.1. There is no statistically significant difference between antibody titers for the fatal vs nonfatal cases (P = .49). After including age in the regression model as a confounder, the association between antibody titer and outcome remained nonsignificant (P = .57). Figure 1. Antibody titers against influenza A(H7N9) virus measured by hemagglutination inhibition (HI) assay as given in Yang et al's Supplementary Table 1B [1], and the fitted model.The crosses correspond to patients with H7N9 who subsequently died, and the circles ... Our observation should not be taken as evidence against the potential role of convalescent sera in treatment of influenza A(H7N9) or other severe influenza virus infections. Passive immunotherapy may still have an important role to play in the response to A(H7N9) and other novel influenza viruses [2], and we agree with the authors that studies on such treatment approaches are urgently needed [3].

Bacterially Produced Recombinant Influenza Vaccines Based on Virus-Like Particles

Although current influenza vaccines are effective in general, there is an urgent need for the development of new technologies to improve vaccine production timelines, capacities and immunogenicity. Herein, we describe the development of an influenza vaccine technology which enables recombinant production of highly efficient influenza vaccines in bacterial expression systems. The globular head domain of influenza hemagglutinin, comprising most of the protein's neutralizing epitopes, was expressed in E. coli and covalently conjugated to bacteriophage-derived virus-like particles produced independently in E.coli. Conjugate influenza vaccines produced this way were used to immunize mice and found to elicit immune sera with high antibody titers specific for the native influenza hemagglutinin protein and high hemagglutination-inhibition titers. Moreover vaccination with these vaccines induced full protection against lethal challenges with homologous and highly drifted influenza strains.

Susceptibility of human and avian influenza viruses to human and chicken saliva

Oral cavity can be an entry site of influenza virus and saliva is known to contain innate soluble anti-influenza factors. Influenza strains were shown to vary in their susceptibility to those antiviral factors. Whether the susceptibility to the saliva antiviral factors plays any role in the host species specificity of influenza viruses is not known. In this study, the antiviral activity of human and chicken saliva against human and the H5N1 avian influenza viruses were investigated by hemagglutination inhibition (HI) and neutralization (NT) assays. In comparison to human influenza viruses, H5N1 isolates showed reduced susceptibility to human saliva as measured by HI and NT assays. Interestingly, an H5N1 isolate that bind to both α2,3- and α2,6-linked sialic acid showed much higher HI titers with human saliva, suggesting that the susceptibility profile was linked to the receptor-binding preference and the presence of α2,6-linked sialic in human saliva. On the other hand, the H5N1 isolates showed increased HI titers but reduced NT titers to chicken saliva as compared to human influenza isolates. The human salivary antiviral components were characterized by testing the sensitivity to heat, receptor destroying enzyme (RDE), CaCl₂/EDTA dependence, and inhibition by mannan, and shown to be α- and γ-inhibitors. These data suggest that the H5N1 HPAI influenza virus had distinctive susceptibility patterns to human and chicken saliva, which may play some roles in its infectivity and transmissibility in these hosts.

An H5N1 influenza DNA vaccine for South Africa

The highly pathogenic avian influenza virus H5N1 is a potent pandemic threat because of its frequent transmission from birds to humans and the increasing possibility of human to human transmission. During the 2009 H1N1 pandemic it was clear that rapid influenza vaccine production is a problem worldwide. Additionally, developing countries like South Africa generally cannot produce their own influenza vaccines because the traditional egg-based vaccine production method currently employed is too lengthy and too difficult to establish. As part of an exercise aimed at exploring the feasibility of producing emergency response influenza vaccines, we investigated an experimental DNA vaccine to the H5N1 influenza virus. We focused on the virion haemagglutinin, because it elicits the primary neutralising immune response following infection. Accordingly, we developed an H5N1 DNA vaccine with full-length and truncated versions of the haemagglutinin gene, to match previously developed protein candidates. Vaccinated mice developed a strong antibody response to the haemagglutinin protein. In addition, the full-length H5 gene elicited high haemagglutination inhibition titres in mice, indicating that it has potential as a candidate pandemic vaccine for South Africa.

Factors influencing infection by pandemic influenza A(H1N1)pdm09 over three epidemic waves in Singapore

Previous influenza pandemics had second and on occasion third waves in many countries that were at times more severe than the initial pandemic waves. This study aims to determine the seroepidemiology of successive waves of H1N1pdm09 infections in Singapore and the overall risks of infection. We performed a cohort study amongst 838 adults, with blood samples provided upon recruitment and at 5 points from 2009 to 2011 and tested by haemagglutination inhibition (HI) with A/California/7/2009 (H1N1pdm09). Surveys on key demographic and clinical information were conducted at regular intervals, and associations between seroconversion and these variables were investigated. After the initial wave from June to September 2009, second and third waves occurred from November 2009 to February 2010 and April to June 2010, respectively. Seroconversion was 13·5% during the first wave and decreased to 6·2% and 6·8% in subsequent waves. Across the three waves, the elderly and those with higher starting HI titres were at lower risk of seroconversion, while those with larger households were at greater risk. Those with higher starting HI titres were also less likely to have an acute respiratory infection. The second and third waves in Singapore had lower serological attack rates than the first wave. The elderly and those with higher HI titres had lower risk, while those in larger households had higher risk of seroconversion.

Development of VAX128, a recombinant hemagglutinin (HA) influenza-flagellin fusion vaccine with improved safety and immune response

BackgroundWe evaluated the safety and immunogenicity profiles of 3 novel influenza vaccine constructs consisting of the globular head of the HA1 domain of the Novel H1N1 genetically fused to the TLR5 ligand, flagellin. HA1 was fused to the C-terminus of flagellin in VAX128A, replaced the D3 domain of flagellin in VAX128B and was fused in both positions in VAX128C. MethodsIn a dose escalation trial, 112 healthy subjects 18–49 and 100 adults ≥65 years old were enrolled in a double blind, placebo controlled clinical trial at two centers. Vaccines were administered IM at doses ranging from 0.5 to 20μg. VAX128C was selected for second study performed in 100 subjects 18–64 years old comparing 1.25 and 2.5μg doses. All subjects were followed for safety and sera collected pre- and post-vaccination were tested by hemagglutination-inhibition (HAI). Serum C-reactive protein and cytokine levels were also measured. ConclusionsIn the first study high HAI titers and high seroconversion and seroprotection rates were observed at doses ≥2.5μg in adults 18–49. In adults ≥65 years, the vaccines doses of ≥4μg were required to induce a ≥4-fold rise in HAI titer, 50% seroconversion and 70% seroprotection. Based on safety, VAX128A was tested up to 8μg, VAX128B to 16μg and VAX128C to 20μg. Dose escalation for VAX128A was stopped at 8μg because one subject had temperature 101.6°F associated with a high CRP response, VAX128B was stopped at 16μg because of a severe AE associated with a high CRP and IL-6 response. VAX128C was not stopped before reaching the 20μg dose. In the second study VAX128C was well tolerated among 100 subjects who received 1.25 or 2.5μg. The peak GMT was 385 (95%CI 272–546), 79% (71–87%) seroconversion and 92% (84–96%) seroprotection. DiscussionFlagellin adjuvanted vaccines can be designed to minimize reactogenicity and retain immunogenicity, thereby representing a promising next generation vaccine technology.

A case study of measles vaccination for university students during the measles outbreak in Tokyo, Japan, 2007

In April 2007, seven students belonging to the same class at Teikyo University developed measles. To prevent the spread of infection, 27 of 106 students in the same class who had low anti-measles antibody titers as measured by hemagglutination inhibition (HI) assay were vaccinated. After the outbreak had subsided, the HI values were investigated in 103 students, and they answered questionnaires about their health condition during the period of the outbreak and their previous clinical histories of measles, including vaccination records. There was no new case of measles after introduction of the vaccination program. However, the HI titers of 42% of the students who were not vaccinated in this program were significantly elevated. Fever and catarrhal signs occurred in 7 of these students with pre-exposure titers of 8 or less. The post-exposure HI titers of 71% of students who were unaffected by measles and had high HI titers (>8) before the epidemic did not increase. These results suggested that people with low HI titers may become potential carriers of measles and that measurement of pre-exposure HI anti-measles antibody titer is a useful method for selection of candidates to undergo vaccination.

Generation of a Genotype VII Newcastle Disease Virus Vaccine Candidate with High Yield in Embryonated Chicken Eggs

To generate a genotype VII Newcastle disease virus (NDV) vaccine with high yield in embryonated chicken eggs, we selected genotype VII NDV strain JS5/05, which possesses a high virus titer in embryos as the parental virus. Using reverse genetics, we generated a genetically tagged derivative (NDV/AI4) of JS5/05 by changing the amino acid sequence of the cleavage site of the F0 protein. Pathogenicity tests showed that NDV/AI4 was completely avirulent. NDV/AI4 was genetically stable and replicated efficiently during 10 consecutive passages in embryos. More importantly, serologic assays showed that oil-emulsion NDV/AI4 induced higher hemagglutination inhibition (HI) titers against the prevalent virus than oil-emulsion LaSota vaccine in chickens and geese. Moreover, NDV/AI4-induced HI titers rose faster than those elicited by LaSota in chickens. Both NDV/AI4 and LaSota provided protection against clinical disease and mortality after the challenge with the genotype VII NDV strain JS3/05. However, NDV/AI4 significantly reduced virus shedding from the vaccinated birds compared to LaSota. Taken together, these results suggest that NDV/AI4 can provide better protection than LaSota and is a promising vaccine candidate against genotype VII NDV.

Evidence of Japanese encephalitis virus infections in swine populations in 8 provinces of Cambodia: Implications for national Japanese encephalitis vaccination policy

Although Cambodia, a Southeast Asian country, is suspected to be highly endemic for Japanese encephalitis virus (JEV), there are no nationally representative data on JEV transmission. Most of the existing data on human disease comes from few sentinel hospitals, and there have been no previous studies or surveillance for JEV transmission among pigs—the amplifying hosts in the natural cycle of JEV transmission. In preparation to develop a nationwide vaccination policy, data are required to show transmission of JEV in all the geographical regions of Cambodia. Analysis of JEV transmission among pigs will provide additional data on geographical scope and intensity of JEV transmission in Cambodia and will help to inform human vaccination policies in Cambodia. In this study, 505 sera obtained from swine bred in familial settings from 8 different provinces in Cambodia were tested by hemagglutination inhibition (HI) and ELISA tests to assess the presence of an immunological response to a JEV infection. Three hundred and thirty two sera (65.7%) were tested positives by HI assay and 321 (63.5%) by ELISA. Our results indicate that pigs particularly older than 6 months (95.2%) were highly infected with JEV in the 8 provinces. The high prevalence of HI antibodies and the high HI titer (>160 in 65.2% of cases and ≥1280 in 24.6% of cases) found in this age group suggest the important role of pigs in the transmission cycle of JEV in nature as they become probably rapidly infected and repeatedly re-exposed to the virus. Since the current pig rearing practices (within the backyard of home) are the same all over Cambodia, the results suggest that the human disease is also likely to be highly prevalent in the other provinces and warrant comprehensive policies for human vaccination and strengthened surveillance for acute meningo-encephalitis.

A novel monoclonal antibody effective against lethal challenge with swine-lineage and 2009 pandemic H1N1 influenza viruses in mice

The HA protein of the 2009 pandemic H1N1 viruses (H1N1pdm) is antigenically closely related to the HA of classical North American swine H1N1 influenza viruses (cH1N1). Since 1998, through mutation and reassortment of HA genes from human H3N2 and H1N1 influenza viruses, swine influenza strains are undergoing substantial antigenic drift and shift. In this report we describe the development of a novel monoclonal antibody (S-OIV-3B2) that shows high hemagglutination inhibition (HI) and neutralization titers not only against H1N1pdm, but also against representatives of the α, β, and γ clusters of swine-lineage H1 influenza viruses. Mice that received a single intranasal dose of S-OIV-3B2 were protected against lethal challenge with either H1N1pdm or cH1N1 virus. These studies highlight the potential use of S-OIV-3B2 as effective intranasal prophylactic or therapeutic antiviral treatment for swine-lineage H1 influenza virus infections.

The Adjuvant Effect of Sophy ¥-Glucan to the Antibody Response in Poultry Immunized by the Avian Influenza A H5N1 and H5N2 Vaccines

Avian influenza virus vaccines produced in oil-emulsified inactivated form with antigen content of at least 160 hemagglutinin units (HAU) induced immunity in birds. However, in addition to enhancing the effect of the adjuvant(s), other additional supplemented biological compounds included in inactivated vaccines could produce higher levels of antibody. We examined in chickens, Vietnamese ducks, and muscovy ducks the adjuvant effect of Sophy β-glucan (SBG), a β-1,3-1,6 glucan produced by the black yeast Aureobasidium pollulans strain AF0-202, when administered with an avian influenza H5 subtype vaccine. In Experiment 1, 40 chickens (ISA Brown hybrid), allocated to four groups of ten each, were immunized with Oil-H5N1(VN), Oil-H5N1(CN), Oil-H5N2(CN), and saline (control group), respectively. In Experiment 2, chickens (ISA Brown hybrid), muscovy ducks (French hybrid), and Vietnamese ducks (indigenous Vietnamese) were used to further assess the effect of SBG on immunogenicity of the Oil-H5N1(VN) Vietnamese vaccine. ELISA and hemagglutination inhibition (HI) assays were used to assess the antibody response. The H5 subtype vaccines initiated significantly higher immune responses in the animals dosed with SBG, with 1.0-1.5 log2 higher HI titers and 10-20% ELISA seroconversion, compared with those not dosed with β-glucan. Notably, some of the animals dosed with SBG induced HI titers higher than 9.0 log2 following boosting immunization. Taken together, our serial studies indicated that SBG is a potential effector, such as enhancing the immune response to the H5 vaccines tested.

Adjuvant effect of liposome in chicken result from induction of nitric oxide

Intranasal delivery of liposome-encapsulated inactivated Newcastle Disease virus (NDV) is known to be an effective vaccine for inducing immunity in the respiratory tract from our previous reports. Four-week-old specific pathogen-free chickens were intranasally immunized with NDV entrapped in phosphatidylcholine-liposomes (PC-Lip). The mucosal levels of anti-NDV s-immunoglobulin A (IgA), serum IgG, a high hemagglutination inhibition titer (1:640), and the high survival rate with the PC-Lip vaccine were comparable to those of our previous report. The immune mechanisms of the PC-Lip adjuvant were determined by in vitro cellular experiments using the NO production of chicken spleen macrophages. The most important finding of this study was proving that macrophages were stimulated by PC-Lip via the extracellular regulated kinase (ERK) 1/2 and nuclear factor (NF)-κB activation pathways. This finding may be useful for developing potent mucosal vaccine delivery systems in the future.

Field Application of the H9M2e Enzyme-Linked Immunosorbent Assay for Differentiation of H9N2 Avian Influenza Virus-Infected Chickens from Vaccinated Chickens

Vaccination for control of H9N2 low-pathogenicity avian influenza (LPAI) in chickens began in 2007 in South Korea where the H9N2 virus is prevalent. Recently, an enzyme-linked immunosorbent assay (ELISA) using the extracellular domain of the M2 protein (M2e ELISA) was developed as another strategy to differentiate between vaccinated and infected chickens. Here, an ELISA using the extracellular domain of the M2 protein of H9N2 LPAI virus (H9M2e ELISA) was applied to differentiate infected from vaccinated chickens using the H9N2 LPAI virus M2 peptide. The specificity and sensitivity of the optimized H9M2e ELISA were 96.1% and 83.8% (the absorbance of the sample to the absorbance for the positive control [S/P ratio] ≥ 0.6), respectively, with the cutoff value (S/P ratio = 0.6), and the criterion of avian influenza (AI) infection in a chicken house was established as >20% reactivity of anti-M2e antibody per house with this cutoff value. After infection in naïve chickens and once-vaccinated chickens with a hemagglutination inhibition (HI) assay titer of 9.25 ± 0.75 log(2) units, the sera from infected chickens were confirmed as AI infected when the chickens were 1 week old in both groups, and AI infection lasted for 24 weeks and 9 weeks in naïve and once-vaccinated chickens, respectively, although in twice-vaccinated chickens with a higher HI titer of 11.17 ± 0.37 log(2) units, anti-M2e antibody in infected sera did not reach a level indicating AI infection. In field application, anti-M2e antibody produced in infected chickens after vaccination or in reinfected chickens could be identified as AI infection, although HI test could not distinguish infected from vaccinated sera. These results indicate the utility of H9M2e ELISA as a surveillance tool in control of H9N2 LPAI infections.

Risk Factors and Immunity in a Nationally Representative Population following the 2009 Influenza A(H1N1) Pandemic

BackgroundUnderstanding immunity, incidence and risk factors of the 2009 influenza A(H1N1) pandemic (2009 H1N1) through a national seroprevalence study is necessary for informing public health interventions and disease modelling.Methods and FindingsWe collected 1687 serum samples and individual risk factor data between November-2009 to March-2010, three months after the end of the 2009 H1N1 wave in New Zealand. Participants were randomly sampled from selected general practices countrywide and hospitals in the Auckland region. Baseline immunity was measured from 521 sera collected during 2004 to April-2009. Haemagglutination inhibition (HI) antibody titres of ≥1∶40 against 2009 H1N1 were considered seroprotective as well as seropositive. The overall community seroprevalence was 26.7% (CI:22.6–29.4). The seroprevalence varied across age and ethnicity. Children aged 5–19 years had the highest seroprevalence (46.7%;CI:38.3–55.0), a significant increase from the baseline (14%;CI:7.2–20.8). Older adults aged ≥60 had no significant difference in seroprevalence between the serosurvey (24.8%;CI:18.7–30.9) and baseline (22.6%;CI:15.3–30.0). Pacific peoples had the highest seroprevalence (49.5%;CI:35.1–64.0). There was no significant difference in seroprevalence between both primary (29.6%;CI:22.6–36.5) and secondary healthcare workers (25.3%;CI:20.8–29.8) and community participants. No significant regional variation was observed. Multivariate analysis indicated age as the most important risk factor followed by ethnicity. Previous seasonal influenza vaccination was associated with higher HI titres. Approximately 45.2% of seropositive individuals reported no symptoms.ConclusionsBased on age and ethnicity standardisation to the New Zealand Population, about 29.5% of New Zealanders had antibody titers at a level consistent with immunity to 2009 H1N1. Around 18.3% of New Zealanders were infected with the virus during the first wave including about one child in every three. Older people were protected due to pre-existing immunity. Age was the most important factor associated with infection followed by ethnicity. Healthcare workers did not appear to have an increased risk of infection compared with the general population.

Hemagglutinin Displayed Baculovirus Protects Against Highly Pathogenic Influenza

Baculovirus (BV) replicating in insect cells can express a foreign gene product as part of its genome. The influenza hemagglutinin (HA) can be expressed from BV and displayed on the surface of baculovirus (HA-DBV). In this study we first generated six recombinant baculoviruses that expressed chimeric HAs with segments of the BV glycoprotein (gp64). The signal peptide (SP) and cytoplasmic tail (CT) domains of gp64 can enhance the display of HA from A/PR8/34 on BV surface, while the transmembrane (TM) domain of gp64 impairs HA display. Different doses of either live or β-propiolactone (BPL)-inactivated HA-DBV were administered to BALB/c mice. Live HA-DBV elicited higher hemagglutination-inhibition (HAI) titers than BPL-inactivated HA-DBV, and provided sterilizing protection. A second generation recombinant BV simultaneously displaying four HAs derived from four subclades of H5N1 influenza viruses was constructed. This tetravalent H5N1 HA-DBV vaccine elicited HAI titers against all four homologous H5N1 viruses, significantly decreasing viral lung titers of challenged mice and providing 100% protection against lethal doses of homologous H5N1 viruses. Moreover, mice vaccinated with HA-DBV had high levels of IFNγ-secreting and HA-specific CD8+ T cells. Taken together, this study demonstrates that HA-DBV can stimulate strong humoral, as well as cellular immune responses, and is an effective vaccine candidate for influenza.

Virus-Like Particle Vaccine Protects against 2009 H1N1 Pandemic Influenza Virus in Mice

BackgroundThe 2009 influenza pandemic and shortages in vaccine supplies worldwide underscore the need for new approaches to develop more effective vaccines.Methodology/Principal FindingsWe generated influenza virus-like particles (VLPs) containing proteins derived from the A/California/04/2009 virus, and tested their efficacy as a vaccine in mice. A single intramuscular vaccination with VLPs provided complete protection against lethal challenge with the A/California/04/2009 virus and partial protection against A/PR/8/1934 virus, an antigenically distant human isolate. VLP vaccination induced predominant IgG2a antibody responses, high hemagglutination inhibition (HAI) titers, and recall IgG and IgA antibody responses. HAI titers after VLP vaccination were equivalent to those observed after live virus infection. VLP immune sera also showed HAI responses against diverse geographic pandemic isolates. Notably, a low dose of VLPs could provide protection against lethal infection.Conclusion/SignificanceThis study demonstrates that VLP vaccination provides highly effective protection against the 2009 pandemic influenza virus. The results indicate that VLPs can be developed into an effective vaccine, which can be rapidly produced and avoid the need to isolate high growth reassortants for egg-based production.