Inactivated Influenza Virus Research Articles

Reduction of spiked porcine circovirus during the manufacture of a Vero cell-derived vaccine

Porcine circovirus-1 (PCV1) was recently identified as a contaminant in live Rotavirus vaccines, which was likely caused by contaminated porcine trypsin. The event triggered the development of new regulatory guidance on the use of porcine trypsin which shall ensure that cell lines and porcine trypsin in use are free from PCV1. In addition, manufacturing processes of biologicals other than live vaccines include virus clearance steps that may prevent and mitigate any potential virus contamination of product. In this work, artificial spiking of down-scaled models for the manufacturing process of an inactivated pandemic influenza virus vaccine were used to investigate inactivation of PCV1 and the physico-chemically related porcine parvovirus (PPV) by formalin and ultraviolet-C (UV-C) treatment as well as removal by the purification step sucrose gradient ultracentrifugation. A PCV1 infectivity assay, using a real-time PCR infectivity readout was established. The formalin treatment (0.05% for 48h) showed substantial inactivation for both PCV1 and PPV with reduction factors of 3.0log10 and 6.8log10, respectively, whereas UV-C treatment resulted in complete PPV (≥5.9log10) inactivation already at a dose of 13mJ/cm but merely 1.7log10 at 24mJ/cm2 for PCV1. The UV-C inactivation results with PPV were confirmed using minute virus of mice (MVM), indicating that parvoviruses are far more sensitive to UV-C than PCV1. The sucrose density gradient ultracentrifugation also contributed to PCV1 clearance with a reduction factor of 2log10. The low pH treatment during the production of procine trypsin was investigated and showed effective inactivation for both PCV1 (4.5log10) and PPV (6.4log10). In conclusion, PCV1 in general appears to be more resistant to virus inactivation than PPV. Still, the inactivated pandemic influenza vaccine manufacturing process provides for robust virus reduction, in addition to the already implemented testing for PCV1 to avoid any contaminations.

Prevention of influenza infection – a Polish perspective

Influenza is a viral respiratory illness that causes high morbidity and significant mortality in humans. Costs associated with influenza in terms of human suffering are immeasurable and the economic costs are very high. Every year, according to the World Health Organization (WHO), 5-25% of the global population suffers from infection with influenza and influenza-like viruses and between 500 thousand and one million individuals of all ages die from multiple organ complications, irrespective of the geographic location. Influenza vaccination is still neglected and the percentage of the global population vaccinated remains low. The first authorization for the use of influenza vaccines in humans was issued in 1941. Currently, many varieties of influenza vaccine are available, containing either fragments of inactivated influenza virus or live vaccine which consists of attenuated virus. The influenza vaccine is most often developed in chick embryos or less frequently in tissue culture such as MDCK and Vero. A variety of inactivated vaccines are registered in Poland. Due to the mutability of the virus, it is not yet possible to develop a universal vaccine, nor can the disease be eradicated; however, prevention is possible by inoculating the greatest percentage of the global population. According to the WHO, Poland is in the penultimate position in Europe in terms of the percentage of the population vaccinated. In the last epidemic season of 2012/2013 only 3.75% of the Polish population was immunized.

Intranasal Vaccination Promotes Detrimental Th17-Mediated Immunity against Influenza Infection

Influenza disease is a global health issue that causes significant morbidity and mortality through seasonal epidemics. Currently, inactivated influenza virus vaccines given intramuscularly or live attenuated influenza virus vaccines administered intranasally are the only approved options for vaccination against influenza virus in humans. We evaluated the efficacy of a synthetic toll-like receptor 4 agonist CRX-601 as an adjuvant for enhancing vaccine-induced protection against influenza infection. Intranasal administration of CRX-601 adjuvant combined with detergent split-influenza antigen (A/Uruguay/716/2007 (H3N2)) generated strong local and systemic immunity against co-administered influenza antigens while exhibiting high efficacy against two heterotypic influenza challenges. Intranasal vaccination with CRX-601 adjuvanted vaccines promoted antigen-specific IgG and IgA antibody responses and the generation of polyfunctional antigen-specific Th17 cells (CD4+IL-17A+TNFα+). Following challenge with influenza virus, vaccinated mice transiently exhibited increased weight loss and morbidity during early stages of disease but eventually controlled infection. This disease exacerbation following influenza infection in vaccinated mice was dependent on both the route of vaccination and the addition of the adjuvant. Neutralization of IL-17A confirmed a detrimental role for this cytokine during influenza infection. The expansion of vaccine-primed Th17 cells during influenza infection was also accompanied by an augmented lung neutrophilic response, which was partially responsible for mediating the increased morbidity. This discovery is of significance in the field of vaccinology, as it highlights the importance of both route of vaccination and adjuvant selection in vaccine development

Immunological Responses against Different Lineages of Influenza B Antigen in School Children during Two Consecutive Seasons

While Influenza B viruses currently circulating worldwide are of two distinct evolutionary hemagglutinin lineages, current trivalent inactivated influenza virus vaccines (TIV) contain only a single component. Single doses of TIV containing B antigen of B/Florida/4/2006 (Yamagata-like) or B/Brisbane/60/2008 (Victoria-like) were administered during 2008/2009 and 2009/2010 influenza seasons, respectively. The objective of this study was to evaluate the immunological response against different lineages of B antigens in school-aged children. A non-randomized sero-epidemiological study was conducted and the immunogenicity responses based on sero-protection rate and geometric mean titre ratio (GMTR) of hemagglutination inhibition (HI) antibodies were measured before and after immunization as well as post-influenza season. Our results suggested that school-aged children under the age of 9 years receiving TIV vaccination induced and retained higher level of sero-protection rate (66.7% and 69% for the 2008-09 and 2009-10 season, respectively) to the homologous lineage than the heterologous lineage post-vaccination (19.4% and 27.6% for the 2008-09 and 2009-10 season, respectively). The need for the quadrivalent TIV by including both lineages of influenza B viruses is recommended in this study, particularly for children under the age of 9 years.

Pre-Existing Cross-Reactive Antibodies to Avian Influenza H5N1 and 2009 Pandemic H1N1 in US Military Personnel

We studied cross-reactive antibodies against avian influenza H5N1 and 2009 pandemic (p) H1N1 in 200 serum samples from US military personnel collected before the H1N1 pandemic. Assays used to measure antibodies against viral proteins involved in protection included a hemagglutination inhibition (HI) assay and a neuraminidase inhibition (NI) assay. Viral neutralization by antibodies against avian influenza H5N1 and 2009 pH1N1 was assessed by influenza (H5) pseudotyped lentiviral particle-based and H1N1 microneutralization assays. Some US military personnel had cross-neutralizing antibodies against H5N1 (14%) and 2009 pH1N1 (16.5%). The odds of having cross-neutralizing antibodies against 2009 pH1N1 were 4.4 times higher in subjects receiving more than five inactivated whole influenza virus vaccinations than those subjects with no record of vaccination. Although unclear if the result of prior vaccination or disease exposure, these pre-existing antibodies may prevent or reduce disease severity.

Influenza vaccination in pregnant women: a systematic review.

Objective. To assess the effects of the inactivated influenza virus vaccine on influenza outcomes in pregnant women and their infants. Methods. We performed a systematic review of the literature. We searched for randomized controlled trials and cohort studies in the MEDLINE, Embase, and other relevant databases (inception to September 2013). Two researchers selected studies and extracted the data independently. We used the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach to assess the quality of the evidence. Results. We included eight studies out of 1,967 retrieved records. Influenza vaccination in pregnant women significantly reduced the incidence of influenza-like illness in mothers and their infants when compared with control groups (high-quality evidence) and reduced the incidence of laboratory-confirmed influenza in infants (moderate-quality evidence). No difference was found with regard to influenza-like illness with fever higher than 38°C (moderate-quality evidence) or upper respiratory infection (very-low-quality evidence) in mothers and infants. Conclusions. Maternal vaccination against influenza was shown to prevent influenza-like illness in women and infants; no differences were found for other outcomes. As the quality of evidence was not high overall, further research is needed to increase confidence and could possibly change these estimates.

Effects of different CpG oligodeoxynucleotides with inactivated avian H5N1 influenza virus on mucosal immunity of chickens

Oligodeoxynucleotide containing unmethylated CpG motifs (CpG-ODN) has been proved to be a potent and safe vaccine adjuvant. However, the application of CpG-ODN in poultry vaccines was limited because of its high cost to benefit ratio. The objective of this study was to identify the CpG-ODN with efficient adjuvant activity and low cost in chickens. Four sequences of CpG-ODN were designed based on CpG-ODN 2006, which was used as a template and positive sequence in our study. In the current study, in vitro observations revealed that the designed CpG-ODN had efficient immunostimulatory effects on chicken splenic lymphocytes. The in vivo results showed that the mRNA expressions of IL-6, IL-12, interferon-γ, and Toll-like receptor (TLR) 21 in upper respiratory tract tissues increased significantly in the early period after intranasal immunization with inactivated avian H5N1 influenza virus (IAIV) and CpG-ODN (P < 0.01). In addition, the avian influenza virus (AIV)-specific secretory IgA antibody level in the lavage fluid of upper respiratory tract increased significantly after intranasal immunization with IAIV and CpG-ODN, so did AIV-specific IgG in serum (P < 0.01). Among all the designed CpG-ODN, CpG-ODN F3 with an addition of poly-guanosine strings at the 3′-end not only had the best enhancement on local mucosal immune response but also showed an effective induction of systemic immune response. Most importantly, the virus challenge study showed that prior administration of IAIV with CpG-ODN F3 could protect chickens effectively against live AIV H5N1 challenge. Additionally, among all the CpG-ODN in our study, the cost of the designed CpG-ODN F3 was the lowest because of the partially phosphorothioate backbone. Therefore, we speculated that CpG-ODN F3 with efficient adjuvant activity and a big cost advantage over CpG-ODN F1 (CpG-ODN 2006) might serve as an efficient and affordable nasal adjuvant for inactivated AIV vaccine in chicken.

Formulation and immunological evaluation of a trivalent vaccine comprising emulsified submicron particles and inactivated virions of H5N1/EV71/JEV

Combination vaccines can reduce the number of injections and simplify the immunization schedule required to prevent different diseases. Here we assessed the immunogenicity in a mouse model of a vaccine composition comprising inactivated influenza viruses (H5N1/H1N1), enterovirus 71 (EV71), and/or Japanese encephalitis virus (JEV) and investigated whether the vaccine formulations can overcome the immunologic interference between the individual vaccine components. We demonstrated that the antigenic competition happens between H5N1/H1N1 or H5N1/EV71 inactivated virions when the vaccine combinations either formulated with Alum suspensions or without adjuvant. In the presence of PELC emulsified particles, EV71-specific immune responses before and after incorporating H5N1 virus into EV71 vaccine were detected of no significant difference; in addition, H5N1- and EV71-specific immune responses were found at the same level when H5N1/EV71/JEV consolidating into combination vaccine. Emulsified vaccine formulation was represented as a potential tool that is found to reduce the number of injections required to prevent multiple infectious strains causing the same disease (H5N1/H1N1) and/or that protect against different diseases (H5N1/EV71). Combination vaccines can also include a third component to protect against H5N1/EV71/JEV at the same time.

Comparative analysis of antibody induction and protection against influenza virus infection by DNA immunization with HA, HAe, and HA1 in mice

Plasmid DNA vaccines are considered alternatives to inactivated influenza virus vaccines to control influenza. Vaccination with a hemagglutinin (HA)-, HA ectodomain (HAe)-, or HA subunit 1 (HA1)-based vaccine can stimulate protective immunity in animals. The aim of this study was to compare their capacity to induce an antibody response and protection against influenza virus infection in mice after DNA vaccination. We constructed three expression vectors encoding full-length HA, HAe, or HA1 of the A/California/07/2009 influenza A virus and designed three animal experiments: (i) BALB/c mice were immunized twice with 30μg of the HA, HAe, or HA1 DNA vaccine with high-voltage electroporation (100V), and 3weeks after boosting, they were challenged with a lethal dose of virus. (ii) Immunization and challenge were as in experiment i, but with low-voltage electroporation (10V). (iii) Mice were immunized once with 50μg of DNA and challenged 1week later. The immunogenic effects of the three DNA vaccines were evaluated in terms of antibody titer, survival rate, bodyweight change, and lung viral titer. In all three experiments, both HA and HAe induced higher antibody and neutralization titers than HA1. Following challenge with a lethal mouse-adapted homologous virus, both HA and HAe reduced the viral titers in lung washes or offered better protection from weight loss than HA1 in experiments ii and iii. Thus, HA1 induces a lower immune response than HA or HAe when used as a DNA vaccination. Our data should be valuable in choosing the optimal candidate vaccine when faced with the threat of pandemic influenza.

Tailored Vaccines Targeting the Elderly Using Whole Inactivated Influenza Vaccines Bearing Cytokine Immunomodulators

Influenza and its complications disproportionately affect the elderly, leading to high morbidity and mortality in this ever-increasing population. Despite widespread vaccination efforts, the current influenza vaccines are less effective in the elderly; hence newer vaccine strategies are needed to improve their efficacy in this age group. We have previously shown that co-presentation of cytokines on the surface of inactivated influenza virus particles affords better protection from lethal homotypic viral challenge in young adult mice than conventional non-adjuvanted whole inactivated vaccine. Here, we determined the efficacy of these vaccine formulations in Balb/c mice "aged" to 17 months ("aged mice") along with the addition of a membrane-bound interleukin-12 (IL-12) vaccine formulation. Our investigations found that a single low-dose intramuscular vaccination with inactivated whole influenza vaccine co-presenting IL-12 was sufficient to provide enhanced protection from subsequent influenza challenge as compared with non-adjuvanted whole inactivated vaccine. Our results indicate that incorporation of cytokines such as IL-12 in a membrane-bound formulation in whole inactivated vaccine may provide a means to lower the vaccine dose while eliciting enhanced protective responses in the elderly, an age group that responds poorly to current vaccination regimens.

Impact of Prior Seasonal H3N2 Influenza Vaccination or Infection on Protection and Transmission of Emerging Variants of Influenza A(H3N2)v Virus in Ferrets

Influenza H3N2 A viruses continue to circulate in swine and occasionally infect humans, resulting in outbreaks of variant influenza H3N2 [A(H3N2)v] virus. It has been previously demonstrated in ferrets that A(H3N2)v viruses transmit as efficiently as seasonal influenza viruses, raising concern over the pandemic potential of these viruses. However, A(H3N2)v viruses have not acquired the ability to transmit efficiently among humans, which may be due in part to existing cross-reactive immunity to A(H3N2)v viruses. Although current seasonal H3N2 and A(H3N2)v viruses are antigenically distinct from one another, historical H3N2 viruses have some antigenic similarity to A(H3N2)v viruses and previous exposure to these viruses may provide a measure of immune protection sufficient to dampen A(H3N2)v virus transmission. Here, we evaluated whether prior seasonal H3N2 influenza virus vaccination or infection affects virus replication and transmission of A(H3N2)v virus in the ferret animal model. We found that the seasonal trivalent inactivated influenza virus vaccine (TIV) or a monovalent vaccine prepared from an antigenically related 1992 seasonal influenza H3N2 (A/Beijing/32/1992) virus failed to substantially reduce A(H3N2)v (A/Indiana/08/2011) virus shedding and subsequent transmission to naive hosts. Conversely, ferrets primed by seasonal H3N2 virus infection displayed reduced A(H3N2)v virus shedding following challenge, which blunted transmission to naive ferrets. A higher level of specific IgG and IgA antibody titers detected among infected versus vaccinated ferrets was associated with the degree of protection offered by seasonal H3N2 virus infection. The data demonstrate in ferrets that the efficiency of A(H3N2)v transmission is disrupted by preexisting immunity induced by seasonal H3N2 virus infection.

Comparison of 3 kinds of Toll-like receptor ligands for inactivated avian H5N1 influenza virus intranasal immunization in chicken

To evaluate the effects of co-administration of inactivated avian influenza H5N1 virus (IAIV) and different Toll-like receptor (TLR) ligands in chickens, 10-d-old chickens were immunized intranasally with IAIV and TLR ligand [Bacillus subtilis spores, polyinosinic-polycytidylic acid, and CpG oligodeoxynucleotides (CpG-ODN), respectively]. The results showed that both anti-avian influenza virus (AIV) specific secretory IgA level in respiratory tract and anti-AIV specific IgG level in serum significantly increased, as well as the expressions of IL-12, interferon-γ, IL-6, and TLR in the nasal cavity and trachea after intranasal immunization with IAIV and TLR ligand. Among the used TLR ligands, B. subtilis spores as the adjuvant for nasal IAIV had the strongest effect on the expression of IL-6 and IL-12 (P < 0.01), whereas the CpG-ODN could present an advantageous effect on the induction of anti-AIV specific IgG and neutralization antibodies (P < 0.01). The chickens that were previously co-administrated with IAIV and B. subtilis spores could survive at an improved rate upon challenge by live AIV H5N1 virus. Our study suggested that B. subtilis spores, polyinosinic-polycytidylic acid, or CpG-ODN all could effectively enhance the local and systemic immune responses to IAIV in chickens. Considering of the effects and cost of these TLR ligands, we prospected that B. subtilis spores might serve as a more affordable and efficacious mucosal adjuvant for nasal IAIV in chickens.

Intranasal vaccination with an inactivated whole influenza virus vaccine induces strong antibody responses in serum and nasal mucus of healthy adults

Haemagglutination inhibition (HI) and neutralization (NT) titers as well as haemagglutinin (HA) specific antibody responses were examined in 50 healthy adults aged between 22 and 69 y old after two intranasal administrations of an inactivated whole virus vaccine derived from A/Victoria/210/2009 virus (45 μg HA per dose) at 3 week intervals. Serum HI titers after two-doses of the nasal vaccine showed >2.5-fold rise in the ratio of geometric mean titer upon vaccination, >40% of subjects with a ≥4-fold increase in titer and >70% of subjects with a titer of ≥1:40, all parameters associated with an effective outcome of vaccination in the criteria defined by the European Medicines Agency. Serum neutralizing antibody responses correlated with HI antibody responses, although NT titers were about 2-fold higher than HI titers. These high levels of serum responses were accompanied by high levels of HI and neutralizing antibody responses in nasal mucus as measured in concentrated nasal wash samples that were about 10 times diluted compared with natural nasal mucus. Serum and nasal HI and neutralizing antibody responses consisted of HA-specific IgG and IgA antibody responses, with IgG and IgA antibodies being dominant in serum and nasal responses, respectively.

Heterosubtypic cross‐protection induced by whole inactivated influenza virus vaccine in mice: influence of the route of vaccine administration

Development of influenza vaccines capable of inducing broad protection against different virus subtypes is necessary given the ever-changing viral genetic landscape. Previously, we showed that vaccination with whole inactivated virus (WIV) induces heterosubtypic protection against lethal virus infection in mice. Whole inactivated virus-induced cross-protection was found to be mediated primarily by flu-specific CD8+ T cells. As it has been demonstrated that the route of vaccine administration strongly influences both the quantity and quality of vaccine-induced immunity, in this study, we determined which route of WIV administration induces optimal heterosubtypic cross-protection. We compared the magnitude of the immune response and heterosubtypic protection against lethal A/PR/8/34 (H1N1) infection after subcutaneous (SC), intramuscular (IM), and intranasal (IN) vaccination with A/NIBRG-14 (H5N1) WIV. Subcutaneous and IM administration was superior to IN administration of influenza WIV in terms of flu-specific CD8+ T-cell induction and protection of mice against lethal heterosubtypic challenge. Surprisingly, despite the very low flu-specific CD8+ T-cell responses detected in IN-vaccinated mice, these animals were partially protected, most likely due to cross-reactive IgA antibodies. The results of this study show that the magnitude of WIV-induced flu-specific CD8+ T-cell activity depends on the applied vaccination route. We conclude that parenteral administration of WIV vaccine, in particular IM injection, is superior to IN vaccine delivery for the induction of heterosubtypic cross-protection and generally appears to elicit stronger immune responses than mucosal vaccination with WIV.

Inactivated influenza virus vaccine is efficient and reduces IL‐4 and IL‐6 in allergic asthma mice

Allergic asthma is a globally respiratory inflammatory disease. Influenza virus is a respiratory pathogen that causes yearly epidemics and results in high rates of morbidity and mortality. Patients with allergic asthma had a more severe symptom and a higher mortality when they were infected with influenza virus. Hence, influenza vaccination is recommended for patients with asthma. We evaluated the efficacy and effects of influenza vaccination on allergic asthma in a mouse model. Ovalbumin-immunized mice were inoculated with inactivated influenza virus A/Puerto Rico/8/34 (PR8) as vaccines and morbidity or mortality and allergic asthma features of these mice were analyzed. Mice inoculated with inactivated PR8 induced high levels of anti-PR8 IgG2a and upregulation of Toll-like receptor (TLR) 7. Vaccinated allergic mice were healthy when they were challenged with live influenza virus while none of non-vaccinated allergic mice survived. Furthermore, inactivated influenza virus vaccine induced neither extra airway inflammation nor asthma features such as IgE, airway hyper-reactivity, and eosinophilia in allergic mice. Particularly, decreased frequency of immune cell infiltrated airways and Th2 cytokines IL-4 and IL-6 production in the bronchoalveolar lavage fluid were noted in vaccinated allergic mice. These results suggested that inactivated influenza virus vaccine is efficient to protect allergic mice from further influenza infection, and it does not exacerbate but reduces IL-4 and IL-6 of allergic asthma. Influenza vaccination is essential and efficient for allergic subjects to protect influenza virus infection.

Heterosubtypic Antiviral Activity of Hemagglutinin-Specific Antibodies Induced by Intranasal Immunization with Inactivated Influenza Viruses in Mice

Influenza A virus subtypes are classified on the basis of the antigenicity of their envelope glycoproteins, hemagglutinin (HA; H1–H17) and neuraminidase. Since HA-specific neutralizing antibodies are predominantly specific for a single HA subtype, the contribution of antibodies to the heterosubtypic immunity is not fully understood. In this study, mice were immunized intranasally or subcutaneously with viruses having the H1, H3, H5, H7, H9, or H13 HA subtype, and cross-reactivities of induced IgG and IgA antibodies to recombinant HAs of the H1–H16 subtypes were analyzed. We found that both subcutaneous and intranasal immunizations induced antibody responses to multiple HAs of different subtypes, whereas IgA was not detected remarkably in mice immunized subcutaneously. Using serum, nasal wash, and trachea-lung wash samples of H9 virus-immunized mice, neutralizing activities of cross-reactive antibodies were then evaluated by plaque-reduction assays. As expected, no heterosubtypic neutralizing activity was detected by a standard neutralization test in which viruses were mixed with antibodies prior to inoculation into cultured cells. Interestingly, however, a remarkable reduction of plaque formation and extracellular release of the H12 virus, which was bound by the H9-induced cross-reactive antibodies, was observed when infected cells were subsequently cultured with the samples containing HA-specific cross-reactive IgA. This heterosubtypic plaque reduction was interfered when the samples were pretreated with anti-mouse IgA polyclonal serum. These results suggest that the majority of HA-specific cross-reactive IgG and IgA antibodies produced by immunization do not block cellular entry of viruses, but cross-reactive IgA may have the potential to inhibit viral egress from infected cells and thus to play a role in heterosubtypic immunity against influenza A viruses.

A mechanistic study on the destabilization of whole inactivated influenza virus vaccine in gastric environment.

Oral immunization using whole inactivated influenza virus vaccine promises an efficient vaccination strategy. While oral vaccination was hampered by harsh gastric environment, a systematic understanding about vaccine destabilization mechanisms was not performed. Here, we investigated the separate and combined effects of temperature, retention time, pH, and osmotic stress on the stability of influenza vaccine by monitoring the time-dependent morphological change using stopped-flow light scattering. When exposed to osmotic stress, clustering of vaccine particles was enhanced in an acidic medium (pH 2.0) at ≥25°C. Fluorescence spectroscopic studies showed that hyper-osmotic stress at pH 2.0 and 37°C caused a considerable increase in conformational change of antigenic proteins compared to that in acidic iso-osmotic medium. A structural integrity of membrane was destroyed upon exposure to hyper-osmotic stress, leading to irreversible morphological change, as observed by undulation in stopped-flow light scattering intensity and transmission electron microscopy. Consistent with these analyses, hemagglutination activity decreased more significantly with an increasing magnitude of hyper-osmotic stress than in the presence of the hypo- and iso-osmotic stresses. This study shows that the magnitude and direction of the osmotic gradient has a substantial impact on the stability of orally administrated influenza vaccine.

A novel combined adjuvant strongly enhances mucosal and systemic immunity to low pathogenic avian influenza after oral immunization in ducks

As natural reservoirs of avian influenza viruses, waterfowl play an important role in the generation, spread, and enzootic transmission of avian influenza. To prevent avian influenza in waterfowl through a simple, noninvasive, and needle-free route, ducks were immunized orally with an inactivated avian influenza virus (H9N2, IAIV) combined with CpG DNA and high-dose glucose, and then the local and systemic immune responses of these ducks were investigated. In addition, the immune protection was assayed after viral challenge. After the oral administration of IAIV combined with CpG DNA and glucose, the expression levels of interleukin-2 and interleukin-6 in the small intestine tissues increased significantly in the early period after booster immunization relative to the levels after immunization with IAIV and a single adjuvant. Significant increases were also observed in the IgA and IgG antibody levels in the local intestinal tract tissues and serum at wk 3, 5, and 7 after the first immunization. Furthermore, enhanced hemagglutination inhibition titers were also detected in serum samples taken between the third and seventh weeks after immunization with IAIV and both adjuvants. In the viral challenge and transmission study, the prior administration of IAIV combined with both CpG DNA and glucose reduced the viral titers observed for the cloaca swabs and colon tissues of challenged ducks and prevented virus transmission between ducks. Our study suggests that the combination of CpG DNA and high-dose glucose can improve immunization with inactivated H9N2 virus by enhancing the local and systemic immune responses and reducing viral shedding.

Production of avian influenza virus vaccine using primary cell cultures generated from host organs

The global availability of a therapeutically effective influenza virus vaccine during a pandemic remains a major challenge for the biopharmaceutical industry. Long production time, coupled with decreased supply of embryonated chicken eggs (ECE), significantly affects the conventional vaccine production. Transformed cell lines have attained regulatory approvals for vaccine production. Based on the fact that the avian influenza virus would infect the cells derived from its natural host, the viral growth characteristics were studied on chicken embryo-derived primary cell cultures. The viral propagation was determined on avian origin primary cell cultures, transformed mammalian cell lines, and in ECE. A comparison was made between these systems by utilizing various cell culture-based assays. In-vitro substrate susceptibility and viral infection characteristics were evaluated by performing hemagglutination assay (HA), 50 % tissue culture infectious dose (TCID₅₀) and monitoring of cytopathic effects (CPE) caused by the virus. The primary cell culture developed from chicken embryos showed stable growth characteristics with no contamination. HA, TCID₅₀, and CPE exhibited that these cell systems were permissive to viral infection, yielding 2-10 times higher viral titer as compared to mammalian cell lines. Though the viral output from the ECE was equivalent to the chicken cell culture, the time period for achieving it was decreased to half. Some of the prerequisites of inactivated influenza virus vaccine production include generation of higher vial titer, independence from exogenous sources, and decrease in the production time lines. Based on the tests, it can be concluded that chicken embryo primary cell culture addresses these issues and can serve as a potential alternative for influenza virus vaccine production.

Recombinant IgA Is Sufficient To Prevent Influenza Virus Transmission in Guinea Pigs

A serum hemagglutination inhibition (HAI) titer of 40 or greater is thought to be associated with reduced influenza virus pathogenesis in humans and is often used as a correlate of protection in influenza vaccine studies. We have previously demonstrated that intramuscular vaccination of guinea pigs with inactivated influenza virus generates HAI titers greater than 300 but does not protect vaccinated animals from becoming infected with influenza virus by transmission from an infected cage mate. Only guinea pigs intranasally inoculated with a live influenza virus or a live attenuated virus vaccine, prior to challenge, were protected from transmission (A. C. Lowen et al., J. Virol. 83:2803-2818, 2009.). Because the serum HAI titer is mostly determined by IgG content, these results led us to speculate that prevention of viral transmission may require IgA antibodies or cellular immune responses. To evaluate this hypothesis, guinea pigs and ferrets were administered a potent, neutralizing mouse IgG monoclonal antibody, 30D1 (Ms 30D1 IgG), against the A/California/04/2009 (H1N1) virus hemagglutinin and exposed to respiratory droplets from animals infected with this virus. Even though HAI titers were greater than 160 1 day postadministration, Ms 30D1 IgG did not prevent airborne transmission to passively immunized recipient animals. In contrast, intramuscular administration of recombinant 30D1 IgA (Ms 30D1 IgA) prevented transmission to 88% of recipient guinea pigs, and Ms 30D1 IgA was detected in animal nasal washes. Ms 30D1 IgG administered intranasally also prevented transmission, suggesting the importance of mucosal immunity in preventing influenza virus transmission. Collectively, our data indicate that IgG antibodies may prevent pathogenesis associated with influenza virus infection but do not protect from virus infection by airborne transmission, while IgA antibodies are more important for preventing transmission of influenza viruses.