Pathogenic Avian Influenza H5N1 Research Articles

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

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

A Novel DNA Vaccine Coding For H5 and N1 Genes of Highly Pathogenic Avian Influenza H5N1 Subtype

Control of avian influenza infection requires a good vaccine that could induce both humoral and cell-mediated immune response, specifically IFN-ɣ production, to maintain a high level of protection along with the minimal level of viral shedding after the infection to prevent secondary epidemics. In the current study, deoxyribonucleic Acid (DNA) vaccine coding for full-length H5 and N1 genes have been produced and evaluated in SPF-chicken. Humoral immune response estimated by haemagglutination inhibition (HI) assay revealed that the DNA vaccine gave a high titer of antibodies at the day 28-post vaccination and 14 days post-challenge. However, the shedding level was minimal with the DNA vaccine (0.1 Log 10 EID50). The IFN-ɣ transcript was upregulated at a higher level in the DNA vaccinated group. The results revealed that the DNA vaccine could induce a high level of humoral and IFN-ɣ level that maintains a high level of protection (92%) with the advantage of limiting the shedding level and thus, prevent secondary epidemics.

Re-emergence of Highly Pathogenic Avian Influenza H5N1 in Nigeria, 2014-2016: Role of Social Network and Value Chain Forces in Interstate Transmission.

Since 2004, high pathogenic avian influenza (HPAI) due to H5N1 virus among others has caused a major veterinary health crisis, resulting in the loss of millions of poultry through death or culling in several countries in Asia, the Middle East, Europe, Africa and North America (Alexander 2007; Lee et al. 2016). The first African outbreak was reported from Nigeria in 2006 in domestic poultry (Joannis et al. 2006) and persisted till 2008 (Fusaro et al. 2010). These outbreaks negatively affected animal and public health as well as the economy, and were caused by viruses belonging to genetic clades 2.2, 2.2.1, 2.2.2 and 2.3.2.1c (Aiki-Raji et al. 2008; Fusaro et al. 2010; Monne et al. 2015).

Aptamer-Based Field-Effect Transistor for Detection of Avian Influenza Virus in Chicken Serum.

Early diagnosis of the highly pathogenic H5N1 avian influenza virus (AIV) is significant for preventing and controlling a global pandemic. However, there is no existing electrical biosensor for detecting biomarkers for AIV in clinically relevant samples such as chicken serum. Herein, we report the first use of an aptamer-functionalized field-effect transistor (FET) as a label-free sensor for AIV detection in chicken serum. A DNA aptamer is employed as a sensitive and selective receptor for hemagglutinin (HA) protein, which is a biomarker for AIVs. This aptamer is immobilized on a gold microelectrode that is connected to the gate of a reusable FET transducer. The specific binding of the target protein results in a change in the surface potential, which generates a signal response of the FET transducer. We hypothesize that a conformational change in the DNA aptamer upon specific binding of HA protein may alter the surface potential. The signal of the aptamer-based FET biosensor increased linearly with the increase in the logarithm of HA protein concentration in a dynamic range of 10 pM to 10 nM with a detection limit of 5.9 pM. The selectivity of the biosensor for HA protein was confirmed by employing relevant interfering proteins. The proposed biosensor was successfully applied to the selective detection of HA protein in a chicken serum sample. Owing to its simple and low-cost architecture, portability, and sensitivity, the aptamer-based FET biosensor has potential as a point-of-care diagnosis of H5N1 AIVs in clinical samples.

High Levels of miR-483-3p Are Present in Serum Exosomes Upon Infection of Mice With Highly Pathogenic Avian Influenza Virus.

Exosomes, the extracellular vesicles that contain functional proteins and RNAs, regulate cell-cell communication. Recently, our group reported that levels of various microRNAs (miRNAs) in bronchoalveolar lavage fluid exosomes were highly increased in influenza virus-infected mice and that one of those miRNAs, miR-483-3p, was involved in the potentiation of the innate immune responses to influenza virus infection in mouse type II pneumocytes. Here, we evaluated exosomal miR-483-3p levels in the serum of influenza virus-infected mice and found that miR-483-3p levels were significantly increased during infection with a highly pathogenic avian H5N1 influenza virus. Moreover, miR-483-3p-enriched exosomes derived from type II pneumocytes potentiated the expression of proinflammatory cytokine genes in vascular endothelial cells. Our findings suggest that serum exosomal transfer of miR-483-3p might be involved in the inflammatory pathogenesis of H5N1 influenza virus infection.

Innate immunemodulator containing adjuvant formulated HA based vaccine protects mice from lethal infection of highly pathogenic avian influenza H5N1 virus

The highly pathogenic avian influenza (HPAI) H5N1 viruses and their spillover into the human population pose substantial economic and public health threats. Although antiviral drugs have some effect in treating influenza infection, vaccination is still the most effective intervention to prevent possible pandemic outbreaks. We have developed a novel H5 influenza vaccine to improve the world’s pandemic preparedness. We produced a hemagglutinin (HA) of HPAI H5N1 virus A/Alberta/01/2014 (AB14) using both mammalian (m) and bacterial (b) expression systems. The purified recombinant proteins were formulated with a proprietary adjuvant (TriAdj) and their efficacy as vaccine candidates was evaluated in mice. Intramuscular delivery of two doses of TriAdj formulated mammalian expressed HA (m-HA/TriAdj) was shown to provide full protection against a lethal challenge of AB14 in mice. In contrast, bacterially expressed HA with TriAdj (b-HA/TriAdj), b-HA without adjuvant, and m-HA without adjuvant resulted in no protection in immunized mice. Furthermore, m-HA/TriAdj elicited significantly higher levels of balanced Th1 and Th2 responses and neutralizing antibody titres. All the mice in the m-HA/TriAdj group survived a lethal AB14 H5N1 challenge and showed no signs of disease or infection as demonstrated by no loss of body weight or detectable virus in the lungs. Our results suggest that m-HA formulated with TriAdj has potential to protect against pandemic H5N1 in the event of its cross over to the human host.

Agricultural and geographic factors shaped the North American 2015 highly pathogenic avian influenza H5N2 outbreak.

The 2014–2015 highly pathogenic avian influenza (HPAI) H5NX outbreak represents the largest and most expensive HPAI outbreak in the United States to date. Despite extensive traditional and molecular epidemiological studies, factors associated with the spread of HPAI among midwestern poultry premises remain unclear. To better understand the dynamics of this outbreak, 182 full genome HPAI H5N2 sequences isolated from commercial layer chicken and turkey production premises were analyzed using evolutionary models able to accommodate epidemiological and geographic information. Epidemiological compartmental models embedded in a phylogenetic framework provided evidence that poultry type acted as a barrier to the transmission of virus among midwestern poultry farms. Furthermore, after initial introduction, the propagation of HPAI cases was self-sustainable within the commercial poultry industries. Discrete trait diffusion models indicated that within state viral transitions occurred more frequently than inter-state transitions. Distance and sample size were very strongly supported as associated with viral transition between county groups (Bayes Factor > 30.0). Together these findings indicate that the different types of midwestern poultry industries were not a single homogenous population, but rather, the outbreak was shaped by poultry industries and geographic factors.

Evaluating two approaches for using positive control in standardizing the avian influenza H5 reverse transcription recombinase polymerase amplification assay

Highly pathogenic avian influenza H5N1 virus causes heavy losses in poultry farms worldwide. Molecular diagnostic techniques like RT-PCR and real-time RT-PCR are considered the gold standard for identification of H5 influenza viruses in clinical samples. These techniques are hampered by the need of well-equipped laboratories, large space requirement, and relatively long time-to-result. Recombinase polymerase amplification (RPA) assay represents an excellent alternative to PCR since it is more simple, rapid, economic, and portable. Reverse transcription RPA (RT-RPA) assay was recently developed for sensitive and specific detection of H5N1 virus in 6–10 min. To ensure the accuracy of the developed assay, two approaches for using a positive control were evaluated in this study. These approaches included: 1) all-in-one (internal positive control; IPC), 2) two-tubes-per-one-sample (external positive control; EPC). Sigma virus (SIGV) RNA and turkey mitochondrial DNA were tested as positive controls in both approaches. For all-in-one approach, both targets (H5 and IPC) were strongly inhibited. In contrast, very good amplification signals were obtained for the two types of EPC with no effect on the analytical sensitivity and specificity of H5 RT-RPA assay in two-tubes-per-one-sample approach. The performance of EPC-based H5 RT-RPA was further validated using 13 tracheal swabs. The results were compared to real-time RT-PCR and proved superior specificity in detecting H5N1 but not H5N8 viruses. Inclusion of EPC did not affect the aptitude of both assays in terms of sensitivity, specificity and reproducibility. In conclusion, the two-tubes-per-one-sample approach was more reliable to control the false negative results in H5 RT-RPA assay.

Evaluation of the immune status of birds and domestic and companion animals for the influenza A virus in Eastern Saudi Arabia.

Background and Aim:Influenza type A virus infections are still one of the major concerns for the health of humans and various species of domestic and companion animals. Wild birds play an essential role in the transmission cycle of the virus. Regularly monitoring the spread of the virus is a significant step in its mitigation. Highly pathogenic avian influenza viruses, including H5N1 and H5N8, have been reported in birds in the Arabian Peninsula, including Saudi Arabia, in recent decades. This study aimed to evaluate the immune status of birds, domestic and companion animals for Influenza type A virus in Eastern Province of Saudi Arabia.Materials and Methods:We collected 195 serum samples from dromedary camels, sheep, goats, native breed chickens, doves, dogs, and cats. We tested these sera for the presence of specific antibodies against influenza type A virus using a commercially available enzyme-linked immunosorbent assay.Results:Our results show that 4% of the tested samples had antibodies in sera, including some doves, chickens, and dogs. These data suggest exposure and seroconversion of these animals or birds to the influenza type A virus.Conclusion:The presence of antibodies against influenza type A virus in sera of some animals and birds without a previous vaccination history against the virus indicates a natural exposure history regarding this virus and seroconversion. Further large-scale molecular and epidemiological studies are needed to obtain a better understanding of the dynamics of influenza type A virus among various species of animals and birds.

Determining highly pathogenic H5 avian influenza clade 2.3.2.1c seroprevalence in ducks, Purbalingga, Central Java, Indonesia

Background and Aim:In Indonesia, highly pathogenic avian influenza (HPAI) H5N1 outbreaks in poultry are still reported. The disease causes a decrease in egg production and an increase in mortality; this has an impact on the economic losses of farmers. Several studies have considered that ducks play a role in the HPAI endemicity in the country; however, little is known about whether or not the type of duck farming is associated with HPAI H5 virus infection, particularly within clade 2.3.2.1c, which has been predominantly found in poultry since 2014. A cross-sectional study was conducted to determine the HPAI seroprevalence for H5 subtype clade 2.3.2.1c in laying ducks that are kept intensively and nomadically and to determine the associated risk factors.Materials and Methods:Forty-nine duck farmers were randomly selected from ten sub-districts in Purbalingga District, Central Java, Indonesia; a cross-sectional study was implemented to collect field data. Based on an expected HPAI prevalence level of 10%, estimated accuracy of ± 5%, and 95% confidence interval (CI), the total sample size was calculated at 36 individuals. Samples must be multiplied by 7 to reduce bias; thus, 252 ducks were taken as samples in this study. Considering that the maintenance and duck handling were uniform and farmers complained that the effect of activity to take duck samples would reduce egg production, this study only took samples from 245 ducks (oropharyngeal swabs and serum). Those samples were taken from five birds on each farm. Hemagglutination inhibition tests examined the serum samples for HPAI H5 Clade 2.3.2.1c, and pool swab samples (five swabs in one viral media transport) were examined by real-time reverse transcription-polymerase chain reaction (qRT-PCR) test for influenza Type A and H5 subtype virus. Information regarding farm management was obtained using a questionnaire; face-to-face interviews were conducted with the duck farmers using native Javanese language.Results:Serum and swabs from 245 ducks were collected in total. For individual birds, 54.69% (134/245) of serum samples were H5 seropositive. Seroprevalence among nomadic ducks was 59.28% (95% CI: 0.48-0.61), which was higher than among intensively farmed ducks (48.57%, 95% CI: 0.38-0.58). Farm-level seroprevalence was 50% (95% CI: 0.30-0.69) for nomadic ducks but only 28.57% (95% CI: 0.11-0.51) for intensively farmed ducks. The farm-level virus prevalence (proportion of flocks with at least one bird positive for influenza Type A) was 17.85% (95% CI: 0.07-0.35) for nomadic ducks and 4.76% (1/21) for intensively farmed ducks (95% CI: 0.008-0.23). All influenza Type A positive samples were negative for the H5 subtype, indicating that another HA subtype AI viruses might have been circulating in ducks in the study area. A relationship between duck farms that were H5 seropositive and their maintenance system was present; however, this relationship was not significant, the nomadic duck system detected 2 times higher H5-seropositive ducks than the intensive farming system (OR: 2.16, 95% CI: 0.33-14.31).Conclusion:This study found that the seroprevalence of HPAI in the duck population level in Purbalingga was 54.69% and demonstrated that the nomadic duck farming system was more likely to acquire HPAI H5 infection than the intensive farming duck system. Other risk factors should be further investigated as the diversity of the farming system is partially related to HPAI H5 infection.

Experimental infection and pathology of two highly pathogenic avian influenza H5N1 viruses isolated from crow and chicken in house crows (Corvus splendens).

We investigated the experimental infection of two highly pathogenic avian influenza H5N1 viruses isolated from crow (A/crow/Assam/142119/2008) and chicken (A/chicken/Sikkim/151466/2009) in house crows (Corvus splendens). Both viruses caused infection in crows, where four out of six and three out of six crows succumbed to H5N1 infection within 11 days post challenge by crow and chicken viruses, respectively. The major clinical signs in crows were wing paralysis, circling and torticollis. The virus shedding detected from swabs was not persistent in both crow nor chicken viruses. Both viruses were isolated more frequently from oral swabs than from cloacal swabs. Both virus strains were isolated from brain, lungs, heart, liver, pancreas, spleen, large intestines of crows that succumbed to H5N1 infection. The surviving birds seroconverted in response to H5N1 virus infection. Microscopically, both viruses caused coagulative necrosis in pancreas and kidneys. Brain showed gliosis and neuronal degeneration. This experimental study highlights that crows could be infected with H5N1 viruses from different hosts with minor differences in pathogenicity. Therefore, it is imperative to carry out surveillance of highly pathogenic avian influenza H5N1 virus in synanthropic birds along with biosecurity measures to mitigate the H5N1 spread in poultry population. Keywords: chicken virus; crow virus; highly pathogenic avian influenza; house crows.

Molecular Epidemiology of Avian Influenza Virus (H5N1), Sumatera Indonesia

The highly pathogenic H5N1 avian influenza virus has been causing outbreaks in several regions in Indonesia, which is followed by high human casualties. It is panzootic virus in poultry and continues to spread and pose a major challenge to health in both animal and human. The H5N1 virus is considered as a serious pandemic threat which has potentially to re-endemic over time. This is proven by mutation of the H5N1 avian influenza virus in the globe. This study aims to investigate the distribution, prevalence, and transmission of avian influenza virus (H5N1) in Sumatera. In a retrospective analysis of 136 samples of H5N1 HA (Hemagglutinin) cds between 2004 and 2016. We present a phylogenetic approach with Neighbor-joining (NJ) tree analysis and Bayesian molecular clock-like-lines, to reconstruct the molecular epidemiology of DNA sequences of H5N1 virus isolated from Sumatera. Overall, the distribution was distinguished into 7 clades, with prevalence detected at 9 out of 13 regions in Sumatera, and all the transmission was originated from outside the island. The spread of H5N1 avian influenza on Sumatera is not linear and homogeneous. Therefore, it has shown a significant change in the variant that currently exists with the original species.

Variability of nonpathogenic influenza virus H5N3 under immune pressure.

Mutations arising in influenza viruses that have undergone immune pressure may promote a successful spread of mutants in nature. In order to evaluate the variability of nonpathogenic influenza virus A/duck/Moscow/4182-C/2010(H5N3) and to determine the common epitopes between it and highly pathogenic H5N1 avian influenza viruses (HPAIV), a set of escape mutants was selected due to action of MABs specific against A/chicken/Pennsylvania/8125/83(H5N2), A/Vietnam/1203/04(H5N1) and A/duck/Novosibirsk/56/05(H5N1) viruses. The complete genomes of escape mutants were sequenced and amino acid point mutations were determined in HA, NA, PA, PB1, PB2, M1, M2, and NP proteins. Comprehensive analysis of the acquired mutations was performed using the Influenza Research Database (https://www.fludb.org) and revealed that all mutations were located inside short linear epitopes, in positions characterized by polymorphisms. Most of the mutations found were characterized as substitutions by predominant or alternative amino acids existing in nature. Antigenic changes depended only on substitutions at positions 126, 129, 131, 145 and 156 of HA (H3 numbering). The positions 126, 145 and 156 were common for HA/H5 of different phylogenetic lineages of H5N1 HPAIV (arisen from A/goose/Guangdong/1/96) and low pathogenic American and Eurasian viruses. Additionally, mutation S145P increased the temperature of HA heat inactivation, compared to wild-type, as was proved by reverse genetics. Moreover, nonpathogenic A/duck/Moscow/4182-C/2010(H5N3) and H5N1 HPAI viruses have the same structure of short linear epitopes in HA (145-157) and internal proteins (PB2: 186-200, 406-411; PB1: 135-143, 538-546; PA: 515-523; NP: 61-68; M1: 76-84; M2: 45-53). These facts may indicate that H5 wild duck nonpathogenic virus could be used as vaccine against H5N1 HPAIV. Keywords: avian influenza virus; H5 hemagglutinin; escape mutants; genetic analysis; phenotypic properties; site-specific mutagenesis.

Epidemiological Study on Highly Pathogenic Avian Influenza H5N1 Virus with Modeling the Impact of Climate Variability on Outbreak Occurrence in Some Governorates of Nile Delta, Egypt.

Epidemiological Study on Highly Pathogenic Avian Influenza H5N1 Virus with Modeling the Impact of Climate Variability on Outbreak Occurrence in Some Governorates of Nile Delta, Egypt.

Incorporating heterogeneous sampling probabilities in continuous phylogeographic inference - Application to H5N1 spread in the Mekong region.

The potentially low precision associated with the geographic origin of sampled sequences represents an important limitation for spatially explicit (i.e. continuous) phylogeographic inference of fast-evolving pathogens such as RNA viruses. A substantial proportion of publicly available sequences is geo-referenced at broad spatial scale such as the administrative unit of origin, rather than more precise locations (e.g. geographic coordinates). Most frequently, such sequences are either discarded prior to continuous phylogeographic inference or arbitrarily assigned to the geographic coordinates of the centroid of their administrative area of origin for lack of a better alternative. We here implement and describe a new approach that allows to incorporate heterogeneous prior sampling probabilities over a geographic area. External data, such as outbreak locations, are used to specify these prior sampling probabilities over a collection of sub-polygons. We apply this new method to the analysis of highly pathogenic avian influenza H5N1 clade data in the Mekong region. Our method allows to properly include, in continuous phylogeographic analyses, H5N1 sequences that are only associated with large administrative areas of origin and assign them with more accurate locations. Finally, we use continuous phylogeographic reconstructions to analyse the dispersal dynamics of different H5N1 clades and investigate the impact of environmental factors on lineage dispersal velocities. Our new method allowing heterogeneous sampling priors for continuous phylogeographic inference is implemented in the open-source multi-platform software package BEAST 1.10. Supplementary data are available at Bioinformatics online.

Development of an effective contemporary trivalent avian influenza vaccine against circulating H5N1, H5N8, and H9N2 in Egypt

Low pathogenicity avian influenza (LPAI) H9N2, highly pathogenic avian influenza (HPAI) H5N1, and H5N8 circulate in Egyptian poultry and cause veterinary and public health burdens. In response, AIV vaccines are commonly used. The main objective of this study was to develop a broad, cross-protective, trivalent vaccine based on circulating AIVs in Egypt. We generated highly replicating avirulent AIVs, H5N1, and H5N8, to be used in combination with H9N2 strain for the generation of an inactivated vaccine. Immunogenicity and protective efficacy of this vaccine were tested. Results showed that a single immunization dose enhanced humoral immune responses giving full protection against challenges with LPAI H9N2, HPAI H5N1, and H5N8 viruses. This efficacious vaccine will reduce the cost of vaccination for poultry growers and is expected to be effective in the field as it is based on contemporary viruses currently in circulation among Egyptian poultry.

The Pattern of Highly Pathogenic Avian Influenza H5N1 Outbreaks in South Asia.

Highly pathogenic avian influenza (HPAI) H5N1 has caused severe illnesses in poultry and in humans. More than 15,000 outbreaks in domestic birds from 2005 to 2018 and 861 human cases from 2003 to 2019 were reported across the world to OIE (Office International des Epizooties) and WHO (World Health Organization), respectively. We reviewed and summarized the spatial and temporal distribution of HPAI outbreaks in South Asia. During January 2006 to June 2019, a total of 1063 H5N1 outbreaks in birds and 12 human cases for H5N1 infection were reported to OIE and WHO, respectively. H5N1 outbreaks were detected more in the winter season than the summer season (RR 5.11, 95% CI: 4.28–6.1). Commercial poultry were three times more likely to be infected with H5N1 than backyard poultry (RR 3.47, 95% CI: 2.99–4.01). The highest number of H5N1 outbreaks was reported in 2008, and the smallest numbers were reported in 2014 and 2015. Multiple subtypes of avian influenza viruses and multiple clades of H5N1 virus were detected. Early detection and reporting of HPAI viruses are needed to control and eliminate HPAI in South Asia.

A novel reassortant clade 2.3.4.4 highly pathogenic avian influenza H5N6 virus identified in South Korea in 2018

Since 2017, clade 2.3.4.4b H5N6 highly pathogenic avian influenza viruses (HPAIVs) have been detected over a broad geographic region, including Eurasia. These viruses have evolved through reassortment with Eurasian low pathogenic avian influenza viruses (LPAIVs), resulting in multiple genotypes. Here, we sequenced the full-length genome of 15 H5N6 HPAIVs collected from wild birds and poultry farms in South Korea from January to March 2018. A comparative phylogenetic analysis was then conducted. Three distinct genotypes were identified in South Korea during 2017/2018, including a novel reassortant genotype, H214. The novel reassortant H5N6 viruses isolated in this study possessed PB2, PA, and NP gene segments of Eurasian LPAIV on a genetic backbone of the H35-like genotype, which was identified in Korea and the Netherlands during 2017. Bayesian molecular clock analysis suggested that the novel reassortant viruses were generated most likely during the fall migration/wintering season of migratory waterfowl in 2017. Considering the continued emergence and spread of clade 2.3.4.4 HPAIV, enhanced surveillance of wild waterfowl is needed for early detection of HPAIV incursions.

Highly Pathogenic Avian Influenza H5N1 A/Chicken/France/150169a/2015 Presents In Vitro Characteristics Consistent with Its Predicted Tropism for Avian Species.

Avian influenza A viruses are a major threat to animal and public health. Since 1997, several highly pathogenic H5N1 avian viruses have been directly transmitted from poultry to humans, caused numerous human deaths, and had considerable economic impact on poultry markets. During 2015-2016, a highly pathogenic avian influenza outbreak occurred in southwestern France. Different subtypes circulated, including the A/chicken/France/150169a/2015 H5N1 highly pathogenic virus, which did not possess the full set of genomic determinants known to promote transmission to humans. In order to evaluate the predicted absence of zoonotic potential, a quick method based on in vitro tests was developed to analyze some genetic and phenotypic host restriction determinants. A receptor-binding assay showed that the virus preferentially recognizes avian cell receptors. Temperature sensitivity revealed a cold-sensitive phenotype of the virus at 33 C as virus replication was reduced in contrast with what is expected for human influenza viruses, according to their primary infection sites. Altogether, our quick evaluation method suggests that the A/chicken/France/150169a/2015 H5N1 highly pathogenic virus has an avian phenotype in vitro, in accordance with in silico predictions based on genomic markers.

Influenza A(H5N1) Virus Subunit Vaccine Administered with CaPNP Adjuvant Induce High Virus Neutralization Antibody Titers in Mice.

The highly pathogenic avian influenza H5N1 virus continues to spread globally in domestic poultry with sporadic transmission to humans. The possibility for its rapid transmission to humans raised global fears for the virus to gain capacity for human-to-human transmission to start a future pandemic. Through direct contact with infected poultry, it caused the largest number of reported cases of severe disease and death in humans of any avian influenza strains. For pandemic preparedness, use of safe and effective vaccine adjuvants and delivery systems to improve vaccine efficacy are considered imperative. We previously demonstrated CaPtivate's proprietary CaP nanoparticles (CaPNP) as a potent vaccine adjuvant/delivery system with ability to induce both humoral and cell-mediated immune responses against many viral or bacterial infections. In this study, we investigated the delivery of insect cell culture-derived recombinant hemagglutinin protein (HA) of A/H5N1/Vietnam/1203/2004 virus using CaPNP. We evaluated the vaccine immunogenicity in mice following two intramuscular doses of 3μg antigen combined with escalating doses of CaPNP. Our data showed CaPNP-adjuvanted HA(H5N1) vaccines eliciting significantly higher IgG, hemagglutination inhibition, and virus neutralization titers compared to non-adjuvanted vaccine. Among the four adjuvant doses that were tested, CaPNP at 0.24% final concentration elicited the highest IgG and neutralizing antibody titers. We also evaluated the inflammatory response to CaPNP following a single intramuscular injection in guinea pigs and showed that CaPNP does not induce any systemic reaction or adverse effects. Current data further support our earlier studies demonstrating CaPNP as a safe and an effective adjuvant for influenza vaccines.