Immunostimulant effects of Pituitary Adenylate Cyclase-Activating Polypeptide and double-stranded (ds)RNA in Orconectes propinquus.

Disease outbreaks in crustacean aquaculture caused by opportunistic and obligate pathogens cause severe economic losses to the industry. Antibiotics are frequently used as prophylactic treatments worldwide, although its overuse and misuse has led to microbial resistance, which has driven the search for novel molecules with immunostimulant and antibacterial activities. Antimicrobial peptides (AMP) and double-stranded (ds)RNAs constitute promising immunostimulants in the fight against infectious diseases in aquaculture. Scientists have made significant progress in testing these molecules in aquatic organisms as potential candidates for replacing conventional antibiotics. However, most studies have been conducted in teleost fish, thus little is known about the immunostimulatory effects in crustaceans, especially in freshwater crayfishes. Consequently, in the present work, we evaluate the immunomodulatory effects of the AMP Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) and high molecular weight (HMW) Poly (I:C) in the northern clearwater crayfish Orconectes propinquus. Two bioassays were conducted to evaluate the effects of different doses of PACAP and Poly (I:C) HMW, different administration routes, as well as the effects of the combined treatment on the crayfish immune system. Results showed the immunostimulatory role of PACAP and Poly (I:C) HMW with effects depending on the dose, the site of injection and the treatment assessed. These findings offer new insights into the crayfish immune system and contribute to the development of effective broad-spectrum immune therapies in aquaculture.

Recurring Trans-Atlantic Incursion of Clade 2.3.4.4b H5N1 Viruses by Long Distance Migratory Birds from Northern Europe to Canada in 2022/2023.

In December 2022 and January 2023, we isolated clade 2.3.4.4b H5N1 high-pathogenicity avian influenza (HPAI) viruses from six American crows (Corvus brachyrhynchos) from Prince Edward Island and a red fox (Vulpes vulpes) from Newfoundland, Canada. Using full-genome sequencing and phylogenetic analysis, these viruses were found to fall into two distinct phylogenetic clusters: one group containing H5N1 viruses that had been circulating in North and South America since late 2021, and the other one containing European H5N1 viruses reported in late 2022. The transatlantic re-introduction for the second time by pelagic/Icelandic bird migration via the same route used during the 2021 incursion of Eurasian origin H5N1 viruses into North America demonstrates that migratory birds continue to be the driving force for transcontinental dissemination of the virus. This new detection further demonstrates the continual long-term threat of H5N1 viruses for poultry and mammals and the subsequent impact on various wild bird populations wherever these viruses emerge. The continual emergence of clade 2.3.4.4b H5Nx viruses requires vigilant surveillance in wild birds, particularly in areas of the Americas, which lie within the migratory corridors for long-distance migratory birds originating from Europe and Asia. Although H5Nx viruses have been detected at higher rates in North America since 2021, a bidirectional flow of H5Nx genes of American origin viruses to Europe has never been reported. In the future, coordinated and systematic surveillance programs for HPAI viruses need to be launched between European and North American agencies.

Characterization of neurotropic HPAI H5N1 viruses with novel genome constellations and mammalian adaptive mutations in free-living mesocarnivores in Canada.

The GsGd lineage (A/goose/Guangdong/1/1996) H5N1 virus was introduced to Canada in 2021/2022 through the Atlantic and East Asia-Australasia/Pacific flyways by migratory birds. This was followed by unprecedented outbreaks affecting domestic and wild birds, with spillover into other animals. Here, we report sporadic cases of H5N1 in 40 free-living mesocarnivore species such as red foxes, striped skunks, and mink in Canada. The clinical presentations of the disease in mesocarnivores were consistent with central nervous system infection. This was supported by the presence of microscopic lesions and the presence of abundant IAV antigen by immunohistochemistry. Some red foxes that survived clinical infection developed anti-H5N1 antibodies. Phylogenetically, the H5N1 viruses from the mesocarnivore species belonged to clade 2.3.4.4b and had four different genome constellation patterns. The first group of viruses had wholly Eurasian (EA) genome segments. The other three groups were reassortant viruses containing genome segments derived from both North American (NAm) and EA influenza A viruses. Almost 17 percent of the H5N1 viruses had mammalian adaptive mutations (E627 K, E627V and D701N) in the polymerase basic protein 2 (PB2) subunit of the RNA polymerase complex. Other mutations that may favour adaptation to mammalian hosts were also present in other internal gene segments. The detection of these critical mutations in a large number of mammals within short duration after virus introduction inevitably highlights the need for continually monitoring and assessing mammalian-origin H5N1 clade 2.3.4.4b viruses for adaptive mutations, which potentially can facilitate virus replication, horizontal transmission and posing pandemic risks for humans.

A threat from both sides: Multiple introductions of genetically distinct H5 HPAI viruses into Canada via both East Asia-Australasia/Pacific and Atlantic flyways.

From 2016 to 2020, high pathogenicity avian influenza (HPAI) H5 viruses circulated in Asia, Europe, and Africa, causing waves of infections and the deaths of millions of wild and domestic birds and presenting a zoonotic risk. In late 2021, H5N1 HPAI viruses were isolated from poultry in Canada and also retrospectively from a great black-backed gull (Larus marinus), raising concerns that the spread of these viruses to North America was mediated by migratory wild bird populations. In February and April 2022, H5N1 HPAI viruses were isolated from a bald eagle (Haliaeetus leucocephalus) and broiler chickens in British Columbia, Canada. Phylogenetic analysis showed that the virus from bald eagle was genetically related to H5N1 HPAI virus isolated in Hokkaido, Japan, in January 2022. The virus identified from broiler chickens was a reassortant H5N1 HPAI virus with unique constellation genome segments containing PB2 and NP from North American lineage LPAI viruses, and the remaining gene segments were genetically related to the original Newfoundland-like H5N1 HPAI viruses detected in November and December 2021 in Canada. This is the first report of H5 HPAI viruses' introduction to North America from the Pacific and the North Atlantic-linked flyways and highlights the expanding risk of genetically distinct virus introductions from different geographical locations and the potential for local reassortment with both the American lineage LPAI viruses in wild birds and with both Asian-like and European-like H5 HPAI viruses. We also report the presence of some amino acid substitutions across each segment that might contribute to the replicative efficiency of these viruses in mammalian host, evade adaptive immunity, and pose a potential zoonotic risk.

More tools for our toolkit: The application of HEL-299 cells and dsRNA-nanoparticles to study human coronaviruses in vitro.

Human coronaviruses (HCoVs) are important human pathogens, as exemplified by the current SARS-CoV-2 pandemic. While the ability of type I interferons (IFNs) to limit coronavirus replication has been established, the ability of double-stranded (ds)RNA, a potent IFN inducer, to inhibit coronavirus replication when conjugated to a nanoparticle is largely unexplored. Additionally, the number of IFN competent cell lines that can be used to study coronaviruses in vitro are limited. In the present study, we show that poly inosinic: poly cytidylic acid (pIC), when conjugated to a phytoglycogen nanoparticle (pIC+NDX) is able to protect IFN-competent human lung fibroblasts (HEL-299 cells) from infection with different HCoV species. HEL-299 was found to be permissive to HCoV-229E, -OC43 and MERS-CoV-GFP but not to HCoV-NL63 or SARS-CoV-2. Further investigation revealed that HEL-299 does not contain the required ACE2 receptor to enable propagation of both HCoV-NL63 and SARS-CoV-2. Following 24h exposure, pIC+NDX was observed to stimulate a significant, prolonged increase in antiviral gene expression (IFNß, CXCL10 and ISG15) when compared to both NDX alone and pIC alone. This antiviral response translated into complete protection against virus production, for 4 days or 7 days post treatment with HCoV-229E or -OC43 when either pre-treated for 6h or 24h respectively. Moreover, the pIC+NDX combination also provided complete protection for 2d post infection when HEL-299 cells were infected with MERS-CoV-GFP following a 24h pretreatment with pIC+NDX. The significance of this study is two-fold. Firstly, it was revealed that HEL-299 cells can effectively be used as an IFN-competent model system for in vitro analysis of MERS-CoV. Secondly, pIC+NDX acts as a powerful inducer of type I IFNs in HEL-299, to levels that provide complete protection against coronavirus replication. This suggests an exciting and novel area of investigation for antiviral therapies that utilize innate immune stimulants. The results of this study will help to expand the range of available tools scientists have to investigate, and thus further understand, human coronaviruses.

Oral delivery of a dsRNA-Phytoglycogen nanoparticle complex enhances both local and systemic innate immune responses in rainbow trout.

Salmonids are one of the most farmed fish species worldwide. These aquatic vertebrates rely heavily on their innate immune responses as the first line of defense to defend themselves against invading pathogens. Although commercial vaccines are available against some viral and bacterial pathogens affecting salmonids, their protective efficacy varies. Using a prophylactic inducer of local and systemic innate immune responses to limit infection could have significant implications in salmonid aquaculture. A potent inducer of innate immune responses in fish is double-stranded RNA (dsRNA), a molecule that all viruses make during their replicative cycle. Polyinosinic: polycytidylic acid (polyI:C) is a synthetic dsRNA commonly used to induce type I interferons (IFNs), interferon stimulated genes (ISGs) as well as an antiviral state in vertebrate species. Based on in vitro data it was hypothesized that both local and systemic innate immune responses, in salmonids, would be enhanced by orally delivering high molecular weight polyI:C (HMW polyI:C) using cationic phytoglycogen nanoparticles (NPs) as a delivery method. The present study investigates this hypothesis using two feed delivery methods. In the first in vivo study, to ensure an equal distribution of dose, individual rainbow trout (Oncorhynchus mykiss) were orally gavaged with feed moistened with a solution containing HMW-NP (polyI:C complexed with cationic phytoglycogen nanoparticles) or HMW polyI:C alone. In a second in vivo experiment, to better mimic a more realistic feeding scenario, rainbow trout were fed feed pellets to which HMW, or HMW-NP was added. The expression of IFN1 and ISGs (vig-3, Mx1) were quantified using real-time PCR in the intestine (local response) and head kidney (systemic response). The results of these studies indicate that HMW-NP induced a higher level of IFN1 and ISG expression in the intestine and head kidney compared to the HMW fed fish. The results of this study could lead to new advances in therapeutics for the aquaculture industry by utilizing the innate immune response against invading pathogens using an orally delivered stimulant.

Comparative Susceptibility of Madin-Darby Canine Kidney (MDCK) Derived Cell Lines for Isolation of Swine Origin Influenza A Viruses from Different Clinical Specimens.

Madin-Darby canine kidney (MDCK) cells are commonly used for the isolation of mammalian influenza A viruses. The goal of this study was to compare the sensitivity and suitability of the original MDCK cell line in comparison with MDCK-derived cell lines, MDCK.2, MDCK SIAT-1 and MDCK-London for isolation of swine-origin influenza A viruses (IAV-S) from clinical specimens. One-hundred thirty clinical specimens collected from pigs in the form of nasal swabs, lung tissue and oral fluids that were positive by PCR for the presence of IAV-S RNA were inoculated in the cell cultures listed above. MDCK-SIAT1 cells yielded the highest proportion of positive IAV-S isolations from all specimen types. For nasal swabs, 58.62% of the specimens were IAV-S positive in MDCK-SIAT1 cells, followed by MDCK-London (36.21%), and conventional MDCK and MDCK.2 cells (27.5%). For lung specimens, 59.38% were IAV-S positive in MDCK-SIAT1 cells, followed by MDCK-London (40.63%), and conventional MDCK and MDCK.2 cells (18.75-31.25%). Oral fluids yielded the lowest number of positive virus isolation results, but MDCK-SIAT1 cells were still had the highest rate (35%) of IAV-S isolation, whereas the isolation rate in other cells ranged from 5-7.5%. Samples with lower IAV-S PCR cycle threshold (Ct) values were more suitable for culturing and isolation. The isolated IAV-S represented H1N1-ß, H1N2-α, H1N1pdm and H3N2 cluster IV and cluster IVB viruses. The result of the current study demonstrated the importance of using the most appropriate MDCK cells when isolating IAV-S from clinical samples.

Phytoglycogen Nanoparticle Delivery System for Inorganic Selenium Reduces Cytotoxicity without Impairing Selenium Bioavailability.

PURPOSE: Selenium is an essential trace element that supports animal health through the antioxidant defense system by protecting cells from oxidative-related damage. Using inorganic selenium species, such as sodium selenite (Na Sel), as a food supplement is cost-effective; however, its limitation as a nutritional supplement is its cytotoxicity. One strategy to mitigate this problem is by delivering inorganic selenium using a nanoparticle delivery system (SeNP). METHODS: Rainbow trout intestinal epithelial cells, bovine turbinate cells and bovine intestinal myofibroblasts were treated with soluble Na Sel or SeNPs. Two SeNP formulations were tested; SeNP-Ionic where inorganic selenium was ionically bound to cationic phytoglycogen (PhG) NPs, and SeNP-Covalent, where inorganic selenium was covalently bound to PhG NPs. Selenium-induced cytotoxicity along with selenium bioavailability were measured. RESULTS: SeNPs (SeNP-Ionic or SeNP-Covalent) substantially reduced cytotoxicity in all cell types examined compared to similar doses of soluble inorganic selenium. The SeNP formulations did not affect selenium bioavailability, as selenium-induced glutathione peroxidase (GPx) activity and GPx1 transcript levels were similarly elevated whether cells were treated with soluble Na Sel or SeNPs. This was the case for all three cell types tested. CONCLUSION: Nanoparticle-assisted inorganic selenium delivery, which demonstrated equal bioavailability without causing deleterious cytotoxic side effects, has potential applications for safely supplementing animal diets with inorganic selenium at what are usually toxic doses.

Enhancing innate antiviral immune responses in rainbow trout by double stranded RNA delivered with cationic phytoglycogen nanoparticles.

Innate immunity is induced when pathogen-associated molecular patterns (PAMPs) bind host pattern recognition receptors (PRRs). Polyinosinic:polycytidylic acid [poly(I:C)] is a synthetic analogue of viral dsRNA that acts as a PAMP, inducing type I interferons (IFNs) in vertebrates. In the present study, the immunostimulatory effects of high molecular weight (HMW) poly(I:C) in rainbow trout cells were measured when bound to a cationic phytoglycogen nanoparticle (Nano-HMW). The physical characteristics of the nanoparticle itself, when bound to different lengths of dsRNA and when cell associated was evaluated. Optimal concentration and timing for innate immune stimulation was measured using the RTG-P1 reporter cell line. The immunostimulatory effects of HMW poly (I:C) was compared to Nano-HMW in vitro using the RTgutGC cell line cultured in a conventional monolayer or a transwell culture system. The ability of an activated intestinal epithelium to transmit an antiviral signal to macrophages was evaluated using a co-culture of RTgutGC cells and RTSll (a monocyte/macrophage cell). In all culture conditions, Nano-HMW was a more effective inducer of IFN-related antiviral immune responses compared to HMW poly (I:C) alone. This study introduces the use of cationic phytoglycogen nanoparticles as a novel delivery system for immunomodulatory molecules to enhance immune responses in aquatic vertebrates.

Development of innate immunity in chicken embryos and newly hatched chicks: a disease control perspective.

Newly hatched chickens are confronted by a wide array of pathogenic microbes because their adaptive immune defences have limited capabilities to control these pathogens. In such circumstances, and within this age group, innate responses provide a degree of protection. Moreover, as the adaptive immune system is relatively naïve to foreign antigens, synergy with innate defences is critical. This review presents knowledge on the ontogeny of innate immunity in chickens pre-hatch and early post-hatch and provides insights into possible interventions to modulate innate responses early in the life of the bird. As in other vertebrate species, the chicken innate immune system which include cellular mediators, cytokine and chemokine repertoires and molecules involved in antigen detection, develop early in life. Comparison of innate immune systems in newly hatched chickens and mature birds has revealed differences in magnitude and quality, but responses in younger chickens can be boosted using innate immune system modulators. Functional expression of pattern recognition receptors and several defence molecules by innate immune system cells of embryos and newly hatched chicks suggests that innate responses can be modulated at this stage of development to combat pathogens. Improved understanding of innate immune system ontogeny and functionality in chickens is critical for the implementation of sound and safe interventions to provide long-term protection against pathogens. Next-generation tools for studying genetic and epigenetic regulation of genes, functional metagenomics and gene knockouts can be used in the future to explore and dissect the contributions of signalling pathways of innate immunity and to devise more efficacious disease control strategies.

Characterization of innate responses induced by in ovo administration of encapsulated and free forms of ligands of Toll-like receptor 4 and 21 in chicken embryos.

Toll-like receptors (TLRs) are a family of innate receptors that recognize pathogen-associated molecular patterns, including double-stranded RNA, CpG DNA and lipopolysaccharide (LPS). After interaction with their ligands, TLRs initiate innate responses that are manifested by activating cells and inducing expression of cytokines that help mediate adaptive immune responses. TLR ligands (TLR-Ls) have the potential to be used prophylactically (alone) or as vaccine adjuvants to promote host immunity. Encapsulating TLR-Ls in nanoparticles, such as Poly (d,l-lactic-co-glycolic acid), may prolong responses through sustained release of the ligands. PLGA nanoparticles protect encapsulated TLR-Ls from degradation and extend the half-life of these ligands by reducing their rapid removal from the body. In this study, encapsulated and free forms of LPS and CpG ODN were administered to embryonation day 18 (ED18) chicken embryos. Spleen, lungs and bursa of Fabricius were collected at 6, 18 and 48hour post-stimulation (hps) and cytokine gene expressions were evaluated using quantitative real-time PCR. Results indicate that both the free and encapsulated forms of LPS and CpG ODN induced innate immune responses in ED18 chicken embryos. Innate responses induced in embryos seem similar to those reported in mature chickens. Significant upregulation of cytokine genes generally occurred by 48hps. Further studies are needed to evaluate long term immunomodulatory effects of encapsulated TLR-Ls and their ability to mediate protection against pathogens of young chicks.

Interactions between lactobacilli and chicken macrophages induce antiviral responses against avian influenza virus.

Macrophages are an important cell type of the innate immune system that upon activation, can exert antiviral functions and also can induce virus-specific adaptive immune responses. Macrophage interaction with certain probiotic bacteria such as lactobacilli can enhance antiviral functions of these cells. We have previously shown that administration of lactobacilli to chickens can effectively augment immune response to vaccine antigens. Here, we investigated the effects of representative strains of three Lactobacillus species, L. acidophilus, L. reuteri and L. salivarius used alone or in combination, in enhancing antiviral activity of chicken macrophages against avian influenza virus in an in vitro model using MQ-NCSU cells. Treatment of macrophages with probiotic lactobacilli significantly enhanced the antiviral functions, as determined by the virus titration assay. We also found that lactobacilli stimulation of macrophages induced significantly higher expression of interleukin (IL)-1ß, interferon (IFN)- γ and IFN-α cytokine genes as well as interferon regulatory factor-7 (IRF7), 2',5'-oligoadenylate synthetase (OAS) and interferon-inducible transmembrane protein M3 (IFITM3) genes. Furthermore, macrophages that were treated with lactobacilli had significantly enhanced production of nitric oxide (NO) and IFN-γ protein as well as surface expression of the costimulatory molecule CD40. However, the antiviral and immunostimulatory effects of probiotic lactobacilli largely depended on the Lactobacillus species studied. Collectively, the results from our study using an in vitro model showed that certain Lactobacillus species can effectively augment antiviral responses in chicken macrophages.

The effects of in ovo administration of encapsulated Toll-like receptor 21 ligand as an adjuvant with Marek's disease vaccine.

Marek's Disease Virus (MDV) is the causative agent of a lymphoproliferative disease, Marek's disease (MD) in chickens. MD is only controlled by mass vaccination; however, immunity induced by MD vaccines is unable to prevent MDV replication and transmission. The herpesvirus of turkey (HVT) vaccine is one of the most widely used MD vaccines in poultry industry. Vaccines can be adjuvanted with Toll-like receptor ligands (TLR-Ls) to enhance their efficacy. In this study, we examined whether combining TLR-Ls with HVT can boost host immunity against MD and improve its efficacy. Results demonstrated that HVT alone or HVT combined with encapsulated CpG-ODN partially protected chickens from tumor incidence and reduced virus replication compared to the control group. However, encapsulated CpG-ODN only moderately, but not significantly, improved HVT efficacy and reduced tumor incidence from 53% to 33%. Further investigation of cytokine gene profiles in spleen and bursa of Fabricius revealed an inverse association between interleukin (IL)-10 and IL-18 expression and protection conferred by different treatments. In addition, the results of this study raise the possibility that interferon (IFN)-ß and IFN-γ induced by the treatments may exert anti-viral responses against MDV replication in the bursa of Fabricius at early stage of MDV infection in chickens.

In ovo administration of Toll-like receptor ligands encapsulated in PLGA nanoparticles impede tumor development in chickens infected with Marek's disease virus.

One of the economically important diseases in the poultry industry is Marek's disease (MD) which is caused by Marek's disease virus (MDV). The use of current vaccines provides protection against clinical signs of MD in chickens. However, these vaccines do not prevent the transmission of MDV to susceptible hosts, hence they may promote the development of new virulent strains of MDV. This issue persuaded us to explore alternative approaches to control MD in chickens. Induction of innate responses at the early stage of life in the chicken may help to prevent or reduce MDV infection. Further, prophylactic use of Toll-like receptor ligands (TLR-Ls) has been shown to generate host immunity against infectious diseases. In this regard, encapsulation of TLR-Ls in Poly(d, l-lactic-co-glycolic acid) (PLGA) may further enhance host responses by controlled release of TLR-Ls for an extended period. Hence, in the current study, protective effects of encapsulated TLR4 and TLR21 ligands, LPS and CpG, respectively, were investigated against MD. Results indicated that administration of encapsulated CpG and LPS first at embryonic day (ED) 18, followed by post-hatch at 14 days-post infection (dpi) intramuscularly, diminished tumor incidence by 60% and 42.8%, respectively at 21dpi compared to the MDV only group. In addition, analysis of cytokine gene profiles of interferon (IFN)-α, IFN-ß, IFN-γ, inducible nitric oxide synthase (iNOS), interleukin (IL)-1ß, IL-18 and IL-10 in spleen and bursa of Fabricius at different time points suggests that TLR-Ls possibly triggered host responses through the expression of IL-1ß and IL-18 to reduce tumor formation. However, further studies are needed to explore the role of these pro-inflammatory cytokines and other influencing elements like lymphocytes in the hindrance of tumor development by TLR-Ls treatment in chickens.

Characterization of Innate Responses Induced by PLGA Encapsulated- and Soluble TLR Ligands In Vitro and In Vivo in Chickens.

Natural or synthetic Toll-like receptor (TLR) ligands trigger innate responses by interacting with distinct TLRs. TLR ligands can thus serve as vaccine adjuvants or stand-alone antimicrobial agents. One of the limitations of TLR ligands for clinical application is their short half-life and rapid clearance from the body. In the current study, encapsulation of selected TLR ligands in biodegradable poly(D,L-lactide-co-glycolide) polymer nanoparticles (PLGA NPs) was examined in vitro and in vivo as a means to prolong innate responses. MQ-NCSU cells (a chicken macrophage cell line) were treated with encapsulated or soluble forms of TLR ligands and the resulting innate responses were evaluated. In most cases, encapsulated forms of TLR ligands (CpG ODN 2007, lipopolysaccharide and Pam3CSK4) induced comparable or higher levels of nitric oxide and cytokine gene expression in macrophages, compared to the soluble forms. Encapsulated CpG ODN, in particular the higher dose, induced significantly higher expression of interferon (IFN)-γ and IFN-ß until at least 18 hr post-treatment. Cytokine expression by splenocytes was also examined in chickens receiving encapsulated or soluble forms of lipopolysaccharide (a potent inflammatory cytokine inducer in chickens) by intramuscular injection. Encapsulated LPS induced more sustained innate responses characterized by higher expression of IFN-γ and IL-1ß until up to 96 hr. The ability of TLR ligands encapsulated in polymeric nanoparticles to maintain prolonged innate responses indicates that this controlled-release system can extend the use of TLR ligands as vaccine adjuvants or as stand-alone prophylactic agents against pathogens.

Delivery of an inactivated avian influenza virus vaccine adjuvanted with poly(D,L-lactic-co-glycolic acid) encapsulated CpG ODN induces protective immune responses in chickens.

In poultry, systemic administration of commercial vaccines consisting of inactivated avian influenza virus (AIV) requires the simultaneous delivery of an adjuvant (water-in-oil emulsion). These vaccines are often limited in their ability to induce quantitatively better local (mucosal) antibody responses capable of curtailing virus shedding. Therefore, more efficacious adjuvants with the ability to provide enhanced immunogenicity and protective anti-AIV immunity in chickens are needed. While the Toll-like receptor (TLR) 21 agonist, CpG oligodeoxynucleotides (ODNs) has been recognized as a potential vaccine adjuvant in chickens, poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles, successfully tested as vaccine delivery systems in other species, have not been extensively explored. The present study, therefore, assessed both systemic and mucosal antibody-mediated responses following intramuscular vaccination (administered at 7 and 21days post-hatch) of chickens with PLGA encapsulated H9N2 AIV plus encapsulated CpG ODN 2007 (CpG 2007), and nonencapsulated AIV plus PLGA encapsulated CpG 2007 vaccine formulations. Virus challenge was performed at 2weeks post-secondary vaccination using the oculo-nasal route. Our results showed that chickens vaccinated with the nonencapsulated AIV vaccine plus PLGA encapsulated CpG 2007 developed significantly higher systemic IgY and local (mucosal) IgY antibodies as well as haemagglutination inhibition antibody titres compared to PLGA encapsulated AIV plus encapsulated CpG 2007 vaccinated chickens. Furthermore, chickens that received CpG 2007 as an adjuvant in the vaccine formulation had antibodies exhibiting higher avidity indicating that the TLR21-mediated pathway may enhance antibody affinity maturation qualitatively. Collectively, our data indicate that vaccination of chickens with nonencapsulated AIV plus PLGA encapsulated CpG 2007 results in qualitatively and quantitatively augmented antibody responses leading to a reduction in virus shedding compared to the encapsulated AIV plus PLGA encapsulated CpG 2007 formulation.

Characterization of Immune Responses to an Inactivated Avian Influenza Virus Vaccine Adjuvanted with Nanoparticles Containing CpG ODN.

Avian influenza virus (AIV), a mucosal pathogen, gains entry into host chickens through respiratory and gastrointestinal routes. Most commercial AIV vaccines for poultry consist of inactivated, whole virus with adjuvant, delivered by parenteral administration. Recent advances in vaccine development have led to the application of nanoparticle emulsion delivery systems, such as poly (d,l-lactic-co-glycolic acid) (PLGA) nanoparticles to enhance antigen-specific immune responses. In chickens, the Toll-like receptor 21 ligand, CpG oligodeoxynucleotides (ODNs), have been demonstrated to be immunostimulatory. The objective of this study was to compare the adjuvant potential of CpG ODN 2007 encapsulated in PLGA nanoparticles with nonencapsulated CpG ODN 2007 when combined with a formalin-inactivated H9N2 virus, through intramuscular and aerosol delivery routes. Chickens were vaccinated at days 7 and 21 posthatch for the intramuscular route and at days 7, 21, and 35 for the aerosol route. Antibody-mediated responses were evaluated weekly in sera and lacrimal secretions in specific pathogen-free chickens. The results indicate that nonencapsulated CpG ODN 2007 in inactivated AIV vaccines administered by the intramuscular route generated higher antibody responses compared to the encapsulated CpG ODN 2007 formulation by the same route. Additionally, encapsulated CpG ODN 2007 in AIV vaccines administered by the aerosol route elicited higher mucosal responses compared to nonencapsulated CpG ODN 2007. Future studies may be aimed at evaluating protective immune responses induced with PLGA encapsulation of AIV and adjuvants.

Systemic immune responses to an inactivated, whole H9N2 avian influenza virus vaccine using class B CpG oligonucleotides in chickens.

Commercial vaccines against avian influenza viruses (AIV) in chickens consist mainly of inactivated AIV, requiring parenteral administration and co-delivery of an adjuvant. Limitations in T helper 1 or T helper 2 biased responses generated by these vaccines emphasize the need for alternative, more efficacious adjuvants. The Toll-like receptor (TLR) 21 ligand, CpG oligodeoxynucleotides (ODN), has been established as immunomodulatory in chickens. Therefore, the objective of this study was to investigate the adjuvant potential of high (20µg) and low (2µg) doses of CpG ODN 2007 (CpG 2007) and CpG ODN 1826 (CpG 1826) when administered to chickens with a formalin-inactivated H9N2 AIV. Antibody responses in sera were evaluated in 90 specific pathogen free (SPF) chickens after intramuscular administration of vaccine formulations at 7 and 21 days post-hatch. Antibody responses were assessed based on haemagglutination inhibition (HI) and virus neutralization (VN) assays; virus-specific IgM and IgY antibody responses were evaluated by ELISA. The results suggest that the vaccine formulation containing low dose CpG 2007 was significantly more effective at generating neutralizing (both HI and VN) responses than formulations with high or low doses of CpG 1826 or high dose CpG 2007. Neutralizing responses elicited by low dose CpG 2007 significantly exceeded those generated by a squalene-based adjuvanted vaccine formulation during peak responses. A significantly higher IgM response was elicited by the formulation containing low dose CpG 2007 compared to high and low doses of 1826. Although the low dose of CpG 2007 elicited a higher IgY response than CpG 1826, the difference was not statistically significant. In conclusion, 2µg of CpG 2007 is potentially promising as a vaccine adjuvant when delivered intramuscularly with inactivated H9N2 virus to chickens. Future studies may be directed at determining the mucosal antibody responses to the same vaccine formulations.