Immunogenicity In Chickens Research Articles

Efficiency of natural oils as alternative adjuvants to mineral oils in inactivated avian influenza vaccine formulation

ABSTRACT 1. The effectiveness of inactivated vaccines depends on selecting the suitable adjuvant for vaccine formulation. The potency of vaccines with low antigen content can be improved with the appropriate adjuvant. This could allow production of more doses and lower the production cost. 2. This study evaluated the efficiency of vaccines prepared using oil extracted from natural sources including argan oil, almond oil, sesame seed oil, pumpkin oil, cactus oil and black seed oil as alternative adjuvants for improving the protection capacity of inactivated influenza virus vaccine as compared to commonly used mineral oils. 3. Each vaccine formulation was evaluated for stability, safety and immunogenicity in chickens, as well as for reducing the viral shedding after challenge infection. 4. The cactus, sesame and pumpkin seed oil-based vaccines were found to be potent and successfully induced the production of humoral immunity in vaccinated chickens.

The Immunogenicity of a Novel Chimeric Hemagglutinin-Neuraminidase-Fusion Antigen from Newcastle Disease Virus by Oral Delivery of Transgenic Canola Seeds to Chickens.

Newcastle disease (ND) is considered as one of the most devastating infectious diseases targeting domestic birds and has considerable threat to the commercial poultry production. Two surface glycoproteins, hemagglutinin-neuraminidase (HN) and fusion (F), act as antigens in the virus structure and also play important roles in infecting host cells. In the current study, the expression of the chimeric HN-F protein in canola seeds and its immunogenicity in chickens were investigated. The HN-F gene was cloned downstream of the fatty acid elongase 1 (FAE1) promoter in the binary expression vector, pBI1400-HN-F, and introduced into rapeseed (Brassica napus L.) using Agrobacterium-mediated transformation. The amount of the HN-F glycoprotein was estimated up to 0.18% and 0.11% of the total soluble protein (TSP) in transgenic seeds and leaves of canola, respectively. Confirmatory analyses of 36 transgenic lines revealed that the HN-F gene was integrated into the genome. Subsequently, HN-F protein could be expressed and accumulated in the seed tissue. Specific pathogen-free (SPF) chickens immunized orally with recombinant HN-F showed a significant rise in specific and hemagglutination inhibition (HI) antibodies 35 to 42days post the first administration. The results implied the potential of transgenic canola seed-based expression for oral delivery of NDV immunogenic glycoproteins.

Enhancement in humoral response against inactivated Newcastle disease vaccine in broiler chickens administered orally with plant-derived soyasaponin

The present study aimed to evaluate the immunopotentiating effect of plant-derived soyasaponin and its immunogenicity in chickens challenged with Newcastle disease virus (NDV). Soyasaponin was extracted from soybean seeds and detected using the phytochemical tests, followed by quantification through the dry-weight method. One-day-old broiler chicks (n = 90) were divided into 3 groups, named as A, B, and C. Group A birds were orally administrated with soyasaponin (5 mg/kg), followed by immunization with inactivated ND vaccine intramuscularly (IM), whereas group B birds were vaccinated with inactivated ND vaccine alone. Group C birds were kept unvaccinated. A booster dose on day 21 was also administered IM to group A and B birds. At day 35, all 3 groups were challenged with NDV. To determine the immunogenicity potential of soyasaponin, antibody titer was measured using the hemagglutination inhibition test before and after the NDV challenge. Histochemical examination was performed to determine the pathological changes associated with NDV infection. Foam formation and hemolytic activity confirmed the presence of saponin in soya bean extract. Group A birds showed a higher antibody response compared with group B and C birds. The disease challenge study showed that soyasaponin-adjuvanted NDV vaccine provided complete protection to group A birds against ND. Moreover, no side effects of soyasaponin were observed on the growth performance of birds during the experiment. Therefore, we can conclude that soyasaponin is a potential immunogenic agent and therefore could be a promising candidate to launch a protective humoral response against ND in chickens.

Molecular characterization of fowl adenovirus isolate of Malaysia attenuated in chicken embryo liver cells and its pathogenicity and immunogenicity in chickens.

Fowl adenovirus (FAdV) is the causative agent of inclusion body hepatitis (IBH) in chickens with significant economic losses due to high mortality and poor production. It was objectives of the study to attenuate and determine the molecular characteristic of FAdV isolate (UPM1137) of Malaysia passages in primary chicken embryo liver (CEL) cells. The cytopathic effect (CPE) was recorded and the present of the virus was detected by polymerase chain reaction (PCR). Nucleotide and amino acid changes were determined and a phylogenetic tree was constructed. The pathogenicity and immunogenicity of the virus at passage 35 (CEL35) with virus titre of 106.7TCID50/mL was determined in day old specific pathogen free (SPF) chicks via oral or subcutaneous route of inoculation. The study demonstrated that the FAdV isolate was successfully propagated and attenuated in CEL cells up to 35th consecutive passages (CEL35) with delayed of CPE formation within 48 to 72 post inoculation (pi) from CEL20 onwards. The virus caused typical CPE with basophilic intranuclear inclusion bodies, refractile and clumping of cells. The virus is belong to serotype 8b with substitution of amino acid at position 44, 133 and 185 in L1 loop of hexon gene and in knob of fiber gene at position 348 and 360 at CEL35. It is non-pathogenic, but immunogenic in SPF chickens. It was concluded that the FAdV isolate was successfully attenuated in CEL cells with molecular changes in major capsid proteins which affect its infectivity in cell culture and SPF chickens.

Development of novel subunit vaccine based on truncated fiber protein of egg drop syndrome virus and its immunogenicity in chickens

Egg-drop syndrome virus (EDSV) is an avian adenovirus that causes markedly decrease in egg production and in the quality of the eggs when it infects chickens. In this report, we engineered truncated fiber protein containing the entire knob domain and part of the shaft region as a vaccine candidate. The protein was obtained in the soluble fraction in Escherichia coli (E. coli), and expression level after nickel-affinity purification was 126 mg/L. By means of multiple characterization methods, it is demonstrated that the recombinant protein retains the native trimeric structure. A single inoculation with the structure-stabilized recombinant protein, even at the lowest dose of 2 μg, stimulated hemagglutination inhibition (HI) antibody responses in chickens, for at least 16 weeks. Neutralizing titers in sera from the protein immunized groups was similar to that of inactivated vaccine immunized group. The lymphocyte proliferation response and cytokine secretion were also induced in immunized SPF chickens. In addition, immunization with the fiber protein also significantly reduced the viral load in the liver. Taken together, these results suggest the truncated fiber protein as an effective single dose, long lasting and rapidly effective vaccine to protect against EDSV.

Saponin-adjuvanted vaccine protects chickens against velogenic Newcastle disease virus.

Despite extensive vaccination campaigns, Newcastle disease virus (NDV) remains endemic in many countries worldwide, and factors that contribute to this failure include mismatched vaccines, partial immunization, and poor husbandry practices. In order to overcome the problem of genetic divergence between circulating field strains and vaccine strains, we saponin-adjuvanted an Egyptian field strain and assessed its safety and immunogenicity in chickens. Immunization of chickens with the vaccine followed by challenge with a velogenic reference strain revealed the potential of the saponin-adjuvanted vaccine to induce a strong immune response that resulted in complete protection of chickens. Importantly, in vaccinated chickens, virus shedding was abolished, providing an added advantage over the currently available commercial live-attenuated and inactivated vaccines, which are unable to prevent shedding. A histopathological investigation demonstrated that the vaccinated chickens had less-severe lesions than challenged unvaccinated and mock-vaccinated chickens. We propose using this formulation as an alternative and improved NDV vaccine platform that can be exploited to control disease not only in Egypt but also in other disease-endemic countries.

Immunogenicity in chickens with orally administered recombinant chicken-borne Lactobacillus saerimneri expressing FimA and OmpC antigen of O78 avian pathogenic Escherichia coli.

Avian colibacillosis is responsible for economic losses to poultry producers worldwide. To combat this, we aimed to develop an effective oral vaccine for chicken against O78 avian pathogenic Escherichia coli (APEC) infection through a Lactobacillus delivery system. Eight Lactobacillus strains isolated from the intestines of broiler chickens were evaluated based on their in vitro adherence ability to assess their potential as a delivery vector. Fimbrial subunit A (FimA) and outer-membrane protein C (OmpC) of APEC with and without fusion to dendritic cell-targeting peptide (DCpep) and microfold cell-targeting peptide (Co1) were displayed on the surface of Lactobacillus saerimneri M-11 and yielded vaccine groups (pPG-ompC-fimA/M-11 and pPG-ompC-fimA-Co1-DCpep/M-11, respectively). The colonization of the recombinant strains in vivo was assessed and the immunogenicity and protective efficacy of orally administered recombinant strains in chickens were evaluated. The colonization of the recombinant strains in vivo revealed no significant differences between the recombinant and wild-type strains. Chickens orally administered with vaccine groups showed significantly higher levels of OmpC/FimA-specific IgG in serum and mucosal IgA in cecum lavage, nasal lavage and stool compared to the pPG/M-11 group. After challenge with APEC CVCC1553, better protective efficacy was observed in chickens orally immunized with pPG-ompC-fimA/M-11 and pPG-ompC-fimA-Co1-DCpep/M-11, but no significant differences were observed between the two groups. Recombinant chicken-borne L. saerimneri M-11 showed good immunogenicity in chickens, suggesting that it may be a promising vaccine candidate against APEC infections. However, the activity of mammalian DCpep and Co1 was not significant in chickens.

Immunologic evaluation of 10 different adjuvants for use in vaccines for chickens against highly pathogenic avian influenza virus

Avian influenza viruses (AIV) are a threat to poultry production worldwide. Vaccination is utilized as a component of control programs for both high pathogenicity (HP) and low pathogenicity (LP) AIV. Over 95% of all AIV vaccine used in poultry are inactivated, adjuvanted products. To identify the best formulations for chickens, vaccines were prepared with beta-propiolactone (BPL) inactivated A/British Columbia/314514-1/2004 H7N3 LP AIV using ten commercially available or experimental adjuvants. Each vaccine formulation was evaluated for immunogenicity in chickens. Challenge studies with an antigenically homologous strain of HPAIV were conducted to compare protection against mortality and measure reductions in virus levels in oral swabs. The four best adjuvants from the studies with BPL inactivated antigen were selected and tested identically, but with vaccines prepared from formalin inactivated virus. Mineral and vegetable oil based adjuvants generally induced the highest antibody titers with 100% seroconversion by 3weeks post vaccination. Chitosan induced positive antibody titers in 100% of the chickens, but the titers were significantly lower than those of most of the oil based adjuvants. Antibody levels from calcium phosphate and alginate adjuvanted groups were similar to those of non-adjuvanted virus. All groups that received adjuvanted vaccines induced similar levels of protection against mortality (0–20%) except the groups vaccinated with calcium phosphate adjuvanted vaccines, where mortality was similar (70%) to groups that received non-adjuvanted inactivated virus or no vaccine (60–100% mortality). Virus shedding in oral swabs was variable among the treatment groups. Formalin inactivated vaccine induced similar antibody titers and protection against challenge compared to BPL inactivated vaccine groups. These studies support the use of oil adjuvanted vaccines for use in the poultry industry for control for AIV.

Recombinant Hemagglutinin of Avian Influenza Virus H5 Expressed in the Chloroplast of Chlamydomonas reinhardtii and Evaluation of Its Immunogenicity in Chickens.

Globally, avian influenza (AI) is a serious problem in poultry farming. Despite vaccination, the prevalence of AI in México highlights the need for new approaches to control AI and to reduce the economic losses associated with its occurrence in susceptible birds. Recombinant proteins from avian influenza virus (AIV) have been expressed in different organisms, such as plants. The present study investigated the feasibility of designing and expressing the HA protein of AIV in the transplastomic microalga Chlamydomonas reinhardtii as a novel approach for AIV control and taking advantage of culture conditions, its reproductive range, and safe use in consideration of the generally regarded as safe food ingredient regulatory classification. The results showed that the HA protein of AIV in C. reinhardtii presents antigenic activity by western blot test and through its application in chickens, demonstrating its feasibility as a recombinant antigen against AIV.

Immunization with live nonpathogenic H5N3 duck influenza virus protects chickens against highly pathogenic H5N1 virus.

Development of an effective, broadly-active and safe vaccine for protection of poultry from H5N1 highly pathogenic avian influenza viruses (HPAIVs) remains an important practical goal. In this study we used a low pathogenic wild aquatic bird virus isolate А/duck/Moscow/4182/2010 (H5N3) (dk/4182) as a live candidate vaccine. We compared this virus with four live 1:7 reassortant anti-H5N1 candidate vaccine viruses with modified hemagglutinin from either A/Vietnam/1203/04 (H5N1) or A/Kurgan/3/05 (H5N1) and the rest of the genes from either H2N2 cold-adapted master strain A/Leningrad/134/17/57 (rVN-Len and rKu-Len) or H6N2 virus A/gull/Moscow/3100/2006 (rVN-gull and rKu-gull). The viruses were tested in parallel for pathogenicity, immunogenicity and protective effectiveness in chickens using aerosol, intranasal and oral routes of immunization. All five viruses showed zero pathogenicity indexes in chickens. Viruses rVN-gull and rKu-gull were immunogenic and protective, but they were insufficiently attenuated and caused significant mortality of 1-day-old chickens. The viruses with cold-adapted backbones (rVN-Len and rKu-Len) were completely nonpathogenic, but they were significantly less immunogenic and provided lower protection against lethal challenge with HPAIV A/Chicken/Kurgan/3/05 (H5N1) as compared with three other vaccine candidates. Unlike other four viruses, dk/4182 was both safe and highly immunogenic in chickens of any age regardless of inoculation route. Single administration of 106 TCID50 of dk/4182 virus via drinking water provided complete protection of 30-days-old chickens from 100 LD50 of the challenge virus. Our results suggest that low pathogenic viruses of wild aquatic birds can be used as safe and effective live poultry vaccines against highly pathogenic avian viruses.

Generation of a chickenized catalytic anti-nucleic acid antibody by complementarity-determining region grafting

In contrast to a number of studies on the humanization of non-human antibodies, the reshaping of a non-human antibody into a chicken antibody has never been attempted. Therefore, nothing is known about the animal species-dependent compatibility of the framework regions (FRs) that sustain the appropriate conformation of the complementarity-determining regions (CDRs). In this study, we attempted the reshaping of the variable domains of the mouse catalytic anti-nucleic acid antibody 3D8 (m3D8) into the FRs of a chicken antibody (“chickenization”) by CDR grafting, which is a common method for the humanization of antibodies. CDRs of the acceptor chicken antibody that showed a high homology to the FRs of m3D8 were replaced with those of m3D8, resulting in the chickenized antibody (ck3D8). ck3D8 retained the biochemical properties (DNA binding, DNA hydrolysis, and cellular internalizing activities) and three-dimensional structure of m3D8 and showed reduced immunogenicity in chickens. Our study demonstrates that CDR grafting can be applied to the chickenization of a mouse antibody, probably due to the interspecies compatibility of the FRs.

Identification and Expression of a Gene Encoding an Epitope that Induces Hemagglutination Inhibition Antibody to Avibacterium paragallinarum Serovar A

The aims of this study were the identification, cloning, and expression of a genetic region encoding an epitope that induces hemagglutination inhibition (HI) antibody against Avibacterium paragallinarum serovar A and an evaluation of the recombinant protein for immunogenicity in chickens. Although two monoclonal antibodies (MAbs) with HI activity, designated S24-951 and S7-1716-5C, were generated in this study, no reactive proteins with both MAbs were identified by Western blot analysis. A gene fragment of 5157 bp, designated hpa5. 1, was cloned from genomic DNA, and a recombinant protein expressed by hpa5.1, designated HPA5.1, reacted with both MAbs on dot-blot analysis. HPA5.1 showed no hemagglutinating activity, but significantly absorbed HI antibodies in the chicken immune serum. Analysis using a series of deletion mutants prepared from hpa5.1 indicated that a 4.8 kbp gene in hpa5.1 is essential for the expression epitope recognized by MAb S24-951. In addition, chickens immunized once with HPA5.1 showed a high protection rate with sufficient HI antibody titers against challenge exposure with a virulent strain of A. paragallinarum serovar A strain 221. These results show that hpa5. I1 is responsible for the expression of an epitope that induces HI antibody, and HPA5.1 might be a candidate for the development of a new vaccine against avian infectious coryza caused by A. paragallinarum serovar A.

Identification and immunogenicity of an immunodominant mimotope of Avibacterium paragallinarum from a phage display peptide library

Avibacterium paragallinarum is the causative agent of infectious coryza. The protective antigens of this important pathogen have not yet been clearly identified. In this paper, we applied phage display technique to screen the immunodominant mimotopes of a serovar A strain of A. paragallinarum by using a random 12-peptide library, and evaluated the immunogenicity in chickens of the selected mimotope. Polyclonal antibody directed against A. paragallinarum strain 0083 (serovar A) was used as the target antibody and phage clones binding to this target were screened from the 12-mer random peptide library. More than 50% of the phage clones selected in the third round carried the consensus peptide motif sequence A-DP(M)L. The phage clones containing the peptide motif reacted with the target antibody and this interaction could be blocked, in a dose-dependent manner, by A. paragallinarum. One of the peptide sequences, YGLLAVDPLFKP, was selected and the corresponding oligonucleotide sequence was synthesized and then inserted into the expression vector pFliTrx. The recombinant plasmid was transferred into an expression host Escherichia coli GI826 by electroporation, resulting in a recombinant E. coli expressing the peptide on the bacterial surface. Intramuscular injection of the epitope-expressing recombinant bacteria into chickens induced a specific serological response to serovar A. A. paragallinarum. The chickens given the recombinant E. coli showed significant protection against challenge with A. paragallinarum 0083. These results indicated a potential for the use of the mimotope in the development of molecular vaccines for infectious coryza.

Generation of the transgenic potato expressing full-length spike protein of infectious bronchitis virus

To seek a new delivery system of vaccine for infectious bronchitis virus (IBV), transgenic potato expressing full-length spike (S) protein of IBV was produced and its immunogenicity in chickens was investigated. One to three copies of S gene of IBV were randomly and stably inserted into potato ( Solanum tuberrosum cv. Dongnong 303) genome by Agrobacterium tumefaciens-mediated transformation. Transcription and translation of S gene for IBV were confirmed by Northern blot and Western blot analyses in transgenic plantlets. Chickens immunized orally and intramuscularly with transgenic potato tubers expressing S protein generated the detectable levels of serum neutralizing antibodies and were protected against the challenge with the virulent IBV. In vitro secretion of interleukin 2 and T lymphocyte proliferation of spleen cells from the immunized chickens varied with the dose and the manner of vaccination with S protein derived from transgenic plants. The results indicated that S protein expressed in transgenic plants might be a new source for the production of Coronaviridae IBV vaccine.

Pathogenicity and immunogenicity in chickens of Newcastle disease viruses isolated from wild ducks. Brief report.

Three Newcastle disease viruses (NDV) isolated from wild ducks in Japan were evaluated for their biological activities, pathogenicity and immunogenicity against one-day-old chickens. One isolate was of the mesogenic type and the other two were of the lentogenic type for chicken. The lentogenic isolates could induce enough immunity in chickens to protect them from challenge with a virulent strain of NDV.

Expression in Escherichia coli of cDNA fragments encoding the gene for the host-protective antigen of infectious bursal disease virus

The larger segment of the IBDV genome codes for a 32-kDa host-protective antigen. Inserts from a cDNA library in pBR 322, containing overlapping cDNA fragments of varying sizes and covering the entire large segment of the IBDV genome, were subcloned into a mixture of expression vectors pUR 290, 291, and 292. Clones expressing the host-protective antigen, or parts of it, were identified by an immunoblot assay and the fusion proteins were further characterized by Western blot analysis using a monoclonal antibody specific for the 32-kDa polypeptide. Hybridization of inserts from expressing clones to the original cDNA library led to the identification of the region of the IBDV genome that codes for the 32-kDa host-protective antigen. Clone D1 which encodes approximately 50% and clone D6 which encodes the entire 32-kDa protein were selected for further studies. The fusion proteins from clones D1 and D6 were affinity purified and tested for their immunogenicity in chickens. Both fusion proteins induced the synthesis of antibodies in both primed and unprimed chickens that reacted specifically with denatured 32-kDa viral protein, but less well with intact virus. It was concluded that the response to the fusion proteins was to linear rather than conformational epitopes on the 32-kDa viral protein.

Immune Response of Chickens to Six Strains of Newcastle Disease Virus as Measured by Hemagglutination-Inhibition Test

SUMMARY Six strains of beta-propiolactone-inactivated Newcastle disease virus were compared for their immunogenicity in chickens. Texas GB, Kansas-Manhattan, and B1-Hitchner produced significantly higher HI antibody than NJ-LaSota, Mass-MK-107, and California 11914 strains of NDV, with each group performing about the same. Hanson et al. (3), in a comparison of the immunogenicity of five strains of Newcastle disease virus (NDV) as formalized antigens, showed that chickens immunized with Manhattan-Kansas and Roakin-NJ were more resistant to challenge than chickens receiving GB-Texas, RO and B1 strains. Sullivan et al. (4), using beta-propiolactone-killed preparations of three strains of NDV, demonstrated that a single injection of GB-Texas afforded better protection than a single injection of Roakin or Manhattan-Kansas. After a booster injection, however, chickens receiving any of the three strains showed essentially the same resistance to challenge. A study was made to compare the immune response of chickens to 6 strains of beta-propiolactone-inactivated NDV and to determine whether strain virulence could be correlated with antigenicity. To circumvent the difficulties encountered by other investigators, the quantity of antigen in each strain of virus used for vaccination was standardized and the serum from individual birds was tested for antibody. The hemagglutination-inhibition (HI) antibody titers were then compared both within and between strains of NDV to determine their significance.