Immunomodulatory in vitro effects of Trichinella cystatin-like protein on mouse splenocytes.

Trichinella parasites have developed specific mechanisms allowing successful completion of their life cycle. These mechanisms are in a great part involved in immunomodulation and studying them may provide a valuable insight into the functioning of the immune system. Trichinella products may be also used as potential therapeutic agents to treat immune diseases. This study investigates the immunomodulatory potential of recombinant multi cystatin-like protein (CLP) derived from T. britovi to determine whether CLP has anti-inflammatory properties in vitro. CLP is a highly antigenic glycoprotein present in Trichinella excetory-secretory (ES) products. AlphaFold structure prediction confirms that it consists of three type-two cystatin-like domains. Mouse splenocytes were stimulated in vitro with lipopolysaccharide (LPS) and co-stimulated with recombinant CLP. The culture supernatants were collected and tested for secreted cytokine levels using ELISA. CLP was found to reduce LPS-induced secretion of inflammatory cytokines TNFα and IL-6. On the contrary, in some experimental groups, co-stimulation with CLP resulted in increased secretion of the regulatory cytokine IL-10. The obtained results indicate that CLP has anti-inflammatory properties and future research on its function is advisable, specifically in the context of the therapy of inflammatory disorders.

Trichinella britovi recombinant proteins produced in Pichia pastoris expression system for specific IgG antibody detection in the sera of mice and pigs infected with Trichinella spp.

Trichinellosis, a disease caused by infection with Trichinella spp, poses an economic problem in the animal sector and a recurrent health problem for humans. Discovering the new diagnostic tests may be achieved by identification and production of species- and stage-specific recombinant proteins of Trichinella genus which are recognized by the host antibodies after infection. In this study the T. britovi proteins identified earlier in excretory-secretory (ES) products: CTRL, ES21 and HSP20, were cloned and produced using a eukaryotic Pichia pastoris system. Their immunodiagnostic properties were verified by measuring the abundance of specific IgG antibodies in sera from mice and pigs experimentally infected with T. britovi or T. spiralis. The rTbCTRL and the rTbES21 proteins were more effectively produced and stable than rTbHSP20. The most sensitive protein for serodiagnostic purposes occurred to be CTRL; anti-rTbCTRL IgG level increased at 41 days post infection (dpi) in pigs infected with T. britovi and 45 dpi for those infected with T. spiralis. The rTbES21 protein was the most specific for the T. britovi species, as no antibody titers were observed in pigs infected with T. spiralis. Following the multiple-antigen strategy, the combination of rTbCTRL + rTbES21 was applied in ELISA, but no significant difference in IgG level was detected in the tested conditions.

The first analysis of Trichinella spiralis and Trichinella britovi adult worm excretory-secretory proteins by two-dimensional electrophoresis coupled with LC-MS/MS.

The two most common Trichinella species present in European countries are Trichinella spiralis and Trichinella britovi. The consumption of raw or undercooked meat with invasive larvae results in trichinellosis. Currently, the most commonly used sources for detecting specific anti-Trichinella antibodies is ELISA with the muscle larvae (ML) excretory-secretory (E-S) proteins. However, these serological methods cannot be efficiently applied at early stage of infection. The aim of the current study was to identify the common and species-specific E-S proteins of T. spiralis and T. britovi adult worms which could have potential for accurate diagnosis of Trichinella infection at an early stage of invasion. Different sets of immunoreactive proteins were identified in T. spiralis and T. britovi proteomes by a combination of two-dimensional electrophoresis (2-DE), immunoblot and LC-MS/MS analysis. Polyubiquitin-B, a possible enoyl-CoA hydratase/isomerase YngF or polyubiquitin-like protein was found to be common for both species; gene ontology analysis confirmed its involvement in proteolysis, oxidation-reduction and translation processes, as well as in molecular transport. These molecules, being secreted or excreted at an early stage of parasite development, may play a critical role in the processes occurring during the initial steps of the host invasion and hence be suitable for diagnostic test development.

Exploiting the potential of 2D DIGE and 2DE immunoblotting for comparative analysis of crude extract of Trichinella britovi and Trichinella spiralis muscle larvae proteomes.

The Trichinella genus poses an interesting puzzle for researchers, having diverged very early in the evolution of the nematodes. The Trichinella spiralis proteome is a cosmopolitan and well-studied model of Trichinella; however, Trichinella britovi also circulates in the sylvatic environment and both species infect humans, resulting in the development of trichinellosis. Few experiments have examined the proteins belonging to the T. britovi proteome. The aim of the present study was to compare the protein expression profiles of crude extracts of T. spiralis and T. britovi muscle larvae using a highly-sensitive two-dimensional differential in-gel electrophoresis (2D DIGE) technique coupled with 2DE immunoblotting. Selected immunoreactive protein spots were then identified by liquid chromatography coupled with mass spectrometry analysis (LC-MS/MS), and their function in Trichinella and the host-parasite interaction was determined by gene ontology analysis. Spots common to both T. spiralis and T. britovi, spots with different expressions between the two and spots specific to each species were labelled with different cyanine dyes. In total, 196 protein spots were found in both proteomes; of these 165 were common, 23 expressed exclusively in T. spiralis and 8 in T. britovi. A comparative analysis of volume ratio values with Melanie software showed that among the common spots, nine demonstrated higher expression in T. spiralis, and 17 in T. britovi. LC-MS/MS analysis of 11 selected spots identified 41 proteins with potential antigenic characteristics: 26 were specific for T. spiralis, six for T. britovi, and eight were found in both proteomes. Gene Ontology analysis showed that the identified T. spiralis proteins possess hydrolytic endopeptidase, endonuclease and transferase activities. Similarly, most of the T. britovi proteins possess catalytic activities, such as lyase, hydrolase, isomerase and peptidase activity. The applied 2D DIGE technique visualized Trichinella spp. protein spots with different molecular weights or isoelectric point values, as well as those with different expression levels. The identified immunoreactive proteins participate in multiple processes associated with host muscle cell invasion and larval adaptation to the host environment. Their reactivity with the host immune system makes them possible candidates for the development of a novel trichinellosis diagnostic test or vaccine against helminthiasis caused by T. spiralis or T. britovi.

Immunoproteomic analysis of Trichinella spiralis and Trichinella britovi excretory-secretory muscle larvae proteins recognized by sera from humans infected with Trichinella.

The present study compares the immunogenic patterns of muscle larvae excretory-secretory proteins (ML E-S) from T. spiralis and T. britovi recognized by Trichinella-infected human sera. Samples were analyzed using two-dimensional electrophoresis (2-DE) coupled with 2D-immunoblot and liquid chromatography-tandem mass spectrometry LC-MS/MS analysis, two ELISA procedures and a confirmatory 1D-immunoblot test. Sera were obtained from nine patients with a history of ingestion of raw or undercooked meat who presented typical clinical manifestations of trichinellosis and from eleven healthy people. Specific anti-Trichinella IgG antibodies were detected in all samples tested with the Home-ELISA kits, but in only four samples for the commercially-available kit. The 1D-immunoblot results indicated that all nine serum samples were positive for T. spiralis ML E-S antigens, expressed as the presence of specific bands. In contrast, eight of the serum samples with T. britovi E-S ML antigens were positive, with one serum sample taken from a patient at 33dpi (days post infection) being negative. To identify immunoreactive proteins that are specifically recognized by host antibodies, both species of ML E-S proteins were subjected to 2D-immunoblotting with human serum taken at 49 dpi. The sera recognized 22 protein spots for T. spiralis and 18 for T. britovi in 2D-immunoblot analysis. Their molecular weights (MW) ranged from 50 to 60 kDa. LC-MS/MS analysis identified both common and specifically-recognized immunoreactive proteins: transmembrane serine protease 9, serine protease, antigen targeted by protective antibodies and Actin-1 partial were shared for both Trichinella species; hypothetical protein T01_7775 and P49 antigen, partial those specific to T. spiralis; deoxyribonuclease-2-alpha and hypothetical protein T03_17187/T12_7360 were specific to T. britovi. Our results demonstrate the value of 2-DE and 2D-immunblot as versatile tools for pinpointing factors contributing to the parasite-host relationship by comparing the secretomes of different Trichinella species.

Immunization with a Recombinant Protein of Trichinella britovi 14-3-3 Triggers an Immune Response but No Protection in Mice.

14-3-3 proteins are present in all eukaryotic organisms and are ubiquitously expressed in a broad range of tissues and cellular compartments. They are regulatory adapter proteins that play key roles in a variety of signaling pathways, and have been proposed as suitable targets for the control and detection of certain parasites. Trichinella britovi is a widely-distributed parasitic nematode, transmitted through ingestion of meat products containing invasive larvae. The present study describes the cloning and expression of Tb14-3-3, and investigates the immunological and protective potential of the recombinant protein. Immunization of mice with rTb14-3-3 triggered an IgG response, and significant differences, in the profiles of secreted cytokines observed in vitro, between experimental groups. Nonetheless, neither specific antibodies, nor increased secretion of IFNγ, IL-4, and IL-10 cytokines, conferred greater protection against infection. No reduction in larval burden was observed during recovery at 48 dpi. Additionally, rTb14-3-3 was not recognized by sera from the infected control mice, except for one, suggesting some mismatch between native and recombinant Tb14-3-3 antigenic sites. Therefore, before 14-3-3 can be considered a potential tool for Trichinella detection and vaccination, more research regarding its target proteins, and actual specific function, is needed.

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

BACKGROUND: Avian 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. METHODS: We 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. RESULTS: All 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. CONCLUSION: Broiler chickens showed a higher number and wider range of fold-changes in the transcriptional response than laying hens.

The Immunological Properties of Recombinant Multi-Cystatin-Like Domain Protein From Trichinella Britovi Produced in Yeast.

Trichinellosis is a globally-distributed zoonotic parasitic disease caused by nematode worms of the genus Trichinella. One of the most common species of Trichinella known to affect human health is T. britovi; however, it is relatively poorly investigated. A thorough knowledge of the proteins expressed by Trichinella is important when developing immunological detection methods and vaccines and studying its interactions with the host. The present study uses the Pichia pastoris expression system to produce a soluble TbCLP antigen which induces strong antibody responses in the host during natural infection. Our results demonstrate the feasibility of TbCLP antigen production in yeasts, which are able to carry out post-translational modifications such as glycosylation and disulfide bond formation; they also indicate that the glycosylated TbCLP antigen had immunogenic effects in the tested mice and induced a mixed Th1/Th2 response, and was associated with a reduced larval burden after challenge with T. britovi. Subsequent in vitro stimulation of mice splenocytes revealed that TbCLP most likely possesses immunomodulatory properties and may play a significant role in the early phase of infection, affecting host immunological responses.

Ultrasensitive electrochemical genosensor for direct detection of specific RNA sequences derived from avian influenza viruses present in biological samples.

An electrochemical genosensor based on an epoxy-phenanthroline-Fe(III)-NH2-ssDNA layer for the detection of RNA derived from Avian Influenza is presented. The biosensor preparation consists of: (I) modification of gold electrodes with aminoethanethiol, (II) modification of the self-assembled monolayer of aminoethanethiol with 5,6-epoxy-5,6-dihydro-[1,10]-phenanthroline using "click" chemistry, (III) a first step of complexation of Fe(III) by 5,6-epoxy-5,6-dihydro-[1,10]-phenanthroline, (IV) a second step of complexation of Fe(III) by 5,6-epoxy-5,6-dihydro-[1,10]-phenanthroline, (V) immobilization of the single stranded amino-DNA probe via "click" chemistry between epoxy and amino groups. The interactions between the ssDNA probe and RNA targets were explored with Osteryoung Square Wave Voltammetry. The genosensor showed a remarkable detection limit of 3 copies/µL (5 aM) for RNA extracted from A/swan/Poland/305/06 (H5N1) containing a fully complementary sequence. A linear dynamic range for this sequence was observed from 3.0×103 to 3.0×105 [copies/µl]. RNA extracted from A/mallard/Poland/446/09 (H7N7), containing a non-complementary sequence, generated a much weaker response. Moreover, the developed genosensor allows to distinguish RNA present in biological samples having 2, 3 and 4 mismatches. This biosensing approach can become a potential alternative tool for detecting RNA samples in biomedical research and early clinical diagnosis of avian influenza viruses.

Sequential DNA immunization of chickens with bivalent heterologous vaccines induce highly reactive and cross-specific antibodies against influenza hemagglutinin.

Vaccines against avian influenza are mostly based on hemagglutinin (HA), which is the main antigen of this virus and a target for neutralizing antibodies. Traditional vaccines are known to be poorly efficient against newly emerging strains, which is an increasing worldwide problem for human health and for the poultry industry. As demonstrated by research and clinical data, sequential exposure to divergent influenza HAs can boost induction of universal antibodies which recognize conserved epitopes. In this work, we have performed sequential immunization of laying hens using monovalent or bivalent compositions of DNA vaccines encoding HAs from distant groups 1 and 2 (H5, H1, and H3 subtypes, respectively). This strategy gave promising results, as it led to induction of polyclonal antibodies against HAs from both groups. These polyclonal antibodies showed cross-reactivity between different HA strains in ELISA, especially when bivalent formulations were used for immunization of birds. However, cross-reactivity of antibodies induced against H3 and H5 HA subtypes was rather limited against each other after homologous immunization. Using a cocktail of HA sequences and/or sequential DNA vaccination with different strains presents a good strategy to overcome the limited effectiveness of vaccines and induce broader immunity against avian influenza. Such a strategy could be adapted for vaccinating laying hens or parental flocks of different groups of poultry.

Transcriptional response to a prime/boost vaccination of chickens with three vaccine variants based on HA DNA and Pichia-produced HA protein.

Highly pathogenic avian influenza causes severe economic losses and is a potential threat to public health. Better knowledge of the mechanisms of chicken response to the novel types of vaccines against avian influenza might be helpful in their successful implementation into poultry vaccination programs in different countries. This work presents a comprehensive analysis of gene expression response elicited in chicken spleens by a combined DNA/recombinant protein prime/boost vaccination compared to DNA/DNA and protein/protein regimens. All groups of vaccinated chickens displayed changes in spleen transcriptomes in comparison to the control group with 423, 375 and 212 identified differentially expressed genes in protein/protein, DNA/DNA and DNA/protein group, respectively. Genes with most significantly changed expression belong to immune-related categories. Depending on a group, a fraction of 15-34% of up-regulated and a fraction of 15-42% of down-regulated immune-related genes are shared by all groups. Interestingly, the most upregulated genes encode ß-defensins, short peptides with antimicrobial activity and immunomodulatory functions. Microarray results were validated with RT-qPCR method, which confirmed differential regulation of the selected immune-related genes. Immune-related differentially expressed genes and metabolic pathways identified in this work are compared to the available literature data on gene expression changes in vaccinated and non-vaccinated chickens after influenza infection.

Effective usage of cationic derivatives of polyprenols as carriers of DNA vaccines against influenza virus.

BACKGROUND: Cationic derivatives of polyprenols (trimethylpolyprenylammonium iodides - PTAI) with variable chain length between 6 and 15 isoprene units prepared from naturally occurring poly-cis-prenols were tested as DNA vaccine carriers in chickens and mice. This study aimed to investigate if PTAI could be used as an efficient carrier of a DNA vaccine. METHODS: Several vaccine mixtures were prepared by combining different proportions of the vaccine plasmid (carrying cDNA encoding a vaccine antigen, hemagglutinin from H5N1 influenza virus) and various compositions of PTAI. The vaccines were delivered by intramuscular injection to either chickens or mice. The presence of specific antibodies in sera collected from the immunized animals was analyzed by enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) test. RESULTS: The mixtures of PTAI with helper lipids, such as DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine), DC-cholesterol [{3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol} hydrochloride] or DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) induced strong humoral response to the antigen encoded by the DNA vaccine plasmid. CONCLUSION: The animal immunization results confirmed that PTAI compositions, especially mixtures of PTAI with DOPE and DC-cholesterol, do work as effective carriers of DNA vaccines, comparable to the commercially available lipid transfection reagent.

Immunogenicity of DNA Vaccine against H5N1 Containing Extended Kappa B Site: In Vivo Study in Mice and Chickens.

Influenza is one of the most important illnesses in the modern world, causing great public health losses each year due to the lack of medication and broadly protective, long-lasting vaccines. The development of highly immunogenic and safe vaccines is currently one of the major problems encountered in efficient influenza prevention. DNA vaccines represent a novel and powerful alternative to the conventional vaccine approaches. To improve the efficacy of the DNA vaccine against influenza H5N1, we inserted three repeated kappa B (κB) motifs, separated by a 5-bp nucleotide spacer, upstream of the cytomegalovirus promoter and downstream of the SV40 late polyadenylation signal. The κB motif is a specific DNA element (10pb-long) recognized by one of the most important transcription factors NFκB. NFκB is present in almost all animal cell types and upon cell stimulation under a variety of pathogenic conditions. NFκB is released from IκB and translocates to the nucleus and binds to κB sites, thereby leading to enhanced transcription and expression of downstream genes. We tested the variants of DNA vaccine with κB sites flanking the antigen expression cassette and without such sites in two animal models: chickens (broilers and layers) and mice (BALB/c). In chickens, the variant with κB sites stimulated stronger humoral response against the target antigen. In mice, the differences in humoral response were less apparent. Instead, it was possible to spot several gene expression differences in the spleens isolated from mice immunized with both variants. The results of our study indicate that modification of the sequence outside of the sequence encoding the antigen might enhance the immune response to the target but understanding the mechanisms responsible for this process requires further analysis.

A prime/boost vaccination with HA DNA and Pichia-produced HA protein elicits a strong humoral response in chickens against H5N1.

Highly pathogenic avian influenza viruses cause severe disease and huge economic losses in domestic poultry and might pose a serious threat to people because of the high mortality rates in case of an accidental transmission to humans. The main goal of this work was to evaluate the immune responses and hemagglutination inhibition potential elicited by a combined DNA/recombinant protein prime/boost vaccination compared to DNA/DNA and protein/protein regimens in chickens. A plasmid encoding hemagglutinin (HA) from the A/swan/Poland/305-135V08/2006 (H5N1) virus, or the recombinant HA protein produced in Pichia pastoris system, both induced H5 HA-specific humoral immune responses in chickens. In two independent experiments, anti-HA antibodies were detected in sera collected two weeks after the first dose and the response was enhanced by the second dose of a vaccine, regardless of the type of subunit vaccine (DNA or recombinant protein) administered. The serum collected from chickens two weeks after the second dose was characterized by three types of assays: indirect ELISA, hemagglutination inhibition (HI) and a diagnostic test based on H5 antibody competition. Although the indirect ELISA failed to detect superiority of any of the three vaccine regimens, the other two tests clearly indicated that priming of chickens with the DNA vaccine significantly enhanced the protective potential of the recombinant protein vaccine produced in P. pastoris.

Codon optimization of antigen coding sequences improves the immune potential of DNA vaccines against avian influenza virus H5N1 in mice and chickens.

BACKGROUND: Highly pathogenic avian influenza viruses are a serious threat to domestic poultry and can be a source of new human pandemic and annual influenza strains. Vaccination is the main strategy of protection against influenza, thus new generation vaccines, including DNA vaccines, are needed. One promising approach for enhancing the immunogenicity of a DNA vaccine is to maximize its expression in the immunized host. METHODS: The immunogenicity of three variants of a DNA vaccine encoding hemagglutinin (HA) from the avian influenza virus A/swan/Poland/305-135V08/2006 (H5N1) was compared in two animal models, mice (BALB/c) and chickens (broilers and layers). One variant encoded the wild type HA while the other two encoded HA without proteolytic site between HA1 and HA2 subunits and differed in usage of synonymous codons. One of them was enriched for codons preferentially used in chicken genes, while in the other modified variant the third position of codons was occupied in almost 100 % by G or C nucleotides. RESULTS: The variant of the DNA vaccine containing almost 100 % of the GC content in the third position of codons stimulated strongest immune response in two animal models, mice and chickens. These results indicate that such modification can improve not only gene expression but also immunogenicity of DNA vaccine. CONCLUSION: Enhancement of the GC content in the third position of the codon might be a good strategy for development of a variant of a DNA vaccine against influenza that could be highly effective in distant hosts, such as birds and mammals, including humans.

A biosensor based on electroactive dipyrromethene-Cu(II) layer deposited onto gold electrodes for the detection of antibodies against avian influenza virus type H5N1 in hen sera.

This paper describes the development of a biosensor for the detection of anti-hemagglutinin antibodies against the influenza virus hemagglutinin. The steps of biosensor fabrications are as follows: (i) creation of a mixed layer containing the thiol derivative of dipyrromethene and 4-mercapto-1-butanol, (ii) complexation of Cu(II) ions, (iii) oriented immobilization of the recombinant histidine-tagged hemagglutinin, and (iv) filling free spaces with bovine serum albumin. The interactions between recombinants hemagglutinin from the highly pathogenic avian influenza virus type H5N1 and anti-hemagglutinin H5 monoclonal antibodies were explored with Osteryoung square-wave voltammetry. The biosensor displayed a good detection limit of 2.4 pg/mL, quantification limit of 7.2 pg/mL, and dynamic range from 4.0 to 100.0 pg/mL in buffer. In addition, this analytical device was applied for the detection of antibodies in hen sera from individuals vaccinated and non-vaccinated against the avian influenza virus type H5N1. The limit of detection for the assay was the dilution of sera 1: 7 × 10(6), which is about 200 times better than the enzyme-linked immunosorbent assay.

New redox-active layer create via epoxy-amine reaction - The base of genosensor for the detection of specific DNA and RNA sequences of avian influenza virus H5N1.

This paper concerns the development of a redox-active monolayer and its application for the construction of an electrochemical genosensor designed for the detection of specific DNA and RNA oligonucleotide sequences related to the avian influenza virus (AIV) type H5N1. This new redox layer was created on a gold electrode surface step by step. Cyclic Voltammetry, Osteryoung Square-Wave Voltammetry and Differential Pulse Voltammetry were used for its characterization. This new redox-active layer was applied for the construction of the DNA biosensor. The NH2-NC3 probe (20-mer) was covalently attached to the gold electrode surface via a "click" reaction between the amine and an epoxide group. The hybridization process was monitored using the Osteryoung Square-Wave Voltammetry. The 20-mer DNA and ca. 280-mer RNA oligonucleotides were used as the targets. The constructed genosensor was capable to determine complementary oligonucleotide sequences with a detection limit in the pM range. It is able to distinguish the different position of the part RNA complementary to the DNA probe. The genosensor was very selective. The 20-mer DNA as well as the 280-mer RNA oligonucleotides without a complementary sequence generated a weak signal.

DNA vaccines against influenza.

Genetic vaccine technology has been considerably developed within the last two decades. This cost effective and promising strategy can be applied for therapy of cancers and for curing allergy, chronic and infectious diseases, such as a seasonal and pandemic influenza. Despite numerous advantages, several limitations of this technology reduce its performance and can retard its commercial exploitation in humans and its veterinary applications. Inefficient delivery of the DNA vaccine into cells of immunized individuals results in low intracellular supply of suitable expression cassettes encoding an antigen, in its low expression level and, in turn, in reduced immune responses against the antigen. Improvement of DNA delivery into the host cells might significantly increase effectiveness of the DNA vaccine. A vast array of innovative methods and various experimental strategies have been applied in order to enhance the effectiveness of DNA vaccines. They include various strategies improving DNA delivery as well as expression and immunogenic potential of the proteins encoded by the DNA vaccines. Researchers focusing on DNA vaccines against influenza have applied many of these strategies. Recent examples of the most successful modern approaches are discussed in this review.

Antibody response to DNA vaccine against H5N1 avian influenza virus in broilers immunized according to three schedules.

Broiler type chickens were immunized intramuscularly with a DNA vaccine encoding hemagglutinin (HA) from H5N1 avian influenza virus. The chickens were divided into four groups: control group which was not immunized, a group which obtained only one dose, and two groups which were immunized twice, one group with a boost two weeks after the priming and the other four weeks. Blood samples were collected at several time points and the dynamics of the humoral response to the vaccine was studied. High level of anti-HA antibodies was detected only in the last two groups, that is in chickens immunized according to the prime-boost strategy, regardless of the schedule. An additional interesting observation of this study was detection of the cross-reactivity of an anti-H5 HA positive serum with H5N2 and H1N1 viruses, suggesting that the DNA vaccine tested can induce antibodies of a broad specificity.

A highly sensitive electrochemical genosensor based on Co-porphyrin-labelled DNA.

We report the use of Co-porphyrins as electrochemical tags for a highly sensitive and selective genosensor. An avian influenza virus-based DNA sequence characteristic of H5N1 was detected at femtomolar levels from competing non-complementary sequences through hybridisation with the labeled DNA.