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Biomedical subjects

J Pitcovski

Publications and source records attributed to J Pitcovski.

At least 19 recordsLinked to original sources
PubMed2006

B subunit of E. coli enterotoxin as adjuvant and carrier in oral and skin vaccination.

Mucosal sites are one of the main natural ports of entry into the body. Stimulation of a local response by antibodies as the systemic protection may enhance the efficacy of non-living vaccines, and allow for vaccination by subunit vaccines without the need for injection. Mucosal or skin vaccination necessitates a suitable adjuvant and carrier. Escherichia coli heat-labile enterotoxin (LT) and its B subunit (LTB) have been found to be effective adjuvants. The aim of this study was to efficiently produce and purify recombinant LTB (brLTB), and examine its adjuvant and carrier properties. The gene encoding LTB was cloned and expressed in E. coli, and the product was found to have a pentameric form with the ability to bind the cell receptor, GM1 ganglioside. A one-step method for efficient purification and concentration of brLTB was developed. Both oral and intramuscular vaccination with purified brLTB yielded high antibody titers, which detected the whole toxin. In an attempt to test its adjuvant characteristics, brLTB was mixed with either BSA or a recombinant protein (rKnob of egg drop syndrome adenovirus) and delivered intramuscularly, orally or transcutaneously. The addition of brLTB significantly elevated the antibody response in groups vaccinated orally and transcutaneously, but had no influence in injected groups. Vaccination with another recombinant protein, (viral protein 2 of infectious bursal disease virus) supplemented with brLTB did not elevate the antibody response, as compared to vaccination with the antigen alone. These results demonstrate that the addition of brLTB makes oral and transcutaneous vaccination with protein antigens possible.

Adenoviridae Infections↗
PubMed2005

A subunit vaccine against hemorrhagic enteritis adenovirus.

Hemorrhagic enteritis virus (HEV) is an adenovirus that infects turkeys and causes immunosuppression and mortality. The virus used for the inactivated vaccine is extracted from spleens of infected turkeys, since its propagation in tissue cultures or embryonated eggs is unsuitable for mass production. The aim of this study was to develop a subunit vaccine based on a capsid protein of the virus. The knob protein, together with an adjacent part of the shaft domain pertaining to the fiber protein of HEV, was expressed in Escherichia coli and tested as a vaccine. Vaccination with this recombinant protein conferred protection against challenge in controlled and in floor-pen experiments. This finding suggests that the knob protein may be used as safe and efficient vaccine against hemorrhagic enteritis of turkeys. The possibility that the knob proteins of other adenoviruses may be protective and serve as vaccine is also discussed.

Adenoviridae Infections↗
PubMed2005

Vaccine and adjuvant activity of recombinant subunit B of E. coli enterotoxin produced in yeast.

Escherichia coli heat-labile enterotoxin (LT) and cholera toxin (CT) have been studied intensively as vaccines against diseases caused by those bacteria and as adjuvants for mucosal vaccination. Two major problems interfere with the use of these promising adjuvants: their toxicity and the residual bacterial endotoxins mixed with the desired LT. In this study, subunit B of LT was expressed in Pichia pastoris yeast cells (yrLTB) and the recombinant protein was purified and concentrated by ion-exchange chromatography. The final yield of the recombinant protein was 5-8 mg/l induction medium. The molecule is in pentameric form and binds to GM1 gangliosides. When given orally to chickens, anti-LTB antibodies were produced, exhibiting its ability to cross the digestive system and induce an immune response. The adjuvant activity of yrLTB was proven by fusing it to viral protein 2 (VP2) of infectious bursal disease virus. Birds intramuscularly vaccinated with this molecule exhibit 70-100% protection, in a dose-response-dependent manner. This method eliminated the bacterial endotoxins and enabled the production of large quantities of LTB. Expression in a eukaryotic system allows the production of fusion proteins that require post-translational modifications. This may allow oral vaccination with a protein fused to yrLTB. The approach described in this study will enable the efficient production of a non-toxic, eukaryotically expressed enterotoxin as a vaccine against the toxin itself or as a carrier or adjuvant for foreign vaccine molecules.

Adjuvants, Immunologic↗
PubMed2005

Heat labile enterotoxin of E. coli: a potential adjuvant for transcutaneous cancer immunotherapy.

Escherichia coli heat labile enterotoxin (LT) has been shown to penetrate intact skin and to activate adaptive immunity. A nontoxic mutant, nLT, and its B subunit (LTB), have been evaluated separately for their potential use as a tool for transcutaneous delivery of antigens for cancer immunotherapy. We have shown that FITC-labeled nLT is taken up by human dendritic cells (hDC) in vitro and in mouse skin, and induces maturation and activation of hDC in vitro. hDC matured with nLT enhanced nonspecific melanoma antigen uptake and presentation to autologous CD8+ T cells. In mouse in vivo studies, nLT or LTB were applied on the skin either mixed with recombinant gp100 or genetically fused with a multiepitope polypeptide (MEP). Fused LTB-MEP induced antibody production that was dependent on LTB cell binding. We conclude that LT derivatives may be useful for the transcutaneous delivery of tumor antigens for cancer immunotherapy.

Adjuvants, Immunologic↗
PubMed2005

Genetic variation in major histocompatibility complex class I alpha2 gene among broilers divergently selected for high or low early antibody response to Escherichia coli.

The MHC genes have a profound effect on animal abilities to respond to specific antigens because they play a role in presenting foreign antigens to T cells during the course of the humoral or cellular immune response. In the current study, polymorphism in the MHC class I alpha2 domain was compared in 2 lines divergently selected for high (HH) or low (LL) antibody response to Escherichia coli vaccine. These lines also differ markedly in their antibody response to natural E. coli exposure and to vaccination with Newcastle disease virus, infectious bronchitis virus, and infectious bursa disease virus. Recent trials have shown that the LL chicks exhibit a significantly higher percentage of CD8+ T lymphocytes in their peripheral blood lymphocytes and spleen than HH chicks. Despite symmetrical selection intensity in both lines, polymorphism of the alpha2-domain gene was higher in the LL line than in the HH line. Among 29 single-nucleotide polymorphism positions found, 3 were unique to the HH line, 15 were unique to the LL line, and 11 were polymorphic in both lines. These single nucleotide polymorphism positions were not 100% line specific and were in agreement with the genetic variation in antibody level or cellular response still found within the selection lines. Five amino acid positions showed significant differences in polymorphism between the selection lines. These were located within the antigen-binding cleft, suggesting that these positions might influence the ability of MHC class I to bind foreign antigens and leading to differences in immunocompetence between the lines.

Amino Acid Sequence↗
PubMed2004

Production and purification of melanoma gp100 antigen and polyclonal antibodies.

gp100 is a melanoma-associated antigen found to carry immunogenic epitopes that can induce a CTL response against tumor cells. Production and purification of large quantities of this polypeptide may be important in the context of diagnosis and vaccinating against melanoma. To overcome the hydrophobic nature of gp100, we cloned and expressed only a part of the protein, and obtained a hydrophilic recombinant polypeptide (HR-gp100) that contained most of the immunogenic peptides. High yield was achieved in an Escherichia coli expression system. The protein was purified by AKTA Prime using anionic-columns. Polyclonal antibodies developed in chicken against HR-gp100 were efficient at detecting gp100 in melanoma cells, as determined by Western blot analysis and by immunohistochemistry. HR-gp100 can be used to develop a vaccine against melanoma. Antibodies to HR-gp100 may be used to detect tumors of melanocytic origin or to determine the level of gp100 expression in tumors prior to immunotherapy with the protein or one of its peptides.

Amino Acid Sequence↗
PubMed2003

A subunit vaccine against the adenovirus egg-drop syndrome using part of its fiber protein.

In this study, the effectiveness of antibodies against the hexon, fiber or a fiber fragment of an avian adenovirus egg-drop syndrome (EDS), in neutralizing the virus was tested. The fiber protein is responsible for binding the virus to the target cell. The fiber fragment knob-s comprises the carboxy-terminal knob domain and 34 amino acids of the immediately adjacent shaft domain of the adenovirus fiber protein. The hexon, fiber capsid protein and knob-s were produced in E. coli and injected into chickens. Antibodies that were produced against the whole fiber protein showed some hemagglutination inhibition (HI) activity. Antibodies produced against the knob-s protein showed HI activity and serum neutralization (SN) activity similar to the positive control-whole virus vaccine. We assume that production of only part of the fiber enables the protein produced in E. coli to fold correctly. Antibodies produced against the hexon protein showed no SN activity. In summary, knob-s induced SN and HI antibodies against EDS virus at a rate similar to the whole virus and were significantly more efficient than the full-length fiber. The recombinant knob-s protein may be used as a vaccine against pathogenic adenovirus infections.

Adenoviridae Infections↗
PubMed2001

Immune response and resistance to infectious bursal disease virus of chicken lines selected for high or low antibody response to Escherichia coli.

Two experimental broiler lines were developed by divergent selection for high (HH) and low (LL) antibody response to Escherichia coli. Antibody response of these lines to immunization with a commercial vaccine (whole inactivated virus, WIV) against infectious bursal disease virus (IBDV) or with proteins VP2 and VP3 of that virus, and their resistance to challenge with a virulent IBDV, were tested. The study was performed with 213 male and female chicks from the tenth generation of the HH and LL lines. At 15 d of age, after disappearance of maternal antibodies, chicks from each line were randomly divided into four groups and injected with WIV, VP2, VP3, or adjuvant alone as a negative control. Chicks were bled 18 d postinjection, and antibody titers were determined by ELISA. Ten days later, the chicks were challenged with a virulent strain of the virus and killed after 10 d; the ratio of bursa of Fabricius to 100 g BW was determined for each bird. Significant differences in antibody titers were found among immunized and control chicks. Chicks from the HH line exhibited significantly higher antibody titers than LL chicks in response to WIV and VP2 vaccines but not to VP3 vaccine. Following challenge, bursa weight (relative to BW) of HH and LL chicks vaccinated with WIV and VP2 was significantly higher (P < 0.01) than that of chicks vaccinated with VP3 or the challenged unvaccinated control. No difference was found in this parameter between the latter two groups. Possible explanations for the differences in the line response to VP2 and VP3 are discussed.

Animals↗
PubMed1999

Storage of viruses on filter paper for genetic analysis.

The purpose of this study was to develop a method to store viruses on filter paper without the need for special conditions for future use of the genetic material. Two non-enveloped viruses were used as models. Infectious bursal disease virus (IBDV), a double-stranded RNA virus that infects chickens, belongs to the Birnaviridae family. Hemorrhagic enteritis virus (HEV), with double-stranded DNA, belongs to the Adenoviridae family. Three different solutions were found suitable for loading the virus. The viruses were stored at room temperature or at 37 degrees C for periods of 5-30 days. Direct reverse transcription-polymerase chain reaction (RT-PCR) (without previous extraction of the RNA) was carried out on filter paper loaded with IBDV, and fragments of the expected size were detected. HEV DNA was extracted from filter paper loaded with purified virus or crude tissue. PCR fragments were found to be of similar intensity to those of control virus that was kept in a tube at -20 degrees C. This method permits the storage and transport of viruses from the field or from clinics to a regional laboratory or any laboratory elsewhere, without the need for prior treatment or special environmental conditions.

Animals↗
PubMed1998

The complete DNA sequence and genome organization of the avian adenovirus, hemorrhagic enteritis virus.

Hemorrhagic enteritis virus (HEV) belongs to the Adenoviridae family, a subgroup of adenoviruses (Ads) that infect avian species. In this article, the complete DNA sequence and the genome organization of the virus are described. The full-length of the genome was found to be 26,263 bp, shorter than the DNA of any other Ad described so far. The G + C content of the genome is 34.93%. There are short terminal repeats (39 bp), as described for other Ads. Genes were identified by comparison of the DNA and predicted amino acid sequences with published sequences of other Ads. The organization of the genome in respect to late genes (52K, IIIa, penton base, core protein, hexon, endopeptidase, 100K, pVIII, and fiber), early region 2 genes (polymerase, terminal protein, and DNA binding protein), and intermediate gene IVa2 was found to be similar to that of other human and avian Ad genomes. No sequences similar to E1 and E4 regions were found. Very low similarity to ovine E3 region was found. Open reading frames were identified with no similarity to any published Ad sequence.

Adenoviridae↗
PubMed1997

Identification of a starting point of breast skin tears during chicken plucking.

A site (STP) was identified on the skin of the chicken, during defeathering in the slaughter house, at which about 90% of breast skin tears started. This site is on the ventral side of the pectoral tract area. There was no difference in location of this site with respect to different commercial lines, sexes, flocks, or time of the day. In order to demonstrate the importance of a small skin tear to ultimate damage, defeathered chickens with a minor tear at a particular site and undamaged defeathered chickens were passed through the defeathering machine a second time. Thirty-six percent of the STP chickens were torn further, but only a small percentage (about 4%) of the undamaged chickens were harmed.

Abattoirs↗
PubMed1989

Isolation and characterization of three class II MHC genomic clones from the chicken.

A genomic library was constructed from sperm DNA from an individual of the inbred chicken line G-B2, MHC haplotype B6. The library was screened with a chicken class II probe (beta 2 exon specific) and three MHC class II beta chain genomic clones were isolated. The restriction maps of the three clones showed that each of the three clones was unique. The position of the beta chain sequence was located in each of the three genomic clones by Southern blot hybridization. Subclones containing the beta chain gene were produced from each of the genomic clones and the orientation of the leader peptide, beta 1, beta 2, transmembrane, and cytoplasmic exons was determined by Southern blot hybridization and nucleotide sequencing. The complete nucleotide sequence of two of the three subclones was determined. Comparison of the nucleotide and predicted amino acid sequences of the two subclones with other class II beta chain sequences showed that the B6 chicken beta chain genes are evolutionarily related to the class II beta chain genes from chickens of other MHC haplotypes, and to class II beta chain genes from other species. Analysis of Southern blots of B6 chicken DNA, as well as the isolation of the three beta chain genes, suggests that chickens of the B6 haplotype possess at least three MHC class II beta chain genes.

Amino Acid Sequence↗
PubMed1989

Analysis of B-G and immune response genes in the Iowa State University S1 chicken line by hybridization of sperm deoxyribonucleic acid with a major histocompatibility complex class II probe.

Sperm DNA was isolated from chickens of the Iowa State University S1 line. Birds were from sublines selected for B-G antigen, humoral immune response to glutamic acid-alanine-tyrosine (IrGAT), and response to Rous sarcoma virus-induced (RSV) tumors. The DNA was digested with restriction enzymes and subjected to Southern blot analysis with a DNA probe specific for a class II gene of the chicken major histocompatibility complex (MHC). Restriction fragment length polymorphisms (RFLP) associated with B-G antigen type were found after digestion of DNA with three enzymes, PvuII, BglII, or Sau3A, out of a total of 15 tested. No RFLP were shown to be associated with IrGAT or RSV type. This study shows that RFLP analysis of DNA may be a useful addition to or alternative to serological evaluation of MHC haplotype in the chicken.

Animals↗
PubMed1987

Selection for early responsiveness of chicks to Escherichia coli and Newcastle disease virus.

A broiler chicken population was divergently selected for high or low early immune responses to Escherichia coli and to Newcastle disease virus (NDV) vaccines. Four selection cycles were performed in one replicate, and a single cycle in a second replicate. Selection was based on sire-family averages of a titer index (TI) calculated as the mean titer of antibodies produced by offspring vaccinated with either E. coli or NDV at 18 or 10 days of age, respectively. After the first selection cycle, TI of the early-high (EH) line were 22 and 38% greater than those of the early-low (EL) line in Replicates A and B, respectively. After four selection cycles, the average immune response to E. coli and NDV of Line EH exceeded that of Line EL by 68%. Viability was greater in the EH than in the EL line. Realized family heritabilities were .72 and .67 in Replicates A and B, respectively and the levels of response to the two antigens were not genetically correlated. The immune response of the EH line developed earlier than that in the EL line as shown by fewer nonresponders against E. coli and the higher response of this line against the two antigens at young ages. Mortality after challenge at 18 days of age and general mortality from hatching to 20 wk of age was lower in Line EH than in Line EL. Body weights at 7 wk were higher in EH than EL chicks.

Animals↗
PubMedDate not supplied

Failure of viral protein 3 of infectious bursal disease virus produced in prokaryotic and eukaryotic expression systems to protect chickens against the disease.

In recent years, infectious bursal disease virus (IBDV) has become a serious economic problem as a result of the emergence of new and very virulent strains. Most of the antibodies produced against IBDV are for the structural proteins viral protein (VP) 2 (VP2) and VP3. The purpose of this study was to test the potential of recombinant VP3 to induce protective antibodies. The gene for VP3 was isolated from a virulent strain of the virus and cloned into prokaryotic (Escherichia coli) and eukaryotic (baculovirus) expression systems. The protein expressed by both systems was of the expected size (32 kD) and was detected by anti-IBDV antibodies. Following partial purification, the polypeptides were injected into intact birds and induced the production of high levels of anti-IBDV antibodies, as detected by immunoblot and enzyme-linked immunosorbent assay tests. These antibodies did not prevent changes in the bursa and mortality when birds were challenged with a virulent IBDV strain after vaccination with the recombinant VP3. The results show that VP3 polypeptide cannot be used as a subunit vaccine against IBDV and raise questions concerning the nature of the neutralizing epitope on this structural protein.

Animals↗
PubMedDate not supplied

Viral protein 1 sequence analysis of three infectious bursal disease virus strains: a very virulent virus, its attenuated form, and an attenuated vaccine.

Comparisons between sequences of very virulent, virulent, and attenuated strains of the infectious bursal disease virus (IBDV) may indicate sites on the genome co-inciding with virulence. In an attempt to detect if such sites exist on the coding region of segment B, viral protein 1 (VP1) (encoded for by segment B) of a very virulent Israeli virus, IL3; its attenuated strain, IL4; and the attenuated Winterfield vaccine 2512 were cloned and sequenced. A comparison was made among them and with six other published sequences of segment B. Six nucleic acids distinguished between IL3 and IL4, three of which were predicted to be expressed as amino acids. A striking similarity between the VP1 sequences of 2512 and P2 (an attenuated German strain) was discovered. Although conclusions could not be drawn concerning attenuation sites on VP1, the analysis performed on the VP1 sequences of the two Israeli strains and the Winterfield 2512 strain sheds light on the phylogeny of IBDV and contributes to the accumulating information that may lead to the identification of virulence-related sites of this virus.

Amino Acid Sequence↗
PubMedDate not supplied

Insect cell-derived VP2 of infectious bursal disease virus confers protection against the disease in chickens.

Infectious bursal disease virus (IBDV) has become a major problem in recent years. Conventional vaccines make use of attenuated or inactivated viral strains, but these are gradually losing their effectiveness. We investigated the possibility of using purified VP2, a subunit of IBDV structural protein expressed in insect cells, as a vaccine. The VP2 gene was cloned into pAcYM1. The cloned gene was expressed in a baculovirus system, giving rise to a high quantity of recombinant VP2 (rVP2) protein. The length of the VP2 is 453 amino acids, and it contains two additional amino acids of the baculovirus at the carboxyl terminus. The molecular mass of the protein is about 48 kD. The rVP2 protein reacted with antibodies raised against viral VP2 and had a similar molecular weight. This protein was tested in a controlled vaccination experiment and compared with an inactivated commercial vaccine. High levels of antibodies were raised by the vaccinated birds. The vaccinated birds were challenged with a pathogenic viral strain. rVP2-vaccinated chickens exhibited high resistance to the virus. No mortality or weight changes in the bursa of Fabricius were observed in the vaccinated birds, whereas in the negative control birds, vaccinated with phosphate buffer, up to 50% mortality was found. Higher levels of antibodies were found by enzyme-linked immunosorbent assay in birds vaccinated with rVP2 compared with those vaccinated with the commercial vaccine. This study suggests the potential use of the isolated rVP2 as a subunit vaccine.

Animals↗