Jacob Pitcovski

Development and large-scale use of recombinant VP2 vaccine for the prevention of infectious bursal disease of chickens

Infectious bursal disease virus (IBDV) is the causative agent of Gumboro disease, an infectious disease of global economic importance in poultry. One of the most effective types of inactivated IBDV vaccine is produced by infecting young chickens with a virulent strain, sacrificing them and extracting the virus from the bursa of Fabricius. The goal of this study was to produce an effective subunit vaccine against IBDV thereby providing an effective means of combating the disease. In areas in which the bursa-derived vaccine is in use, this subunit vaccine would eliminate the use of live birds for the production of inactivated vaccines. The gene for viral protein 2 (VP2) of IBDV was cloned into a Pichia pastoris expression system. This efficient system allowed us to meet the need for inexpensive vaccines required by the poultry industry. Following expression and scale-up, the protein was used to vaccinate chickens, against either Gumboro disease alone or in combination with inactivated Newcastle disease virus (NDV). Full protection was conferred against IBDV following vaccination with the subunit recombinant vaccine. No untoward influence on the response to the NDV vaccine was recorded. Over 250 million birds have already been vaccinated with this vaccine. The advantages of a subunit vaccine over an inactivated one are discussed. This approach will enable rapid adjustment to new virulent strains if and when they appear.

Capture of Tumor Cell Membranes by Trogocytosis Facilitates Detection and Isolation of Tumor-Specific Functional CTLs

The success of adoptive cell transfer in the treatment of metastatic cancer in humans is dependent on the selection of highly active tumor-specific cytotoxic T cells. We report here that CTLs capture membrane fragments from their targets while exerting cytotoxic activity and thus gain a detectable functional signature by which they can be identified. Fluorochrome labeling or biotinylation was used to tag tumor cells. CD8(+) T cells were coincubated with the tagged targets, sorted, and functionally evaluated. Our results show that membrane capture by CD8(+) lymphocytes is T-cell receptor dependent, epitope specific, and preferentially associated with highly cytotoxic clonal subsets. CTLs that captured membranes from unmodified melanoma exhibited enhanced cytotoxic activity against tumor cell lines and autologous melanoma. In a human melanoma in vivo model, adoptive transfer of membrane-capturing, peptide-specific T cells, but not noncapturing or bulk CD8(+) T cells, inhibits tumor progression. Membrane capture is therefore a signature of antigen-specific CTLs endowed with high functional avidity and may have direct relevance in the clinical application of adoptive immunotherapy.

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.

The impact of PEGylation on protein immunogenicity

Covalent attachment of PEG (PEGylation) is widely used to improve the pharmaceutical properties of therapeutic proteins. The applicability and safety of this method have been proven by the use of various PEGylated pharmaceutical proteins approved by the Food and Drug Administration (FDA). One of the properties attributed to PEGylation is immunogenicity reduction of the PEGylated protein. In this study, the impact of PEGylation on immunogenicity was tested and compared for two proteins (chicken IgY and horse IgG) in two strains of mice (Balb/c and C57BL/6) for two routes of administration (i.v. and i.m.) and two sizes of PEG (5 kD and 20 kD). The influence of PEG was shown to be inconsistent between the mouse strains and routes of administration, even with the same tested protein. Consequently, immunogenicity reduction by PEGylation cannot be predicted or assumed; it must be tested on an individual case basis.

Genetic and antigenic characterization of sigma C protein from avian reovirus.

Avian reovirus (ARV) causes viral arthritis, tenosynovitis, liver infection and immunosuppression in birds. Live-attenuated and inactivated vaccines for ARV are available, but do not efficiently protect against recent variants. Sigma C, which mediates virus attachment to target cells, is the most variable protein in ARV. Antibodies to this protein neutralize viral infection. The purpose of the present study was to characterize sigma C in isolates of ARV from infected birds, as compared with the vaccine strain. Amino acids 27 to 293 of sigma C from 28 Israeli isolates were compared, classified and analysed using bioinformatics tools. Large variations were found among the isolates, and the vaccine strain was shown to differ from most of the studied strains, which could explain the failure of commonly used vaccinations in protecting birds against ARV infection. Based on sigma C protein sequences from all over the world, ARV can be divided into four groups. Isolates from all groups were found in the field simultaneously, possibly explaining the insufficient protection achieved by the vaccine strain, which is represented in one of the groups. The results point out the need and the difficulty in producing a wide-ranging vaccine. Several conserved regions among all reported ARV sigma C proteins were identified. These peptides were further studied for structural and functional properties, and for antigenic characterization. The results of this study shed light on peptide selection for a broad and efficient vaccine.

Wide-range protection against avian reovirus conferred by vaccination with representatives of four defined genotypes.

Many isolates of the contagious avian reovirus have been characterized, mainly based on the sequence of their sigma C protein. These isolates have been classified into four genotypes. Currently available vaccines are of limited effectiveness, likely due to the existence of many variants. The aim of this study was to test the efficacy of a vaccine consisting of a mixture of prototypes (representatives) of the four defined genotypic groups of avian reovirus. The prototypes were selected based on their distance from the isolates within each genotype. All prototypes were found to be virulent. Antibodies produced against each of the prototypes neutralized all members of its genotype. Birds were then vaccinated with a mixture of the four prototypes. Results suggest that the 4-valent vaccine can prevent disease and confer broad protection against field isolates of avian reovirus.

Attenuation of very virulent infectious bursal disease virus and comparison of full sequences of virulent and attenuated strains

A very virulent strain of infectious bursal disease virus (IBDVks) was isolated from the bursae of Fabricius of IBDV-affected broiler chickens. Following 43 serial passages in specific pathogen-free embryonated eggs, an attenuated strain was established (IBDVmb). Dosages of IBDVmb in the range 102 to 104 embryo infective dose of 50% were found to be safe and protective for commercial chicks. Chickens vaccinated with live vaccine containing IBDVmb responded with precipitating and type-specific neutralizing antibodies, and were immune to subsequent challenge with a very virulent IBDV. IBDVmb has been used as an attenuated vaccine throughout the world since 1993. A comparison of the full sequences of the virulent and attenuated strains (IBDVks and IBDVmb, respectively) revealed seven nucleotides that were different, four of them leading to changes in the amino-acid sequence. Comparison of the protein sequencse of these strains and published sequences of very virulent and attenuated phenotypes lead us to sugget that the novel difference responsible for virulence of the Israeli strains are: residue 272 (VP2, very conserved site) and residue 527 (VP4), both in segment A, and in segment B (VP1) residues 96 and 161 (both conserved). Our study strengthens the possibility that more than one protein is involved in IBDV attenuation. In all reports, including ours, virulence was reduced without affecting antigenicity of the neutralizing epitopes in VP2. This could have practical implications for attenuated-vaccine development.

Overcoming the susceptibility gap between maternal antibody disappearance and auto-antibody production.

In the first 10-14 days of a chicks life, protection is conferred by maternal antibodies. Further broiler protection is achieved by active vaccination. However, the high level of maternal antibodies interferes with the induction of an effective immune response by vaccination at a young age. As a result, there is a gap between the reduction in protective maternal antibodies and elevation of self-produced antibodies following active vaccination. The major aim of this study was to test an approach consisting of passive and active vaccination to overcome this gap and to provide continuous resistance to infectious viral diseases during the broilers growth period. Newcastle disease virus (NDV), which is one of the worlds most prevalent infectious diseases of poultry, was tested as a model. Following subcutaneous injection of 18 hemagglutination-inhibiting (HI) units of anti-NDV immunoglobulin Y per 1-day-old chick, protective log2 antibody titers above 4 could be detected to at least 17 days of age. The combination of passive immunization on day 1 of age with attenuated live vaccination on day 10 led to high protective titers throughout the entire growth period, up to 41 days of age. Moreover, the HI titers in the group of birds immunized with the combined vaccination were significantly more homogeneous than those in the group vaccinated only with live virus. Thus, full protection against NDV of all broilers in flock during their entire growth period was achieved by a vaccination regime that combines passive immunization and live vaccination.

Immune Responses to Mucosal Vaccination by the Recombinant S1 and N Proteins of Infectious Bronchitis Virus

Infectious bronchitis virus (IBV) is prevented primarily by the use of live attenuated vaccines, which are known to have a limited strain range of protection. Alternative vaccines against the emerging new virus strains can improve control of the disease. The aim of this study was to evaluate the immunogenic potential of two recombinant viral proteins, when administered by eyedrop, without the assistance of a vector. The recombinant S1 (rS1) and N (rN) proteins of the M41 strain expressed in E. coli were tested, and the live attenuated vaccine H120 was used as a positive control. Protection was evaluated by re-isolation of virus from tracheas of vaccinated chickens after challenge with strain M41. After three immunizations, rS1 glycoprotein induced 40% protection, while vaccination with rN provided no protection. Vaccination with rS1, rN, or H120 induced a cellular immune response as demonstrated by in vitro ChIFN-γ production by splenocytes of vaccinated birds. Vaccination with H120, and to a lesser extent rS1, induced HI and virus-specific IgG antibody production. These findings indicate that recombinant viral proteins administered through the mucosal route can evoke an immune response without the assistance of a vector.

Transcutaneous immunization with hydrophilic recombinant gp100 protein induces antigen-specific cellular immune response.

The objective of this study was to evaluate the potential of transcutaneous immunization with tumor antigen to induce cell-mediated immunity. For this purpose, hydrophilic recombinant gp100 protein (HR-gp100) was topically applied on human intact skin in vitro, and used as a vaccine in a mouse model. We demonstrate that HR-gp100 permeates into human skin, and is processed and presented by human dendritic cells. In a mouse model, an HR-gp100-based vaccine triggered antigen-specific T cell responses, as shown by proliferation assays, ELISA and intracellular staining for IFN-γ. Transcutaneous antigen delivery may provide a safe, simple and effective method to elicit cell-mediated immunity.