Performing Department
MICROBIOLOGY & IMMUNOLOGY
Non Technical Summary
Equine Herpesvirus Type 1 (EHV-1) may have the potential as an immunization and gene therapy vector. We will test the hypothesis of using EHV-1 as a vector by generating recombinant viruses expressing immunogenic proteins of important animal and human pathogens, i.e. Bovine Viral Diarrhea Virus, Porcine Respiratory and Reproductive Syndrome Virus, Hepatitis C Virus, and Human Immunodeficiency Virus.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The objectives of the proposal are the construction of various recombinant EHV-1 using an infectious bacterial artificial chromosome (BAC) clone of the modified live EHV-1 strain RacH. The recombinant viruses will express immunogens of important animal and human viral pathogens. Mutagenesis of the BAC clone in Escherichia coli cells will be done using RecE/T- and RecA-based cloning. Two different genomic loci will be used for the insertion of the foreign genes, which will help to avoid positional effects on the expression of foreign genes. One insertion site will be in the unique short genomic region and the Eco-gpt (xgpt) locus will be replaced with foreign sequences. The second will be in the middle of the so-called unique long region of the genome, and glycoprotein M will be replaced with the open reading frames (ORF) to be expressed. Synthetic open reading frames, which will be optimized to the herpesvirus codon usage, will be used for expression of both HIV
Pr55gag and BVDV-C_E2. These synthetic ORFs have already been shown to be functional after transient or constitutive transfection in eukaryotic cells under the control of the human cytomegalovirus immediate early promoter/enhancer6. In the case of the HCV C-E2 region and PRRSV Orf5, synthetic sequences are not required, because production, stability and nuclear export of mRNA appears unaffected when the authentic sequences are used. Following the construction of the recombinant viruses, they will be tested for their general growth properties in cultured cells, i.e. the intra- and extracellular virus titers as well as plaque diameters will be determined. These measurements will provide the first evidence for putative growth restrictions (or advantages) of various mutants. In a second step, the humoral immune response against the antigens expressed by EHV-1 will be assessed after inoculation of the different constructs into mice. Inoculations will be done by the intranasal or
intramuscular route. A total of three injections in 30-day intervals are planned. At various times post inoculation, mice will be bled and the antibody titers against the foreign immunogens as well as against EHV-1 in serum will be determined using commercially available enzyme-linked immunosorbent assays (ELISA). Also, EHV-1 virus titers in peripheral blood will be analyzed by co-cultivation experiments and flow cytometry detecting green fluorescent protein (GFP) that is expressed by the virus mutants to be generated.
Project Methods
We will use a series of integrated approaches to evaluate the potential of an avirulent modified live Equine Herpesvirus Type 1 (EHV-1) strain to serve as a universal vector for immunization and gene therapy. The proposed studies include the generation and testing of mutant EHV-1, which are designed to assay for efficient expression of foreign genes of human and animal pathogens, and to evoke an immune response against the inserted proteins in mice. The animal experiments are also designed to demonstrate that the short-lived immunity observed after immunization/infection with EHV-1 can be of great advantage for repeated immunizations using RacH as a vector. In terms of human vaccination, we have chosen to express structural genes of two important human viral pathogens, the human immunodeficiency virus (HIV) gag precursor (Pr55gag) and the Hepatitis C Virus (HCV) serotype 1 capsid to p7 region (C p7). The two pathogens cause devastating diseases and efficacious vaccines
have yet to be generated. In addition, growth of HCV in cell culture is impossible so far. By using a modified-live virus approach for vaccine delivery, which can enter professional antigen-presenting cells and other cells of the immune system, both the humoral and cellular immunity should be elicited and result in a sustained immune response. In order to develop EHV-1 as a tool to rationally present antigens to the immune system of livestock, we will concentrate on Bovine Viral Diarrhea Virus (BVDV), an important pathogen affecting both beef cattle and dairy cows, and the Porcine Respiratory and Reproductive Syndrome Virus (PRRSV), an Arterivirus causing severe economic problems in the pig industry. Expression of the BVDV serotype Ia structural protein region, which is very similar to that of HCV, will be performed. In contrast to HCV, which encodes a capsid protein, the p7 protein, and two glycoproteins (E1 and E2), BVDV encodes three glycoproteins, referred to as Erns (E0), E1 and
E2, in addition to the capsid and p7 proteins. The latter - in the case of BVDV - is not considered a structural protein. The PRRSV Orf5 encoding GP5, the major target of anti-PRRSV neutralizing antibodies, will also be inserted into EHV-1 strain RacH. In the case of the introduction of the BVDV and HIV genes, synthetic nucleotide sequences have to be used, because the two viruses have a codon usage that will not allow efficient nuclear expression. These synthetic genes are already available. The insertions of the foreign genes into the EHV-1 strain RacH genome will be performed using a BAC clone of RacH that has been established in the laboratory. The insertion will be performed by exploiting the DNA repair and mutagenesis apparatus of Escherichia coli cells, and will predominantly use a method referred to as RecA mutagenesis. The generated mutant viruses will be tested both in vitro and in vivo. The in vitro testings include the determination of virus growth properties and the
assessment of foreign gene expression. After the in vitro testings, the induction of virus- or antigen-specific antibodies in mice after application of the recombinant virus will be analyzed.