Systemic DNA immunization against ovine lentivirus using particle-mediated epidermal delivery and modified vaccinia Ankara encoding the gag and/or env genes

Abstract

To determine whether systemic immunization with plasmid DNA and virus vector against visna/maedi virus (VMV) would induce protective immune responses, sheep were immunized with VMV gag and/or env sequences using particle--mediated epidermal bombardment and injection of recombinant modified vaccinia Ankara. The results showed that immunization induced both humoral and cell--mediated responses prior to and after virus challenge. The vaccination protocol did not prevent infection, but immunization with the gag gene or a combination of gag and env genes resulted in significantly reduced provirus loads in blood and mediastinal lymph node, respectively. Provirus loads in lung and draining lymph node were unaffected, but p25 expression was undetectable in lungs of animals immunized with a combination of gag and env genes. Analysis of target tissues for lesions at post--mortem showed that immunization with the env gene caused a significant increase in lesion score, while the gag gene or a combination of gag and env genes had no effect. Inclusion of the ovine interferon--γ gene in the initial priming mixture had minimal effect on immune responses, provirus load, or lesion development, although it resulted in a decreased p25 expression in the lung. The results thus show that systemic immunization with gag or a combination of gag and env genes reduces provirus load in blood and lymphoid tissue, respectively whereas env immunization has no effect on provirus load but increased lesion development.

Development and comparison of strain specific gag and pol real-time PCR assays for the detection of Visna/maedi virus

“…Previous identification of sheep infected successfully was based on the measurement of anti--VMV antibodies in serum, T cell proliferation and anti--VMV T cell reactivity and determination of histopathological changes in lungs and mediastinal lymph nodes. A total of 222 DNAs from PBMCs of 74 uninfected sheep were used as negative controls to determine the specificity of the real--time assays (Niesalla et al, 2009). …”

“…Evaluation of specificity and sensitivity of gag and pol dual labelled probe PCR assays: analysis of PBMC samples A panel of 204 DNAs extracted from PBMCs of 68 sheep infected experimentally and 222 DNAs from PBMCs of 74 uninfected sheep (Niesalla et al, 2009) was analyzed with both dual labelled probe real--time PCR assays. A set volume of 5 μl of DNA was used as template irrespective of DNA concentration of the samples.…”

“…The British VMV strain EV1 was isolated from a sheep displaying symptoms of arthritis and pneumonia and its complete genome was cloned and sequenced ( Sargan et al, 1991). This strain has been and is currently used for in vivo and in vitro studies focusing on diverse topics such as the identification of viral promoter elements, or cytokines that control the expression and replication of the virus , Sutton et al, 1997and Zhang et al, 2002, the dissection of the initial steps of the infection ( Bird et al, 1993, Blacklaws et al, 1995and Ryan et al, 2000, the characterization of the immune response ( Bird et al, 1993 andSingh et al, 2006), the routes of viral entry and dissemination ( Blacklaws et al, 1995, McNeilly et al, 2007 and the development of immunization strategies ( Gonzalez et al, 2005, Niesalla et al, 2009and Reina et al, 2008. In some of these studies the presence of the EV1 genome has been investigated by conventional PCR ( Bird et al, 1993and Ryan et al, 2000 or by quantitative competitive PCR (QC PCR) ( Zhang et al, 2000).…”

Use of B7 costimulatory molecules as adjuvants in a prime-boost vaccination against Visna/Maedi ovine lentivirus

“…In goats, SRLV immunization with env plasmids has led to decreased viral replication and load and diminished disease [11] whilst gag gene or peptide immunization has resulted in enhancement of proviral load or disease [12] and [13]. In sheep, immunization may lead to protection against early lesion development or against viral infection (decreased load) [14], [15] and [16]. Vaccination with inactivated virus leads to disease [12], [17] and [18], but using an attenuated VMV clone results in a diminished number of viral isolations after challenge [19].…”

“…Mucosal immunization using plasmids containing VMV env confers a protective effect seen by decreased proviral load [14] and [16], whilst the use of the gag gene enhances protection against early lesion development [16]. When the same plasmids are delivered intradermally and this is followed by modified vaccinia Ankara virus (MVA) boosting, only the gag gene (or gag in combination with env) gives rise to partial protection against infection [15], but when the vaccination protocol is changed [20], pre--existing immune responses to GAG proteins do not prevent infection. Thus, immunization routes and viral genes used clearly affect the results of vaccination.…”