Evaluation of lentivirus dna vaccination strategies in sheep

Animals immunised systemically with gag + gagIFN showed a significant reduction in pro-virus load in blood. The pro-virus load in lung tissues in these animals was not significantly different from controls. Pro-virus loads in blood and tissues of animals immunised with gp150env were not different from controls. The results suggest that virus dissemination from the lung tissue was reduced by gag immunization. Thus, systemic DNA immunisation did not prevent infection but induced a reduction in pro-virus load in blood in animals immunised with the gag gene. The end users of the information are other scientists working in the field of SRLV control, or working with other lentiviruses, or working with other plasmid DNA vaccines, or in the general area of vaccines and immune responses, or in the general area of lentivirology. Commercial companies are unlikely to be interested in the information unless it could be shown in larger numbers of animals that the reduced pro-virus load following gag immunisation was significant. Further work is required using larger group sizes to show that gag immunisation results in significantly reduced pro-virus load in blood.

Animals immunised systemically with p55gag showed a significant reduction in lesion scores in the lungs and MLN as determined by histopathological analysis. Animals immunised with gp150env did not show such a reduction. Inclusion of IFN in the priming and first boost did not enhance the protective effect of gag immunisation. Thus, systemic gag immunisation protects against lesion formation but not against infection. The end users of the information are other scientists working in the field of SRLV control, or working with other lentiviruses, or working with other plasmid DNA vaccines, or in the general area of vaccines and immune responses, or in the general area of lentivirology. Commercial companies may be interested in the information that gag immunisation protects against lesion formation and severity. However, further work using larger group sizes is necessary to improve on the observed protective effect before any commercial activity would be warranted.

MVV EV1 was grown in tissue culture and purified by sucrose density centrifugation. The purified EV1 was used as antigen to develop a whole virus ELISA to detect and quantify antibodies to MVV in serum samples. The ELISA can be used to detect seroconversion and antibody titre in experimental animals (as in this project) or to detect antibodies for routine diagnostic purposes. The end users of the information are other scientists working in the field of SRLV control, or working with other lentiviruses, or working with other plasmid DNA vaccines, or in the general area of vaccines and immune responses, or in the general area of lentivirology. Commercial companies may be interested in the whole virus ELISA, but a whole virus ELISA is available commercially already and the ELISA developed within the project has no particular advantage in terms of sensitivity or specificity over the commercial ELISA. Since there are 4 commercially available ELISAs for SRLV diagnosis the potential market is small and it is therefore unlikely that the project ELISA would be commercialised.

Antibody responses were elevated prior to challenge in animals immunised systemically with gag, gagenv, or gagenvIFN. In contrast, T cell proliferative responses were elevated after systemic immunisation with gag, env, or gag+env before challenge. Cytotoxic T cell responses were only observed in animals immunized with gag+env, while IFNg production was not elevated in any group. After challenge, elevated antibody responses were found when gagenv and gagenvIFN immunized animals were compared to controls. All immunised animals showed elevated T cell proliferative responses following challenge, while cytotoxic responses were elevated only in env and gagenv immunized groups. IFNg production by T cells was elevated after gag or env immunization, but not gag+env immunization. The results show that systemic MVV DNA immunisation primes for T cell responses but is less efficient at priming humoral responses. The end users of the information are other scientists working in the field of SRLV control, or working with other lentiviruses, or working with other plasmid DNA vaccines, or in the general area of vaccines and immune responses, or in the general area of lentivirology. Commercial companies may be interested in the information that T cell proliferation to MVV EV1 antigens was not associated with IFNg production, but without the identity of the cytokine(s) produced by the proliferating T cells being known it is unlikely that anything of commercial value can be pursued. Further work is required to determine the identity of the cytokines produced by the primed T cells.

The genes encoding ovine IFNg and IL4 were cloned and ligated into a eukaryotic plasmid expression vector that expresses the IFNg and IL4 proteins in mammalian cells both in vitro and in vivo. These plasmid vectors can be used as immunomodulators in vaccine studies (as in this project) using plasmid DNA vaccination methods. Small ruminant IFNg and IL4 genes have been used previously in other vaccine studies and represent pre-existing knowledge. The end users of the information are other scientists working in the field of SRLV control, or working with other lentiviruses, or working with other plasmid DNA vaccines, or in the general area of vaccines and immune responses, or in the general area of lentivirology. Commercial companies may be interested in the plasmid reagents, but the market for these is likely to be restricted to the small ruminant research community and therefore very small.

In mucosally immunized animals, antibody responses were elevated in env and envIFN groups after plasmid immunization. The antibodies were of the IgG1 subclass, with no IgG2 antibody responses being observed. There was no evidence that MVA boosted the response after week 7. After challenge, most animals sero-converted; though there were no significant differences in antibody titres between immunized and control groups. T cell proliferative responses were found in the groups receiving gag, gagenv, or gagenvIFN immunization before challenge. Immunization with gag induced significantly elevated IFNg production by T cells compared to controls at week 7-post DNA immunization. Increased proliferative responses were observed following MVV challenge in all immunized groups at some point compared to controls. There were no significant differences in IFNg production between immunized and control groups after challenge. Very few T cell cytotoxic responses were observed prior to and after challenge following mucosal immunization. No significant differences were observed. The end users of the information are other scientists working in the field of SRLV control, or working with other lentiviruses, or working with other plasmid DNA vaccines, or in the general area of vaccines and immune responses, or in the general area of lentivirology. Commercial companies are unlikely to be interested in the information.

Animals immunised mucosally with EV1 gag gene showed a lower frequency of moderate/severe lesions in the lungs and MLN. This protective effect was significantly weaker than that observed with systemic immunisation with the gag gene. No protective effect was observed with the env gene and inclusion of IFNg in the priming and first booster did not enhance the protective effect. The end users of the information are other scientists working in the field of SRLV control, or working with other lentiviruses, or working with other plasmid DNA vaccines, or in the general area of vaccines and immune responses, or in the general area of lentivirology. Commercial companies may be interested in the information though they are unlikely to consider commercialisation.

The genes encoding MVV EV1 p55gag precursor and gp160 env precursor were cloned and ligated into a commercially available live attenuated adenovirus vector that express the recombinant proteins in mammalian cells in vitro and in vivo. In addition, the gene encoding MVV EV1 p55gag precursor was cloned and ligated into a commercially available modified vaccinia Ankara (MVA) live attenuated virus vector. An MVA vector encoding the MVV gp150 env precursor was already available before the project started and represents prior knowledge. The adenovirus vectors encoding p55gag or gp150env were unstable and require further manipulation to render them functional. The MVA vectors are functional. These recombinant vectors can used as immunogens for priming or boosting in vaccine studies (as in this project), or for studying the function of the recombinant proteins in cells, or for producing recombinant p55gag or gp150env precursor recombinant proteins in mammalian cells. The end users of the information are other scientists working in the field of SRLV control, or working with other lentiviruses, or working with other plasmid DNA vaccines, or in the general area of vaccines and immune responses, or in the general area of lentivirology. Commercial companies may be interested in the MVA vectors, but until an effective vaccine strategy is produced it is unlikely that there would be a market for the vectors other than for the SRLV research community, which is very small. Similarly, using these vectors to produce recombinant proteins for diagnostic purposes is unlikely since the amounts produced would be too small to be commercially viable. Also, the gag components p25, p17, and p14 can all be produced in large quantities using bacterial expression systems. The gp150 protein would be of potential interest for diagnostic purposes, but the amounts that could be produced are likely to be too small.

Real-time PCR tests to detect and quantify MVV EV1 pro-virus DNA and mRNA in blood and tissues were developed. The primers and dual-labeled probes were designed on the gag and pol genes of EV1. Comparison of the relative sensitivities of the gag and pol assays showed that the gag assay was superior. The sensitivity of the gag real-time assay is approximately 3 copies/reaction. The real-time PCR tests can be used to detect and quantify pro-virus DNA or mRNA (after reverse transcription) in the blood and tissues of experimental animals involved in vaccines studies (as in this project) or in pathogenesis studies. The assays could also be used to detect MVV in routine diagnostic settings, though the assays are specifically designed to detect the EV1 strain and have not been assessed for sensitivity and specificity in relation to field strains. The end users of the information are other scientists working in the field of SRLV control, or working with other lentiviruses, or working with other plasmid DNA vaccines, or in the general area of vaccines and immune responses, or in the general area of lentivirology. Commercial companies may be interested in the real-time PCR tests, but further work is required to determine if the assays have any value in routine diagnosis. Also, the gag and pol PCR tests were designed specifically to detect MVV EV1 strain for the purposes of the project, and were not designed with the routine diagnosis of SRLV in the field in mind. It is unlikely that the assays will be commercialised without an assessment of their value in the field.

Salmonella abortus ovis Rv6 was found not to support stable transformation of eukaryotic expression plasmids encoding MVV gag and env genes. When the plasmids were conditioned by prior transformation into a low endonuclease strain of S. typhimurium, stable transformation of the Rv6 strain was achieved. However, no expression was observed from these plasmids in vitro or in vivo. An Aro- strain of S. typhimurium also failed to give expression of the transgenes. It was concluded that the plasmids used were not functional inside the salmonella strains chosen for the work, and that further work would have to be performed to identify suitable plasmid/salmonella strain combinations that would give expression. The end users of the information are other scientists working in the field of SRLV control, or working with other lentiviruses, or working with other plasmid DNA vaccines, or in the general area of vaccines and immune responses, or in the general area of lentivirology, or in the general area of bacterial vectors. Commercial companies may be interested in the information though they are unlikely to consider commercialisation.

Real-time PCR tests to detect and quantify ovine IFNg and IL4 mRNA in peripheral blood mononuclear cells were developed. The primers were based on published sequences and represent pre-existing knowledge. The real-time PCR tests can be used to detect and quantify mRNA specific for IFNg and IL4 in peripheral blood cells in experimental animals involved in vaccine studies (as in this project) or in pathogenesis studies. The tests could be used as diagnostic reagents in routine diagnosis of SRLV or other pathogens. This would be performed by measuring production of IFNg or IL4 mRNA by T cells after stimulation with antigens specific to the pathogen (MVV EV1 in this project, but any suitable antigen in the case of other pathogens). The end users of the information are other scientists working in the field of SRLV control, or working with other lentiviruses, or working with other plasmid DNA vaccines, or in the general area of vaccines and immune responses, or in the general area of lentivirology, or in the general area of animal disease. Commercial companies may be interested in the IFNg or IL4 real-time PCR tests, but several similar assays to detect bovine IFN? or IL4 mRNA have been published. The market for such assays is likely to be small unless high throughput modifications were developed which allowed the assays to be performed on large numbers of samples. Further work is required to determine if the assays have any value in routine diagnosis.

The p55gag and gp150env precursor genes, which encode the core and envelope proteins of maedi visna virus have been cloned and ligated into a eukaryotic plasmid expression vector that expresses the gag and env recombinant proteins in mammalian cells both in vitro and in vivo. These plasmid vectors can be used as immunogens in vaccine studies (as in this project) using plasmid DNA vaccination methods. The vectors have other potential uses. They could also be used to study the gag and env precursor virus proteins in terms of their basic structure, their interaction with cellular proteins, their processing into individual virus proteins (i.e. p25, p17, p14 core proteins from the gag precursor; and gp135, gp46 envelope proteins from the env precursor), and their use as diagnostic reagents. The env precursor gene from MVV has not been expressed from a plasmid construct to date and this represents an innovative successful element of the project. The end users of the information are other scientists working in the field of SRLV control, or working with other lentiviruses, or working with other plasmid DNA vaccines, or in the general area of vaccines and immune responses, or in the general area of lentivirology. Commercial companies and organisations concerned with SRLV control or regulation may be interested in the plasmid reagents. However, the market for these is likely to be restricted to the SRLV research community and therefore very small. These end users are unlikely to be involved until there is a usable effective vaccine in place.

The genes encoding MVV EV1 p25, p17, and p14 were cloned and ligated into a commercially available prokaryotic expression vector that expresses the recombinant proteins in E. coli. The proteins were purified using a combination of GST and His tags, with the GST being cleaved from the protein. These recombinant proteins can used as antigens in immunological assays such as T cell proliferation, T cell cytokine production, and ELISA (as in the current project). They could also be used as immunogens in vaccine studies, or as antigens in other immunoassays such as western blotting. All three proteins have been produced before and used in immunoassays and consequently they represent prior knowledge. The genes encoding the envelope proteins gp150, gp135, and gp46 were ligated into eukaryotic expression vectors and these were transformed into a variety of mammalian cells. No significant expression was observed. Eventually, fragments of sequence from gp135 and gp46 were codon-optimised and the corresponding DNA synthesised. These fragments were ligated into the expression vectors and transformed into mammalian cells and expression was observed. Thus, efficient expression of the env genes requires codon-optimisation. The end users of the information are other scientists working in the field of SRLV control, or working with other lentiviruses, or working with other plasmid DNA vaccines, or in the general area of vaccines and immune responses, or in the general area of lentivirology. Commercial companies may be interested in the recombinant proteins, but ELISAs that utilise p25 are currently available commercially and the protein produced in the project is not different in any significant way. The p17 and p14 proteins have been used previously in ELISAs produced for research purposes, but the available information suggests that these proteins do not have an advantage over p25 as diagnostic reagents. The market for these is likely to be restricted and very small. The recombinant env proteins may have potential as vaccine candidates or as diagnostic reagents but further work is required to investigate these possibilities.

Animal immunized mucosally with env but not gag showed significantly reduced pro-virus loads in blood but not tissues as measured by real-time PCR. The immunising method utilised plasmid DNA complexed to polyethyleneimine. This method gives transgene expression in sheep lungs in other studies, and therefore immunological responses were expected locally in tissues. The results suggest that virus dissemination from the lung tissue was reduced by env immunization. The end users of the information are other scientists working in the field of SRLV control, or working with other lentiviruses, or working with other plasmid DNA vaccines, or in the general area of vaccines and immune responses, or in the general area of lentivirology. Commercial companies may be interested in the information though they are unlikely to consider commercialisation.

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