Research Accomplishment Reports 2007

Control of Equine Infectious Anemia (EIA)

C.J. Issel, R.F. Cook, S.J. Cook
Department of Veterinary Sciences

 

Project Description

Equine infectious anemia virus (EIAV) is a lentivirus closely related to HIV. These viruses have evolved to persist in their hosts despite the presence of strong immune responses and have proven to be remarkably refractory to the development of effective vaccines. Lentiviruses can undergo considerable antigenic variation, particularly in the surface unit (SU) component of the envelope glycoproteins. Therefore, variation in SU is believed to be a major contributor to viral persistence and one of the primary impediments to the design of fully protective vaccines. However, the effect of progressively increasing levels of env gene variation on vaccine efficacy has not been evaluated.

To address this fundamental problem three groups of eight ponies were immunized with a live-attenuated EIAV vaccine strain previously demonstrated to provide protection against a virulent challenge virus containing homologous env gene sequences. The first immunized pony group was challenged with a virus containing homologous SU sequences (0%), while the second and third groups were challenged with viruses that differed at the amino acid level in SU by 6% and 13% respectively. The challenge viruses were all derived from our standard EIAVuk3 pathogenic infectious molecular clone and were homologous in all sequences except those encoding SU. While, clinical signs were undetectable in 87.5% of the 0% challenge group, this figure declined to 62.5% and 37.5% in the 6% and 13% challenge groups, demonstrating an inverse linear correlation between divergence in SU and protection from disease. These studies, performed in collaboration with scientists at the University of Pittsburgh, show for the first time that relatively minor variation in SU has a significant impact on vaccine efficacy.

We have also collaborated with the Irish Equine Centre on molecular characterization of the strain responsible for the 2006 EIA outbreak in Ireland. To date the nucleotide sequence of the viral gag gene has been determined. Comparison to North American and Asian EIAV isolates revealed that although sequences encoding the p26 major capsid antigen were 90% homologous, regions encoding p15 and p9 displayed extensive diversity with only 75% and 50% homology respectively.

Another component of the EIA control program is to develop methods to broaden immune responses. Immunodominance restricts immune responses to a small minority of the potential epitopes present within infectious agents and therefore might be a weakness in the case of highly mutable pathogens such as EIAV. Therefore, we have produced expression enhanced versions of EIL-7 and EIL-15 as under certain circumstances these cytokines are capable of circumventing the immunodominance mechanism(s). During molecular cloning of EIL-7 from normal horse tissue, 3 splice variants were identified, similar to those detected in some cancerous human tissues. Although the function of these variants is unknown there is speculation they might act as IL-7 antagonists. Contrary to this belief, we have demonstrated that all forms of EIL-7 stimulate 3H thymidine incorporation by horse PBMC.

Impact

The study on the effects of env gene variation on vaccine efficacy has tremendous potential implications for future research on the immunological control of all lentiviral infections including HIV. Although numerous immunization strategies have been evaluated in the EIAV/horse system, the best evidence for induction of fully protective immune responses has been seen with live attenuated vaccines. However, our results demonstrate that in many horses even these responses are unable to provide protection against relatively modest variation in SU. An important aspect of this study is that with the exception of SU all major structural antigens (Gag, Pol and the transmembrane component of the envelope glycoprotein) were identical between the challenge viruses and the vaccine strain. Therefore, it might be predicted that immune responses directed against all the shared homologous viral proteins, representing 82% of the viral coding capacity and/or against conserved regions within SU should have been sufficient to provide protection in most cases. Surprisingly, this was not observed, as the number of animals succumbing to disease rose from approximately one third to nearly two thirds as variation in SU increased from 6% to 13%. Although, it remains to be determined if the proportion of protected animals would continue to decline by further increases in the diversity of SU, our results demonstrate that for over 60% of the horse population, SU variation is a primary determinant of lentiviral vaccine efficacy. This information will not only dictate the future of EIAV vaccine research but will have far reaching consequences for other lentiviral systems including HIV.

Nucleotide sequence analysis of an isolate from the 2006 outbreak of EIAV in Ireland has demonstrated more extensive variation in the viral gag gene than postulated originally, particularly within Gag p15 and Gag p9. However, overall variation in Gag p26 was limited to under 10% which is an important finding considering this is a major antigen component of all current USDA approved AGID and ELISA tests for the serological detection of EIAV infections. Finally, as a result, of our work on equine cytokines, the University of Kentucky is currently pursuing patent protection for the EIL-7 and EIL-15 enhanced expression constructs.

Publications

Tagmyer, T., Craigo, J.K., Cook, S.J., Issel, C.J. and Montelaro, R.C. (2007). Characterization of Broadly Reactive T-Helper and CTL Epitopes in the Envelope Proteins of Equine Infectious Anemia Virus, Journal of General Virology, 88: 1324-1336.

Issel, C.J., Cordes, T.R., and Halstead, S. (2007). Control of EIA Should Take Some New Directions. Article#8787 on TheHorse.com (accessed 2/28/2007 at http://www.thehorse.com/ViewArticle.aspx?ID=8787).

Issel, C.J. (2007). Equine Infectious Anemia, The Horse 24(3): 114.

Issel, C.J., and Anderson, G. (2007). The Stigma of EIA is Misplaced, Article#9038 on TheHorse.com (accessed 3/2/2007 at http://www.thehorse.com/ViewArticle.aspx?ID=9038).

Issel, C.J., and Sadlier, M. (2007). Reducing the Risks of Infection in Veterinary Practices: Recent Lessons Learned with Equine Infectious Anemia (EIA), Page 89 to 102 in Proc. Hagyard Bluegrass Equine Symposium 2007, Lexington, KY.

Craigo, J.K., Zhang, B., Barnes, S., Tagmyer, T.L., Cook, S.J., Issel, C.J., Montelaro, R.C. (2007). Envelope Variation as a Primary Determinant of Lentiviral Vaccine Efficacy, Proc. National Acadent of Sciences, 104: 15105-15110.

Quinlivan, M., Cook, R.F., and Cullinane, A. (2007). Real-Time Quantitative RT-PCR and PCR Assays for a Novel European Field Isolate of Equine Infectious Anaemia Virus Based on Sequence Determination of the gag Gene, Veterinary Record, 160: 611-618.