News and Publications

Viral Pathogenesis, DNA Vaccines, and Ribozymes

J.L. Whitton, S. Harkins, D.E. Hassett, I. Mena, F. Rodriguez, M.K. Slifka, J. Zhang

Ongoing studies in this laboratory are concerned with making life as difficult for viruses as they do for us. To this end, we focus on 3 aspects of the interaction between viruses and their hosts. First, we study antiviral vaccination to understand how vaccines work and how they might be improved. Second, we study viral pathogenesis to determine how these agents cause disease. Third, we are evaluating ribozymes as a therapeutic measure to intervene in ongoing infection.

ANTIVIRAL VACCINES AND DNA IMMUNIZATION

Antiviral vaccines are a landmark in medical care and have led to the eradication of smallpox; the approaching eradication of poliomyelitis; and the diminution, at least in developed countries, of a variety of microbial diseases. Nevertheless, new challenges (e.g., HIV disease) have arisen, and old foes (e.g., tuberculosis, measles) threaten to reappear. A recent approach to immunization is the direct inoculation of plasmid DNA.

This approach has great promise but, surprisingly, the precise mechanism that underlies this technology remains unclear. We showed that inoculation of DNA into an immune host results in protective immunity and a florid local inflammatory response, rich in CD4⁺ and CD8⁺ T cells. We also found that DNA immunization can be improved by covalently attaching the viral protein to ubiquitin, a cellular protein that "marks" other proteins for degradation. The ubiquitinated protein is much better than the unmodified viral protein at inducing protective cytotoxic T lymphocytes, but it cannot induce antibodies. DNA immunization also works well in newborn animals, as early as the day of birth. In other ongoing studies, we are evaluating the use of minigenes--isolated T- and B-cell epitopes--to induce protective immunity, determining subdominant epitopes of cytotoxic T lymphocytes, and investigating the capacity of soluble proteins to induce cytotoxic T lymphocytes.

VIRAL PATHOGENESIS

Coxsackievirus B3 is an important human pathogen that causes a variety of clinical syndromes, including myocarditis. It is strongly implicated as an underlying cause of idiopathic dilated cardiomyopathy, the most common indicator for heart transplantation in young males. We previously showed the importance of CD4⁺ and CD8⁺ T cells in the control of virus-induced myocarditis and in its immunopathologic cause. We recently began to study the role of the cytolytic protein perforin in this abnormality.

Myocarditis is markedly reduced in the absence of perforin, but viral clearance is unaffected. Removal of perforin therefore enables us to uncouple the antiviral and immunopathologic consequences of infection with coxsackievirus B3 and indicates that perforin is a promising therapeutic target. We are evaluating DNA immunization in the coxsackievirus B3 model and are constructing recombinant coxsackieviruses to more precisely understand the immunologic and immunopathologic characteristics of this infection.

ANTIVIRAL RIBOZYMES

Antibiotic therapy for bacterial infections is conceptually simple, because these organisms in general replicate extracellularly. In contrast, viruses replicate intracellularly, subverting the machinery of host cells. The intimate interaction between viruses and host proteins in part explains the difficulty in producing specific antiviral agents. Antisense approaches, which rely on the uniqueness of the viral nucleic acid sequence, offer a tantalizing alternative approach to antiviral therapy.

Previously, we showed that antiviral ribozymes expressed in tissue culture can sometimes, but not always, diminish viral titers. To determine whether ribozymes expressed in the right place, at the right time, and in a good concentration can interfere with viral infection and disease in vivo, we are attempting to express antiviral ribozymes in the CNS of trans-genic mice.

PUBLICATIONS

Bell, A., Whitton, J.L., Fujinami, R.S. Antisense mediated resistance to measles virus infection in HeLa cells. J. Infect. Dis. 176:258, 1997.

Gebhard, J.R., Perry, C.M., Harkins, S., Lane, T.E., Mena, I., Asensio, V.C., Campbell, I.L., Whitton, J.L. Coxsackievirus B3-induced myocarditis: Perforin exacerbates disease, but plays no detectable role in virus clearance. Am. J. Pathol., in press.

Hassett, D.E., Zhang, J., Whitton, J.L. Neonatal DNA immunization with an internal viral protein is effective in the presence of maternal antibodies and protects against subsequent viral challenge. J. Virol. 71:7881, 1997.

Rodriguez, F., An, L.L., Harkins, S., Zhang, J., Yokoyama, M., Widera, G., Fuller, J.T., Kincaid, C., Campbell, I.L., Whitton, J.L. DNA immunization with minigenes: Low frequency of memory CTL and inefficient antiviral protection are rectified by ubiquitination. J. Virol., in press.

Rodriguez, F., Zhang, J., Whitton, J.L. DNA immunization: Ubiquitination of a viral protein enhances CTL induction, and antiviral protection, but abrogates antibody induction. J. Virol. 71:8497, 1997.

van der Most, R.G., Murali-Krishna, K., Whitton, J.L., Oseroff, C., Alexander, J., Southwood, S., Sidney, J., Chesnut, R.W., Sette, A., Ahmed, R. Identification of D^b and K^b restricted subdominant cytotoxic T-cell responses in lymphocytic choriomeningitis virus infected mice. Virology 240:158, 1998.

Whitton, J.L. Infection: Immunological barriers. In: Encyclopedia of Life Sciences. Robertson, S. (Ed.). McMillan Reference Ltd., London, in press.

Whitton, J.L. An overview of antigen presentation and its central role in the immune response. In: Antigen Presentation. Whitton, J.L. (Ed.). Springer-Verlag, New York, 1998, p. 1.

Whitton, J.L., Gebhard, J.R., Mena, I. Antisense and ribozyme approaches to the control of virus infection. In: Persistent Viral Infections. Ahmed, R., Chen, I. (Eds.). Wiley, New York, in press.