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J Virol, June 1998, p. 5174-5181, Vol. 72, No. 6
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
DNA Immunization with Minigenes: Low Frequency of
Memory Cytotoxic T Lymphocytes and Inefficient Antiviral Protection Are
Rectified by Ubiquitination
Fernando
Rodriguez,1
Ling Ling
An,1
Stephanie
Harkins,1
Jie
Zhang,1
Masayuki
Yokoyama,2
Georg
Widera,3
James T.
Fuller,3
Carrie
Kincaid,1
Iain L.
Campbell,1 and
J.
Lindsay
Whitton1,*
Department of Neuropharmacology, The Scripps
Research Institute, La Jolla, California
920371;
PowderJect Vaccines Inc.,
Madison, Wisconsin 537113; and
Institute
of Biomedical Engineering, Tokyo Women's Medical College,
Shinjuku-ku, Tokyo 162, Japan2
Received 29 December 1997/Accepted 17 March 1998
Our previous studies have shown that isolated cytotoxic T
lymphocyte (CTL), B-cell, and T-helper epitopes, for which we coined the term minigenes, can be effective vaccines; when expressed from
recombinant vaccinia viruses, these short immunogenic sequences confer
protection against a variety of viruses and bacteria. In addition, we
have previously demonstrated the utility of DNA immunization using
plasmids encoding full-length viral proteins. Here we combine the two
approaches and evaluate the effectiveness of minigenes in DNA
immunization. We find that DNA immunization with isolated minigenes
primes virus-specific memory CTL responses which, 4 days following
virus challenge, appear similar in magnitude to those induced by
vaccines known to be protective. Surprisingly, this vigorous CTL
response fails to confer protection against a normally lethal virus
challenge, although the CTL appear fully functional because, along with
their high lytic activity, they are similar in affinity and cytokine
secretion to CTL induced by virus infection. However this DNA
immunization with isolated minigenes results in a low CTL precursor
frequency; only 1 in ~40,000 T cells is epitope specific. In
contrast, a plasmid encoding the same minigene sequences covalently
attached to the cellular protein ubiquitin induces protective immunity
and a sixfold-higher frequency of CTL precursors. Thus, we show that
the most commonly employed criterion to evaluate CTL responses
the
presence of lytic activity following secondary stimulation
does not
invariably correlate with protection; instead, the better correlate of
protection is the CTL precursor frequency. Recent observations indicate
that certain effector functions are active in memory CTL and do not require prolonged stimulation. We suggest that these early effector functions of CTL, immediately following infection, are critical in
controlling virus dissemination and in determining the outcome of the
infection. Finally, we show that improved performance of the
ubiquitinated minigenes most probably requires polyubiquitination of
the fusion protein, suggesting that the enhancement results from more
effective delivery of the minigene to the proteasome.
*
Corresponding author. Mailing address: Dept. of
Neuropharmacology, CVN-9, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Phone: (619) 784-7090. Fax: (619) 784-7380. E-mail: lwhitton{at}scripps.edu.

Manuscript 11246-NP from The Scripps Research Institute.
J Virol, June 1998, p. 5174-5181, Vol. 72, No. 6
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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