Genetically engineered simian immunodeficiency viruses (SIV) that is limited to a single cycle of infection was evaluated as a nonreplicating AIDS vaccine approach for rhesus macaques. Four Mamu-A*01؉ macaques were inoculated intravenously with three concentrated doses of single-cycle SIV (scSIV). Each dose consisted of a mixture of approximately equivalent amounts of scSIV strains expressing the SIV mac 239 and SIV mac 316 envelope glycoproteins with mutations in nef that prevent major histocompatibility complex (MHC) class I downregulation. Viral loads in plasma peaked between 10 4 and 10 5 RNA copies/ml on day 4 after the first inoculation and then steadily declined to undetectable levels over the next 4 weeks. SIV Gag-specific T-cell responses were detected in peripheral blood by MHC class I tetramer staining (peak, 0.07 to 0.2% CD8 ؉ T cells at week 2) and gamma interferon (IFN-␥) enzyme-linked immunospot (ELISPOT) assays (peak, 50 to 250 spot forming cells/10 6 peripheral blood mononuclear cell at week 3). Following the second and third inoculations at weeks 8 and 33, respectively, viral loads in plasma peaked between 10 2 and 10 4 RNA copies/ml on day 2 and were cleared over a 1-week period. T-cell-proliferative responses and antibodies to SIV were also observed after the second inoculation. Six weeks after the third dose, each animal was challenged intravenously with SIV mac 239. All four animals became infected. However, three of the four scSIV-immunized animals exhibited 1 to 3 log reductions in acute-phase plasma viral loads relative to two Mamu-A*01 ؉ control animals. Additionally, two of these animals were able to contain their viral loads below 2,000 RNA copies/ml as late as 35 weeks into the chronic phase of infection. Given the extraordinary difficulty in protecting against SIV mac 239, these results are encouraging and support further evaluation of lentiviruses that are limited to a single cycle of infection as a preclinical AIDS vaccine approach.

We have devised a novel approach for producing simian immunodeficiency virus (SIV) strains and, potentially, human immunodeficiency virus type 1 (HIV-1) strains that are limited to a single cycle of infection. Unlike previous lentiviral vectors, our single-cycle SIV is capable of expressing eight of the nine viral gene products and infected cells release immature virus particles that are unable to complete subsequent rounds of infection. Single-cycle SIV (scSIV) was produced by using a two-plasmid system specifically designed to minimize the possibility of generating replication-competent virus by recombination or nucleotide reversion. One plasmid carried a full-length SIV genome with three nucleotide substitutions in the gag-pol frameshift site to inactivate Pol expression. To ensure inactivation of Pol and to prevent the recovery of wild-type virus by nucleotide reversion, deletions were also introduced into the viral pol gene. In order to provide Gag-Pol in trans, a Gag-Pol-complementing plasmid that included a single nucleotide insertion to permanently place gag and pol in the same reading frame was constructed. We also mutated the frameshift site of this Gag-Pol expression construct so that any recombinants between the two plasmids would remain defective for replication. Cotransfection of both plasmids into 293T cells resulted in the release of Gag-Pol-complemented virus that was capable of one round of infection and one round of viral gene expression but was unable to propagate a spreading infection. The infectivity of scSIV was limited by the amount of Gag-Pol provided in trans and was dependent on the incorporation of a functional integrase. Single-cycle SIV produced by this approach will be useful for addressing questions relating to viral dynamics and viral pathogenesis and for evaluation as an experimental AIDS vaccine in rhesus macaques.

Rtsl is a high-molecular-weight (126 x 106) plasmid encoding resistance to kanamycin. It expresses unusual temperature-sensitive phenotypes, which affect plasmid maintenance and replication, as well as host cell growth. We have cloned the essential replication region of Rtsl from pAK8, a smaller derivative which is phenotypically similar to Rtsl. Restriction endonuclease digests of isolated pAK8 deoxyribonucleic acid were allowed to "self-ligate" (ligation without an additional cloning vector) and subsequently were used to transform Escherichia coli strain 20S0 to kanamycin resistance. Screening of these strains for the phenotypes of thermosensitive host growth and temperature-dependent plasmid elimination demonstrated that these two properties were expressed independently. Furthermore, it was shown that the Rtsl replication locus per se is not necessarily responsible for altered host growth at the nonpermissive temperature. The kanamycin resistance fragment of pAK8 was also cloned into pBR322. Electrophoretic analysis of BamHI restriction enzyme digests of this plasmid and similar digests of an Rtsl miniplasmid has allowed the identification of an 18.6-megadalton fragment carrying the replication locus and a 14.1-megadalton fragment carrying the kanamycin resistance gene. Rtsl is a high-molecular-weight R factor of the T-incompatibility group (4) which confers upon its host resistance to kanamycin. When examined in Escherichia coli 20S0, the Rtsl plasmid copy number is stringently regulated (10), and the molecular size is approximately 126 megadaltons (Mdal) (10, 11). The unique feature of Rtsl is the thermosensitivity of strains harboring this plasmid during incubation at 42.5°C. At 42.5°C, the efficiency of conjugal transfer is markedly diminished (30), and the Rtsl-mediated resistance to T-even bacteriophages is not expressed (12, 17, 36). In addition, there is a detrimental effect on the growth of cells carrying Rtsl (27, 35), and significant membrane lesions have been observed (12). Furthermore, an alteration occurs in the control of autonomous maintenance, causing the subsequent appearance of Rsegregants (11). The ability of Rtsl DNA to replicate as a closed covalent circle (CCC) is impaired at 42.50C (11, 33), which may correlate with the effect of Rtsl on host growth at the nonpermissive temperature. Recently, two groups have selected strains