Identification of highly attenuated mutants of simian immunodeficiency virus

Abstract

Deletion mutants of the pathogenic clone of simian immunodeficiency virus isolate 239 (SIVmac239) were derived that are missing nef, vpr, and upstream sequences (US) in the U3 region of the LTR (SIVmac239 delta3), nef, vpx, and US (SIVmac239 delta3x), and nef, vpr, vpx, and US (SIVmac239 delta4). These multiply deleted derivatives replicated well in the continuously growing CEMx174 cell line and were infectious for rhesus monkeys. However, on the basis of virus load measurements, strength of antibody responses, and lack of disease progression, these mutants were highly attenuated. Measurements of cell-associated viral load agreed well with assays of plasma viral RNA load and with the strengths of the antibody responses; thus, these measurements likely reflected the extent of viral replication in vivo. A derivative of SIVmac239 lacking vif sequences (SIVmac239 delta vif) could be consistently grown only in a vif-complementing cell line. This delta vif virus appeared to be very weakly infectious for rhesus monkeys on the basis of sensitive antibody tests only. The weak antibody responses elicited by SIVmac239 delta vif were apparently in response to low levels of replicating virus since they were not elicited by heat-inactivated virus and the anti-SIV antibody responses persisted for greater than 1 year. These results, and the results of previous studies, allow a rank ordering of the relative virulence of nine mutant strains of SIVmac according to the following order: delta vpr > delta vpx > delta vpr delta vpx approximately delta nef > delta3 > delta3x > or = delta4 > delta vif > delta5. The results also demonstrate that almost any desired level of attenuation can be achieved, ranging from still pathogenic in a significant proportion of animals (delta vpr and delta vpx) to not detectably infectious (delta5), simply by varying the number and location of deletions in these five loci.

FIG. 1

Cell-associated virus loads. The numbers of infectious cells in PBMC are expressed in code on the y axis as a function of weeks after inoculation of virus. Each curve represents the average of multiple animals. For SIVmac239, averages of 16 and 18 animals were used for weeks 1 and 2; averages of 5 to 9 animals were used for the subsequent times due to the lack of sampling of individual animals at those times or due to the planned sacrifice of the animal. Five animals from experiment B were used for Δ3, two animals from experiment C were used for Δ3x, and two animals from experiment D were used for Δ4. Four animals that received SIVmac239Δ4 at TSI Mason Laboratories yielded very similar results (22a). Code for PBMC load: 0, virus was not recovered even when 10⁶ PBMC were used; 1, virus was recovered with an average of 10⁶ but not fewer PBMC; 2, 333,333 PBMC; 3, 111,111 PBMC; 4, 37,037 PBMC; 5, 12,345 PBMC; 6, 4,115 PBMC; 7, 1,371 PBMC; 8, 457 PBMC.

FIG. 2

Plasma SIV RNA levels at the indicated weeks postinoculation for animals infected with wild-type SIVmac239 (A), Δ3 (B), Δ3x (C), and Δ4 (D) for the 12 animals in experiment A and two controls analyzed separately. Dashed lines indicate threshold sensitivity of the assay, 300 copy eq/ml.

FIG. 3

Early plasma SIV RNA loads. The median plasma RNA values at week 1 postinoculation (A) and at peak (B) for each group of infected animals are derived from the data shown in Fig. 2. The dashed lines indicate the threshold sensitivity of the assay.

FIG. 4

Antibody responses. Plasma samples from the 12 rhesus monkeys infected in parallel in experiment A were tested for the development of anti-SIV antibody responses by ELISA reactivity to whole lysed virus. All of the samples in panels A to C were tested on the same day under identical conditions. The samples from the Δ4-infected monkeys were retested under the high-sensitivity ELISA conditions (D).

FIG. 5

Replication of SIVmac239Δvif. Stock viruses containing 10 ng of p27 were tested in parallel for replication in CEMx174 cells and in the vif-completing CEMx174 cell line.

FIG. 6

Synthesis of vpx and vpr by SIVmac239Δvif. Virion proteins of SIVmac239open (wild type [WT]), SIVmac239Δvpr (ΔVPR), and SIVmac239Δvif (Δvif) containing 200 ng of p27^gag were separated in an SDS–15% polyacrylamide gel and electroblotted onto membrane filters. Vpr, Vpx, and p27^gag proteins were detected by anti-Vpr (αVPR), anti-Vpx (αVPX), and anti-p27^gag (αp27) antisera, respectively, using the Amersham ECL detection system. Sizes are indicated in kilodaltons.

FIG. 7

Antibody responses to SIV in rhesus monkeys inoculated with SIVmac239Δvif. Sequential plasma samples from the rhesus monkeys inoculated with SIVmac239Δvif containing 360 ng of p27 were tested for the presence of antibodies to SIV under high-sensitivity conditions.

FIG. 8

Effect of heat inactivation of SIVmac239Δvif on antibody responses in inoculated rhesus monkeys. Sequential plasma samples were analyzed as described in the legend to Fig. 7.

FIG. 9

Western blot reactivity of selected sera. WT, wild type.