Immunization with Single-Cycle SIV Significantly Reduces Viral Loads After an Intravenous Challenge with SIVmac239

Strains of simian immunodeficiency virus (SIV) that are limited to a single cycle of infection were evaluated for the ability to elicit protective immunity against wild-type SIVmac239 infection of rhesus macaques by two different vaccine regimens. Six animals were inoculated at 8-week intervals with 6 identical doses consisting of a mixture of three different envelope variants of single-cycle SIV (scSIV). Six additional animals were primed with a mixture of cytoplasmic domain-truncated envelope variants of scSIV and boosted with two doses of vesicular stomatitis virus glycoprotein (VSV G) trans-complemented scSIV. While both regimens elicited detectable virus-specific T cell responses, SIV-specific T cell frequencies were more than 10-fold higher after boosting with VSV G trans-complemented scSIV (VSV G scSIV). Broad T cell recognition of multiple viral antigens and Gag-specific CD4+ T cell responses were also observed after boosting with VSV G scSIV. With the exception of a single animal in the repeated immunization group, all of the animals became infected following an intravenous challenge with SIVmac239. However, significantly lower viral loads and higher memory CD4+ T cell counts were observed in both immunized groups relative to an unvaccinated control group. Indeed, both scSIV immunization regimens resulted in containment of SIVmac239 replication after challenge that was as good as, if not better than, what has been achieved by other non-persisting vaccine vectors that have been evaluated in this challenge model. Nevertheless, the extent of protection afforded by scSIV was not as good as typically conferred by persistent infection with live, attenuated SIV. These observations have potentially important implications to the design of an effective AIDS vaccine, since they suggest that ongoing stimulation of virus-specific immune responses may be essential to achieving the degree of protection afforded by live, attenuated SIV

Comparison of Plasma Viremia and Antibody Responses in Macaques Inoculated with Envelope Variants of Single-Cycle Simian Immunodeficiency Virus Differing in Infectivity and Cellular Tropism▿

Molecular differences in the envelope glycoproteins of human immunodeficiency virus type 1 and simian immunodeficiency virus (SIV) determine virus infectivity and cellular tropism. To examine how these properties contribute to productive infection in vivo, rhesus macaques were inoculated with strains of single-cycle SIV (scSIV) engineered to express three different envelope glycoproteins with full-length (TMopen) or truncated (TMstop) cytoplasmic tails. The 239 envelope uses CCR5 for infection of memory CD4+ T cells, the 316 envelope also uses CCR5 but has enhanced infectivity for primary macrophages, and the 155T3 envelope uses CXCR4 for infection of both naive and memory CD4+ T cells. Separate groups of six rhesus macaques were inoculated intravenously with mixtures of TMopen and TMstop scSIVmac239, scSIVmac316, and scSIVmac155T3. A multiplex real-time PCR assay specific for unique sequence tags engineered into each virus was then used to measure viral loads for each strain independently. Viral loads in plasma peaked on day 4 for each strain and were resolved below the threshold of detection within 4 to 10 weeks. Truncation of the envelope cytoplasmic tail significantly increased the peak of viremia for all three envelope variants and the titer of SIV-specific antibody responses. Although peak viremias were similar for both R5- and X4-tropic viruses, clearance of scSIVmac155T3 TMstop was significantly delayed relative to the other strains, possibly reflecting the infection of a CXCR4+ cell population that is less susceptible to the cytopathic effects of virus infection. These studies reveal differences in the peaks and durations of a single round of productive infection that reflect envelope-specific differences in infectivity, chemokine receptor specificity, and cellular tropism

MHC class I alleles present in the rhesus macaques selected for this study.

<p>MHC class I typing was performed by allele-specific PCR as described in Kaizu <i>et al.</i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000272#ppat.1000272-Kaizu1" target="_blank">[80]</a>.</p

Boosting with VSV G trans-complemented single-cycle SIV significantly increases virus-specific CD8⁺ T cell frequencies.

<p>Responses are shown for Mm 182-99, Mm 89-00, and Mm 377-03 in Group A (A), and Mm 383-02, Mm 328-02, and Mm 295-00 in Group B (B). Whole blood was stained with monoclonal antibodies to CD3 and CD8, and one of the following MHC class I tetramers; Mamu-A*01-Gag_181–189, Mamu-A*01-Tat_28–35, or Mamu-A*02-Nef_159–167. Samples were analyzed by flow cytometry, and the percentage of tetramer-positive cells was determined at each time point after gating on the CD3⁺CD8⁺ lymphocyte population. Responses greater than 0.05% (dashed line) are considered positive.</p

Both immunization regimens elicited antibody responses capable of neutralizing lab-adapted, but not primary isolates of SIV.

<p>Binding antibody responses were measured for Group A (A) and Group B (B) animals at a 1/20 dilution of plasma on ELISA plates coated with whole-virus lysate. The mean and standard deviation (error bars) of the antibody responses in each group are plotted in gray. Arrows indicate the time points of each inoculation, and the dashed line indicates the mean level of non-specific binding for pre-immune plasma at the time of the first inoculation. (C) SIV-specific neutralizing antibody titers at the time of challenge. Serial two-fold dilutions of plasma were tested for the ability to inhibit infection of target cells harboring a Tat-inducible SEAP reporter gene by lab-adapted SIV_mac251 (SIV_mac251_LA), SIV_mac316, SIV_mac155T3, and SIV_mac239. The dashed line in each plot indicates 50% neutralization of virus infectivity.</p

Broad T cell recognition of each of the eight viral gene products expressed by single-cycle SIV.

<p>IFNγ ELISPOT responses are shown for Group B animals one week after each boost with VSV G trans-complemented scSIV, week 13 (A) and week 25 (B). Total responses to all 8 viral proteins are indicated in the center of each plot in SFC per million PBMC.</p

Immunization with single-cycle SIV elicits virus-specific CD4⁺ T helper cell responses.

<p>SIV Gag-specific CD4⁺ T cell responses were measured by intracellular cytokine staining. (A) CD4⁺ T cell responses are shown for each of the animals in Group B two weeks after the first boost (week 14). The gated populations indicate CD4⁺ T cells that have upregulated CD69 and TNFα after a 6-hour incubation in medium alone (R10) or in medium containing a set of overlapping Gag peptides (Gag). (B) Changes in the frequency of Gag-specific CD4⁺ T cells after each boost are summarized as bar graphs. The percentages of activated CD69⁺TNFα⁺ cells reflect the difference between responses to the Gag peptide pool and background responses in R10 medium. The arrows indicate booster inoculations on weeks 12 and 24.</p

Viral RNA loads were detectable in plasma after each dose of single-cycle SIV.

<p>(A) Animals in Group A were inoculated with 6 identical doses of the same cryopreserved stocks of scSIV_mac239 TM_open, scSIV_mac316 TM_open, and scSIV_mac155T3 TM_open at 8-week intervals. (B) Animals in Group B were primed with a mixture of scSIV_mac239 TM_stop, scSIV_mac316 TM_stop, and scSIV_mac155T3 TM_stop and boosted with VSV G trans-complemented scSIV_mac239 TM_open on weeks 12 and 24. Viral loads were measured independently for each strain of scSIV using a quantitative multiplex real-time RT-PCR assay specific for unique sequence tags (<i>ggr</i>, <i>cao</i>, and <i>gsa</i>) engineered into each viral genome <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000272#ppat.1000272-DeGottardi1" target="_blank">[32]</a>. The threshold of detection for this assay was 30 RNA copy eq./ml (dotted line).</p