Immune clearance of highly pathogenic SIV infection

Abstract

Established infections with the human and simian immunodeficiency viruses (HIV and SIV, respectively) are thought to be permanent with even the most effective immune responses and antiretroviral therapies only able to control, but not clear, these infections. Whether the residual virus that maintains these infections is vulnerable to clearance is a question of central importance to the future management of millions of HIV-infected individuals. We recently reported that approximately 50% of rhesus macaques (RM; Macaca mulatta) vaccinated with SIV protein-expressing rhesus cytomegalovirus (RhCMV/SIV) vectors manifest durable, aviraemic control of infection with the highly pathogenic strain SIVmac239 (ref. 5). Here we show that regardless of the route of challenge, RhCMV/SIV vector-elicited immune responses control SIVmac239 after demonstrable lymphatic and haematogenous viral dissemination, and that replication-competent SIV persists in several sites for weeks to months. Over time, however, protected RM lost signs of SIV infection, showing a consistent lack of measurable plasma- or tissue-associated virus using ultrasensitive assays, and a loss of T-cell reactivity to SIV determinants not in the vaccine. Extensive ultrasensitive quantitative PCR and quantitative PCR with reverse transcription analyses of tissues from RhCMV/SIV vector-protected RM necropsied 69-172 weeks after challenge did not detect SIV RNA or DNA sequences above background levels, and replication-competent SIV was not detected in these RM by extensive co-culture analysis of tissues or by adoptive transfer of 60 million haematolymphoid cells to naive RM. These data provide compelling evidence for progressive clearance of a pathogenic lentiviral infection, and suggest that some lentiviral reservoirs may be susceptible to the continuous effector memory T-cell-mediated immune surveillance elicited and maintained by cytomegalovirus vectors.

Figure 1. Virologic analysis of early RhCMV/SIV vector-mediated protection

a, Plasma viral load (pvl) profiles of 5 RhCMV/SIV vector-vaccinated RM with complete control of viremia following IR SIVmac239 challenge. All 5 RM controlled viremia to below the 30 c. eq./ml limit of quantification for the standard pvl assay used for all pre-necropsy samples, and to below the 1–5 c. eq./ml limit of detection for the ultrasensitive pvl assay used on necropsy samples (individual detection limits for each terminal sample shown). b, Frequencies of peripheral blood memory CD8+ T cells specific for SIV proteins that were (Gag + Pol) or were not (Vif) included in the RhCMV/SIV vectors, shown before and after the onset of the controlled SIV infection. The response frequencies (plotted as mean ± SEM) were normalized to the response frequencies immediately prior to SIV infection for the vaccine-elicited SIVgag- and SIVpol-specific responses, and to the peak frequencies following SIV infection for the de novo SIVvif-specific responses. c, Analysis of tissue-associated SIV DNA and RNA in the 5 RhCMV/SIV vector-protected RM at necropsy using ultrasensitive quantitative PCR/RT-PCR (PID, post-infection day).

Figure 2. Longitudinal analysis of RhCMV/SIV vector-mediated protection after IVag challenge

a, Plasma viral load profiles of Groups A–C RM after infection by repeated, limiting dose, IVag SIVmac239 challenge, with the day of infection defined as the challenge prior to the first above-threshold plasma viral load. The fraction of infected RM that met controller criteria (see Full Methods) in Group A (9 of 16) vs. Groups B and C (0 of 18) was significantly different (p = 0.0002) by two-sided Fisher’s exact test. Note that Rh20363 initially manifested aviremic protection, but then relapsed with productive, albeit controlled, infection at week 31 post-infection. b, Mean (± SEM) frequencies of peripheral blood memory CD8+ T cells specific for SIV proteins that were (Gag + Pol) or were not (Vif) included in the RhCMV/SIV vectors, measured before and after the onset of SIV infection in the 9 Group A RM with initial aviremic control (response frequencies normalized as described in Fig. 1b). The asterisk indicates n = 8 (minus Rh20363 post-relapse) and the plus sign indicates n = 7 (minus Rh20363 and Rh20347, the latter used in the CD8+ cell depletion study described in Suppl. Fig. 14). c, Quantification of tissue-associated SIV RNA (left panel) and DNA (right panel) in the designated longitudinal samples of the 9 Group A controllers vs. 2 representative viremic progressors. All sample types were analyzed at weeks 5, 9 and 17 in all RM. All sample types were analyzed a 4^th time in all controller RM between post-infection weeks 30–40, and PBMC, BM and LN samples were analyzed a 5^th time in 8 of 9 controller RM between post-infection weeks 42 and 55. Each symbol represents a single determination from the designated tissue, except when a multiplication factor is shown (e.g., x 7 indicates a total of 7 samples from different RM with below threshold measurements for that time point).

Figure 3. Virologic analysis of medium- to long-term RhCMV/SIV vector-mediated protection

a,b, Plasma viral load profiles of 10 RhCMV/SIV vector-vaccinated RM that controlled SIV infection after IR challenge (8 long-term; 2 medium-term). The limit of detection for all pre-terminal plasma viral load assays is 30 c. eq./ml; the limit of detection for the ultrasensitive assay used on the terminal sample of the study was ≤1 c. eq./ml. Note that one of the RM with medium-term protection (Rh26467) was CD8+ cell-depleted 10 days prior to the terminal sample. c,d, Quantification of tissue-associated SIV DNA and RNA in 4 long-term and 2 medium-term protected RhCMV/SIV-vaccinated RM studied at necropsy, including the CD8+ cell-depleted RM (Rh26467). e, Assessment of residual replication-competent, cell-associated SIV in medium- and long-term protected RM by adoptive transfer of 6 × 10⁷ hematolymphoid cells (3 × 10⁷ blood leukocytes and 3 × 10⁷ LN cells or, in one transfer from Rh26467, represented by the open symbol, 3 × 10⁷ BM leukocytes and 3 × 10⁷ spleen cells) to SIV-naïve RM with SIV infection in the recipient RM delineated by plasma viral load. Cell transfers from RM with conventional elite SIV control and ART-suppressed SIV infection resulted in rapid onset of SIV infection in the recipient RM, but no SIV infection was observed in RM receiving cells from medium- to long-term RhCMV/SIV vector-protected RM (including Rh26467, analyzed both before and after CD8+ cell depletion).