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Immune clearance of highly pathogenic SIV infection
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Nature Oct 2013
"In summary, the ability of CMV vectors to implement continuous, long-term, and potent antipathogen immune surveillance makes them promising candidates for vaccine strategies intended to prevent and cure HIV/AIDS, as well as other chronic infections."
"In the past 5 years, the HIV/AIDS vaccine field has concluded that a prophylactic HIV/AIDS vaccine must prevent or eliminate HIV infection, as it is thought that any residual infection runs a high risk of eventual progression13. Our demonstration here that the virus-specific, effector memory T cells maintained by a persistent vector can shut down productive SIV infection, and by maintaining immune surveillance over time, functionally cure and possibly eradicate this infection, indicates that an effector memory T-cell-targeted HIV/AIDS vaccine could (by itself, or combined with antibody-targeted approaches) provide meaningful long-term efficacy. Our results also suggest that an effector memory T-cell-targeted vaccine might contribute to HIV cure strategies. Although the SIV reservoirs that initially develop in RhCMV/SIV vector-vaccinated controllers are smaller in size, and possibly different in character from HIV/SIV reservoirs in the setting of ART administration initiated in chronic infection, it is conceivable that the indefinitely persistent, unconventionally targeted10, viral-specific T cells elicited and maintained by CMV vectors-alone or in combination with agents designed to activate HIV gene expression1, 2, 12-might exert potent immune pressure on cells with any HIV protein expression (including expression of viral antigen by stochastically activated, latently infected cells) and thereby facilitate depletion of residual HIV reservoirs in patients on suppressive ART. It is also possible that these responses might stringently control recrudescent 'rebound' infection after ART withdrawal in a manner analogous to their control of primary SIV infection in this study. In summary, the ability of CMV vectors to implement continuous, long-term, and potent antipathogen immune surveillance makes them promising candidates for vaccine strategies intended to prevent and cure HIV/AIDS, as well as other chronic infections."
Immune clearance of highly pathogenic SIV infection
Nature Oct 2013
Scott G.Hansen1*, Michael Piatak Jr2*, Abigail B. Ventura1, Colette M.Hughes1, Roxanne M. Gilbride1, Julia C. Ford1, KelliOswald2,
Rebecca Shoemaker2, Yuan Li2, Matthew S. Lewis1, Awbrey N. Gilliam1, Guangwu Xu1, Nathan Whizin1, Benjamin J. Burwitz1,
Shannon L. Planer1, John M. Turner1, Alfred W. Legasse1, Michael K. Axthelm1, Jay A. Nelson1, Klaus Fruh1, Jonah B. Sacha1,
Jacob D. Estes2, Brandon F. Keele2, Paul T. Edlefsen3, Jeffrey D. Lifson2 & Louis J. Picker1
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 infections1, 2, 3, 4. 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.
Clinical and experimental observations have suggested that HIV and SIV infections might be vulnerable to immune control or pharmacological clearance in the first few hours to days of infection, before both the viral amplification needed for efficient mutational escape and the establishment of the highly resilient viral reservoir that sustains the infection4, 6, 7, 8. Cytomegalovirus (CMV) vectors were designed to exploit this putative window of vulnerability, based on their ability to elicit and indefinitely maintain high frequency, effector-differentiated, and broadly targeted virus-specific T cells in potential sites of early viral replication5, 9, 10. Indeed, the pattern of protection observed in approximately 50% of RhCMV/SIV vector-vaccinated RM after intrarectal SIVmac239 challenge was consistent with early immunological interception of the nascent SIV infection at the portal of viral entry and immune control before irreversible systemic spread5. Protected RM manifested a very transient viraemia at the onset of infection, followed by control of plasma SIV levels to below the threshold levels of quantification, except for occasional plasma viral 'blips' that waned over time, and after one year, demonstrated only trace levels of tissue-associated SIV RNA and DNA at necropsy using ultrasensitive assays. The occurrence of plasma viral blips and the recurrence of 'breakthrough' progressive SIV infection in 1 of the 13 RhCMV/SIV vector-protected RM at day 77 after infection indicated that SIV was not immediately cleared from these protected RM, but the failure to find more than trace levels of SIV nucleic acid in systemic lymphoid tissues was consistent with the productive infection being largely contained at the portal of entry with the possibility of eventual clearance. Given the crucial importance of understanding the degree to which a highly pathogenic lentivirus can be contained or even cleared by adaptive immunity, we sought to define more precisely the spread and dynamics of SIV infection in RM that controlled the infection as a consequence of RhCMV/SIV vector vaccination, and in particular, the extent to which residual SIV was eventually cleared from these animals.
To establish the extent of SIV spread early after the onset of RhCMV/SIV vector-mediated control, we studied a group of five RM vaccinated with RhCMV vectors containing SIV Gag, Rev/Tat/Nef, Env and Pol (but not Vif) inserts that were taken to necropsy within 24 days of controlling plasma viraemia after intrarectal inoculation with SIVmac239. All of these RM had measureable SIV RNA in plasma for one or two weekly time points after challenge, followed by at least three consecutive weekly samples with plasma SIV RNA below 30 copy equivalents (equiv.) per ml, and at the time of necropsy, below 5 copy equiv. ml-1, as measured by an ultrasensitive assay (Fig. 1a). Infection was confirmed by the de novo development of T-cell responses against SIV Vif (not included in the vaccine) in all RM (Fig. 1b and Supplementary Fig. 1a). As previously described5, protection occurred without anamnestic boosting of vaccine-elicited SIV-specific CD8+ T-cell responses in blood (Fig. 1b), and at necropsy, robust CD4+ and CD8+ T-cell responses to the SIV proteins included in the RhCMV/SIV vaccine vectors were identified (Supplementary Fig. 1b). We then used ultrasensitive, nested quantitative PCR (qPCR) and quantitative PCR with reverse transcription (qRT-PCR) assays to quantify SIV DNA and RNA, respectively, in the tissues of these protected RM, in comparison with tissues from three unchallenged, RhCMV/SIV vector-vaccinated RM (SIV- controls), two unvaccinated RM with productive SIV infection (one progressor and one elite controller) and three RM with SIV infection suppressed with antiretroviral drug treatment (ART) (Fig. 1c, Supplementary Figs 2-4 and Supplementary Table 1). Two of the five RhCMV/SIV vector-protected RM showed levels of SIV DNA and RNA approaching the very low level background signal observed for SIV- control RM. However, the other three showed readily measurable SIV RNA, not only in rectal/colonic mucosa (portal of entry), but also in lymph nodes draining the portal of entry (iliosacral and mesenteric lymph node groups), as well as in sites of presumed haematogenous spread: bone marrow, spleen and liver. The level of SIV RNA in the tissues of these three RM was less than that seen in progressive infection, but comparable to that in the elite SIV controller and in ART-suppressed SIV infection. Notably, however, levels of tissue-associated SIV DNA in the RhCMV/SIV vector-protected RM were all substantially lower than in the RM with elite control and ART suppression, probably reflecting virological control before, rather than after, peak viral replication in the RhCMV/SIV vector-protected RM, and the limited time for SIV DNA+ cells to accumulate in these RM before necropsy. Although these data suggest a much smaller SIV reservoir in the RhCMV/SIV vector-protected RM than in the SIV+ controls, including the RM with ART-suppressed SIV infection, we were able to recover replication-competent SIV from iliosacral lymph nodes and spleen in all five of the RhCMV/SIV-protected RM taken to early necropsy (and from bone marrow and mesenteric lymph nodes in three of these five RM), including the two RM with near background levels of SIV RNA by nested qRT-PCR (Table 1). This replication-competent SIV was found in tissues manifesting only minimal interferon-stimulated gene expression, significantly less than found in either progressive or ART-suppressed SIV infection (Supplementary Fig. 5). Taken together, these data demonstrate that in RhCMV/SIV vector-protected RM, SIV can escape the portal of entry and establish infection in draining lymph nodes, as well as bone marrow, spleen and liver, before stringent control.
After intrarectal inoculation, SIV infection has been reported to spread to draining lymph nodes within 4 h (ref. 11), a rate of dissemination that may preclude SIV-specific effector memory T cells from containing the infection within the mucosa. By contrast, the development of SIV infection after intravaginal inoculation has been reported to require local amplification, with distal spread only after 4-5 days6. To determine whether RhCMV/SIV vector-elicited T-cell responses might locally control and perhaps clear an intravaginal SIV challenge, we compared the outcome of repeated, limiting dose intravaginal SIVmac239 challenge in cycling female RM vaccinated twice (weeks 0 and 14) with RhCMV/SIV vectors (group A) versus similar RM vaccinated twice with RhCMV vectors encoding non-SIV inserts (group B) or left unvaccinated (group C), with challenge 78 weeks after initial vaccination (Supplementary Fig. 6). The immunogenicity of RhCMV/SIV vectors in these female RM was similar to that described for male RM with robust, effector memory-biased SIV-specific CD4+ and CD8+ T-cell responses to all SIV inserts (Supplementary Figs 7 and 8), but little to no SIV Env-specific antibody responses (Supplementary Fig. 9). As previously described for intrarectal SIV challenge of male RM5, RhCMV/SIV vector vaccination did not significantly affect the number of intravaginal SIV challenges required to achieve infection relative to control-vaccinated and unvaccinated RM (Supplementary Fig. 10), but did markedly alter the course of SIV infection with 9 out of 16 RhCMV/SIV vector-vaccinated female RM manifesting stringent (MHC class I allele-independent) control of plasma viraemia compared with none of 18 infected female control RM (Fig. 2a and Supplementary Table 2). Five of these nine protected female RM manifested a second episode of transient plasma viraemia within the first 12 weeks after initial control, but overall, the fraction of protected female RM (followed for at least 30 weeks) with such plasma viral blips (56% versus 100%; P = 0.02 by Fisher's exact test) and the number of blips per RM (0.7 versus 6.0; P < 0.0001 by two-sided Wilcoxon rank sum test) were less than that observed in RhCMV/SIV vector-vaccinated male RM protected after intrarectal challenge5. Other characteristics of protection in these intravaginally challenged, RhCMV/SIV vector-vaccinated female RM were identical to those previously reported for RhCMV/SIV vector-mediated protection of male RM against intrarectal challenge5, including development of de novo SIV Vif-specific CD4+ and CD8+ T-cell responses, lack of an anamnestic boost of the vaccine-elicited SIV-specific CD4+ or CD8+ T cells, lack of SIV Env seroconversion, and lack of CD4+ T-cell depletion at mucosal effector sites (Fig. 2b and Supplementary Figs 9, 11 and 12).
To determine whether SIV infection spread from the cervical/vaginal mucosa in the nine RhCMV/SIV vector-protected female RM, we biopsied bone marrow, peripheral lymph nodes (axillary/inguinal) and small intestinal mucosa for nested qRT-PCR and qPCR analysis of SIV RNA and DNA, respectively, at 5, 9, 17 and >30 weeks after infection. Notably, in the first 9 weeks of infection, five of these nine RM manifested levels of SIV RNA in bone marrow comparable to levels seen in uncontrolled SIV infection, but, whereas in uncontrolled infection SIV RNA levels were similarly high in peripheral blood mononuclear cells (PBMCs), lymph nodes and intestinal mucosa, SIV RNA was either not detected or detected only at very low levels in these sites in the RhCMV/SIV vector-protected RM (Fig. 2c). Moreover, in contrast to uncontrolled infection, SIV DNA was inconsistently detected in the samples from the RhCMV/SIV vector-protected RM, and by 40 weeks after infection, all nine of the RhCMV/SIV vector-protected RM had at least one sample set in which both SIV RNA and DNA were below the level of detection in all sites. In eight of these RM (excluding Rh20363, see below), all samples obtained subsequent to 30 weeks after infection showed SIV RNA and DNA levels below the level of detection, with the exception of one PBMC sample with low-level SIV RNA (454 copy equiv. per 108 cells). The differences in the frequency of SIV detection in samples obtained at 5, 9 and 17 weeks versus >30 weeks after infection from these eight RM were highly significant (P = 0.002 for all samples, P = 0.0006 for bone marrow by two-sided Wilcoxon rank sum tests).
The ability to detect tissue-associated SIV early, but not late, after infection in these eight stably protected female RM, particularly in bone marrow, is consistent with initial spread and subsequent control and progressive clearance of SIV. In accordance with this, the frequencies of circulating SIV Vif-specific T cells, which are elicited and maintained by antigen derived from SIV infection (rather than the vaccine), progressively declined in these RM until these responses were no longer detectable (Fig. 2b and Supplementary Fig. 11).
However, despite having no detectable SIV RNA or DNA in PBMCs and tissue samples at week 17, and declining SIV Vif-specific T-cell responses, one animal (Rh20363) showed the emergence of low-level productive SIV infection at week 31 after infection (Fig. 2a). The boosting of SIV-specific CD4+ and CD8+ T-cell responses (Fig. 2b and Supplementary Fig. 11), including de novo CD8+ T-cell responses to canonical Mamu-A*01-restricted SIV epitopes (Supplementary Fig. 13), the appearance of cell-associated RNA and DNA in subsequent PBMCs, lymph node and intestinal samples (Fig. 2c), and the induction of increased plasma and PBMC-associated SIV loads with experimental in vivo CD8+ lymphocyte depletion (Supplementary Fig. 14) indicates that this RM spontaneously converted from a unique state of stringent viral containment with little or no continuing viral replication to a different state characterized by continuing, but low-level SIV replication (consistent with conventional 'elite' immunological control). In keeping with this, sequence analysis of the breakthrough virus 3 weeks after initial viral rebound showed little evolution from the initial SIVmac239 sequence except, notably, a putative escape mutation in the Tat-SL8 epitope sequence, consistent with early escape from the Tat-SL8-specific T-cell responses that developed after viral rebound at week 31 (Supplementary Figs 13 and 15). Given the enormous breadth of RhCMV/SIV vector-elicited CD8+ T-cell responses10, this limited sequence evolution suggests that the loss of aviraemic control in Rh20363 was more likely due to inadequate immune surveillance of residual infection than mutational escape. Experimental CD8+ lymphocyte depletion was also performed on three RhCMV/SIV vector-protected female RM that retained aviraemic control, and in keeping with previous analysis of CD8+ lymphocyte depletion of RhCMV/SIV vector-vaccinated male RM protected after intrarectal challenge5, 9, this treatment did not induce detectable plasma viraemia (Supplementary Fig. 14). However, one of these RM (Rh21176) transiently manifested unequivocal detection of SIV RNA (10 out of 10 replicates positive) and replication-competent SIV (7 out of 20 co-cultures positive) in lymph nodes at day 10 after CD8+ lymphocyte depletion, demonstrating the presence of at least local, very low level residual SIV infection in this RM after 52 weeks of stringent control. In contrast to Rh20363, Rh21176 maintained aviraemic control, indicating that this RM's immune system either controlled or eliminated residual foci of SIV replication.
The finding that RhCMV/SIV vector-protected RM are able to control haematogenous SIV dissemination after both intrarectal and intravaginal challenge suggested that the immune responses elicited by these vectors might provide protection even when mucosal surfaces are bypassed. To assess this possibility, we challenged six RhCMV/SIV-vaccinated RM with low dose, intravenous SIVmac239, and found that two of these six RM manifested the same pattern of control observed after mucosal challenge-a transient, low-level viraemia associated with the development of an SIV Vif-specific T-cell response, and detection of SIV RNA in bone marrow (high level) and/or PBMCs (low level) early, but not late, after infection (Supplementary Fig. 16). Taken together, these data indicate that (1) RhCMV/SIV vector-elicited immune responses can mediate protection regardless of the route of SIV challenge, (2) viral control is both local and systemic, and (3) replication-competent SIV can persist in several sites for weeks to months in protected RM (even when aviraemic), but seems to decline over time.
To determine the ultimate fate of residual SIV in RhCMV/SIV vector-protected RM, we followed a total of ten protected RM for 69-180 weeks after infection (Fig. 3a, b). In all of these RM, plasma viral blips became increasingly infrequent over time, with no blips observed after 70 weeks. The frequency of the SIV infection-dependent, SIV Vif-specific CD8+ T cells in blood also progressively declined in all RM until these responses were no longer detectable (Supplementary Fig. 17). In contrast to the SIV Vif-specific CD8+ T-cell responses, the SIV-specific CD8+ T-cell responses elicited by the RhCMV/SIV vectors remained stable, including high frequencies of CD8+ T cells capable of recognizing autologous SIV-infected CD4+ T cells (Supplementary Fig. 18). Analysis of six of these medium- to long-term protected RM at necropsy, including one RM that was CD8+ lymphocyte-depleted 10 days before necropsy (Supplementary Fig. 19), confirmed the systemic loss of SIV Vif-specific T cells, and the maintenance of RhCMV vector-elicited, SIV-specific T cells (Supplementary Fig. 20). Most importantly, ultrasensitive, nested qRT-PCR and qPCR analysis of ≥54 tissues per animal (ten replicates per tissue, including extensive sampling of all tissues shown to contain SIV in the short-term RhCMV/SIV vector-protected RM) revealed extremely low to absent levels of SIV RNA and DNA that were indistinguishable from measurements in unchallenged RhCMV/SIV-vaccinated (SIV-) controls (Fig. 3c, d, Supplementary Figs 2 and 21 and Supplementary Tables 1 and 3). Moreover, despite extensive sampling (>240 cultures per animal), no replication-competent SIV was isolated by co-culture analysis from the lymphoid tissues of these RM (Table 1). Finally, we asked whether the adoptive transfer of a total of 6 X 107 haematolymphoid cells (3 X 107 each of peripheral blood leukocytes and lymph node cells, or 3 X 107 each of bone marrow leukocytes and spleen cells) from three SIV+ control RM (two with ART-suppressed infection and one elite controller), and five medium- or long-term RhCMV/SIV vector-protected RM (including one RM tested before and after CD8+ cell depletion) would initiate infection in SIV-naive RM. Remarkably, although cells from the SIV+ controls, including ART-suppressed RM, rapidly initiated SIV infection in the SIV-naive recipients (manifested by the onset of SIV replication and induction of SIV Vif-specific T-cell responses), no evidence of SIV infection was observed in the SIV-naive recipients receiving cells from the medium- and long-term RhCMV/SIV vector-protected RM (Fig. 3e and Supplementary Fig. 22). Taken together, these data provide strong evidence that after being unequivocally infected with SIV, these RhCMV/SIV vector-vaccinated RM cleared detectable infection, such that by all measured criteria (lack of plasma viral blips, absence of Vif-specific T-cell responses, extensive ultrasensitive qRT-PCR and qPCR and co-culture analysis, and adoptive transfer) these RM were indistinguishable from RhCMV/SIV vector-vaccinated controls that had never been exposed to SIV. Although we cannot rule out residual virus below our level of detectability, or in tissues not examined, these data strongly support progressive immune-mediated clearance of an established lentivirus infection, leading to a situation meeting criteria for a functional cure12 and consistent with possible viral eradication.
In the past 5 years, the HIV/AIDS vaccine field has concluded that a prophylactic HIV/AIDS vaccine must prevent or eliminate HIV infection, as it is thought that any residual infection runs a high risk of eventual progression13. Our demonstration here that the virus-specific, effector memory T cells maintained by a persistent vector can shut down productive SIV infection, and by maintaining immune surveillance over time, functionally cure and possibly eradicate this infection, indicates that an effector memory T-cell-targeted HIV/AIDS vaccine could (by itself, or combined with antibody-targeted approaches) provide meaningful long-term efficacy. Our results also suggest that an effector memory T-cell-targeted vaccine might contribute to HIV cure strategies. Although the SIV reservoirs that initially develop in RhCMV/SIV vector-vaccinated controllers are smaller in size, and possibly different in character from HIV/SIV reservoirs in the setting of ART administration initiated in chronic infection, it is conceivable that the indefinitely persistent, unconventionally targeted10, viral-specific T cells elicited and maintained by CMV vectors-alone or in combination with agents designed to activate HIV gene expression1, 2, 12-might exert potent immune pressure on cells with any HIV protein expression (including expression of viral antigen by stochastically activated, latently infected cells) and thereby facilitate depletion of residual HIV reservoirs in patients on suppressive ART. It is also possible that these responses might stringently control recrudescent 'rebound' infection after ART withdrawal in a manner analogous to their control of primary SIV infection in this study. In summary, the ability of CMV vectors to implement continuous, long-term, and potent antipathogen immune surveillance makes them promising candidates for vaccine strategies intended to prevent and cure HIV/AIDS, as well as other chronic infections.
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