The goal of this study was to comprehensively assess the level of viral control and virus induced immunity after a third, heterologous high dose mucosal SIV challenge in three rhesus macaque elite controllers. In contrast to other elite controller macaques studied, our macaques were unique in possessing no MHC haplotypes and TRIM5α polymorphisms known to be associated with elite control. Even given the complicated immunization/challenge history of these macaques, they provide an opportunity to undercover alternative, non-MHC-restricted immune responses associated with protection. The myriad array of positive immune responses we observed in these macaques points to multiple immune protective mechanisms, especially potent humoral immunity. Coupled with comprehensive assessments of viremia throughout the body and mucosal gene transcription, this study has implicated a broad range of factors involved in maintenance of strong viremia control.
Of the three original macaques, only macaque #5 continued to maintain strict, elite control, as defined by undetectable plasma viremia, of the heterologous SIVsmE660
mucosal challenge. However, examination of viral loads in mesenteric and axillary lymph nodes showed that this macaque, while negative pre-challenge, became infected. Both SIVsmE660
, reactivated at low level from previous exposures, were undetected in blood, but present and contained locally in the lymph node (). When SIV is administered across a mucosal barrier, virus establishes an initial localized infection in a small number of cells, the founder population (Li et al., 2005
; Miller et al., 2005
). Dissemination to draining lymph nodes can occur within 24 hours, but until viremia hits a threshold level in the mucosa – usually 5–6 days – viremia in the distal sites and periphery is unsustainable. We did not assess the level of viremia at the rectal mucosa, the site of mucosal challenge, but SIVsmE660
was disseminated to draining lymph nodes and remarkably, subsequently contained. This control of viremia was mediated at the local site of infection, in the lymph node itself, or at both sites.
It was surprising that before rechallenge, a low level SIVmac251
infection (108 copies) was detected in the jejunum more than 3 years after CD8 depletion and re-appearance of viremia in macaque #5 (). Interestingly, SIVsmE660
was never detected in the jejunum, although a critical 2-week timepoint could not be evaluated due to lack of sufficient sample. However, the original SIVmac251
infection was reactivated in the jejunum following SIVE660
challenge, increased 10-fold, and thereafter remained stable. To our knowledge, this is the first detection of long term viremia sequestration and control at a mucosal site. Whatever the mechanism(s) of mucosal immune control, it did not prevent SIVE660
acquisition. Nevertheless, SIVE660
replication was subsequently controlled in the lymph node to a level that precluded spread of viremia to the blood over the entire post challenge period. It was recently shown that in human elite suppressors, low level, ongoing viral replication did not reseed the latent reservoir, and most likely originated from an as yet unknown cryptic reservoir (O’Connell et al., 2010
). It is known that upon removal of HAART therapy and necropsy of SIV-infected rhesus macaques, virus can be detected in numerous tissue sanctuaries, in particular lymph nodes, spleen, and gastrointestinal sites (North et al., 2010
), similar to sites where viremia was detected here. While we have learned much regarding initial viral infection at mucosal sites and its eventual systemic spread, greater understanding of the immune processes that contain it mucosally and control its dissemination is needed.
In spite of the strong control of SIVmac251
in jejunal tissue and the inability to detect SIVE660
at that site, we were not able to associate local cellular immune responses with viremia control. Virus-specific responses in the jejunum were consistently negative by intracellular cytokine staining. Continued stimulation by the low level viremia at this mucosal effector site might have resulted in already highly activated cells becoming anergic and unresponsive to further in vitro
stimulation. However, in a study in which a large amount of jejunal tissue was obtained for analysis by necropsy of macaques immunized with live attenuated virus, cytokine secreting cells were correlated with protection (Genesca et al., 2008
). Furthermore, a recent study in which virus-specific effector cells were visualized in situ
(Li et al., 2009
) supports the notion that these cells exist at this site. We may have missed detection of cytokine-positive cells due to sampling issues. Alternatively, other mechanisms may have played a role in the protection observed.
In contrast to results obtained with jejunal tissue, macaque #5 exhibited outstanding cellular immune responses in terms of breadth and frequency, particularly in blood and BAL. SIV-specific IFN-γ secreting PBMC in addition to broad, potent T cell proliferative responses existed prior to SIVE660
challenge and were maintained throughout the post-challenge phase. Polyfunctional, CD4 and CD8 central and effector memory cells specific for both Env and Gag were present at extremely high frequency pre-challenge and were boosted post-challenge in both peripheral blood and BAL (, ). The expansion post-challenge of vaccine-specific EnvsmH4
CD4 memory cells in PBMC to an effector phenotype during the acute phase of infection followed by contraction during set point to polyfunctional cells () is of note. The complex nature of viremia control in macaque #5 makes definitive conclusions difficult, but these effector cells may have been contributing to control of SIVE660
infection, especially if they homed back to the rectal mucosa. The concomitant disappearance of CD4 memory cells from the lung at week 2 () could also have provided for a transient influx of effector cells to the rectal mucosa. Vaccine elicited effector memory T cell responses have recently been shown to be associated with protection from SIV mucosal challenge (Hansen et al., 2009
Macaques #9 and #7 exhibited lesser control of SIVsmE660 viremia in comparison to macaque #5, although both showed blunted acute viremia and subsequent control to undetectable levels during chronic infection. Macaque #7 rapidly controlled viremia by week 6 and maintained this control until week 36 when a sharp increase in viral load indicated immune escape. We plan to sequence viruses around this time point after single genome amplification in order to determine if a recombination event occurred and shed light on a possible mechanism of escape. The kinetics of viremia control for animal #9 differed. This macaque exhibited undetectable viremia beginning at week 36, however, after week 75 viral loads became positive and increased slowly (data not shown). Therefore, escape from immune control may be occurring for this macaque as well. SIVmac251 was never detected before or after SIVsmE660 challenge in plasma or tissues of either of these macaques. Unlike animal #5, both macaques controlled the early SIVmac251 challenges to undetectable levels and therefore were not exposed to transient SIVmac251 viremia. We speculate that the low-level SIVmac251 infection of macaque #5, kept under control by potent immune responses, may be playing a significant role in its maintenance of stronger viremia control by providing a persistent immunologic stimulus.
Although macaques #7 and #9 lacked SIV-specific cells in PBMC pre- or post-SIVsmE660 challenge, both macaques displayed pre-existing virus specific memory cells in BAL which were boosted following SIVsmE660 exposure. Since we consistently detected SIV-specific CD8+ effector cells at this site for all three macaques, we speculate that long term memory cells might reside in the upper respiratory tract (URT), possibly in the lymph nodes draining the lungs. Our Ad5hr-SIV recombinant vaccines replicate in the URT after intranasal/intratracheal immunization in the rhesus macaque model. Therefore, it is likely that durable vaccine-induced immune cells would reside there.
Functionally diverse, mucosal and plasma antibody responses were uniquely observed here for all macaques, but especially for #5. Macaque #5 exhibited serum antibodies able to mediate multiple activities prior to SIVE660
challenge. The antibodies, both binding and neutralizing, were generally of high titer and were maintained post-challenge. In addition to neutralization of primary and TCLA-SIV, sera of macaque #5 neutralized HIV-2, with enhanced titers seen with addition of sCD4, implying recognition of CD4-induced epitopes. As SIVsmE660
was not neutralized, activities mediated by non-neutralizing antibodies such as ADCC and ADCVI were assessed. Both activities were mediated by sera of macaque #5, and both have been correlated with reduced viremia (Gomez-Roman et al., 2005
; Xiao et al., 2010
). Most of these functional antibody activities were detected post-challenge for both macaques #9 and #7 too, albeit at lower levels. Macaque #9 showed more potent SIVsmE660
-specific ADCVI activity and a more rapid SIVE660
-specific ADCC anamnestic response than #7, which may have contributed to the better viremia control of this macaque during chronic infection.
IgG memory B cells specific for SIV Env were present in bone marrow of all three macaques, indicative of their long vaccination and challenge history. Macaque #5 again exhibited the highest levels. With regard to mucosal antibody, anamnestic SIV Env-specific IgG responses were seen for all three macaques, and IgA responses for macaques #5 and #9. Again, macaque #5 had the highest rectal antibody levels. The mucosal antibodies elicited mediated transcytosis inhibition against SIVmac251
(macaque #5) and SIVsmE660
(macaques #5 and #9). Inhibition of transcytosis has been associated with protection both in macaques (Hidajat et al., 2009
; Xiao et al., 2010
) and in humans (Devito et al., 2000
; Shen et al., 2010
). We have observed that transcytosis inhibitory activity is significantly correlated with reduced chronic viremia in the SHIV89.6P
rhesus macaque model (Xiao et al., 2010
) suggesting that it might not only be important in protecting against acquisition, but might also play a role in protection against cell to cell transmission. This would be relevant here regarding control of viremia at tissue sites.
The evaluation by microarray of intestinal gene expression over time has provided a number of novel observations. Firstly, humoral immune response genes at this site were upregulated post virus exposure for macaque #5. Although we detected mucosal antibodies in rectal secretions, we can now link these functional responses to gene expression directly after virus exposure since transcriptional analysis was performed at week 2 post-challenge.
Secondly, numerous genes involved in induction of immune responses were upregulated in macaque #5 immediately following challenge. These were related to processes such as innate immunity, antigen presentation, chemotaxis and T cell responses. A lesser upregulation of inflammatory response genes, in particular interferon known to be induced to high levels in SIV unprotected macaques (Bosinger et al., 2009
), was observed for macaque #5. In addition, tight junction and epithelial repair and regeneration genes associated with protection from SIV infection (George et al., 2003
; George et al., 2005
) were upregulated in all three macaques, but more so for macaque #5, reflecting greater control of viremia. Further post challenge analysis of clear differences seen between the elite controller macaque #5 and macaques #7 and #9, which did not control viremia to undetectable levels, might in the future, yield new clues to SIV protection in the rhesus macaque model.
It is likely that the stronger protection seen in macaque #5 resulted in part from the cryptic exposure to SIVmac251. In this animal, vaccine-elicited anti-envelope antibody was boosted by exposure to SIVmac251 and also augmented by development of anti-gag cellular responses. The resultant local containment of virus by potent immunity had the effect of creating a “live-attenuated virus” from the pathogenic SIVmac251 challenges, leading to subsequent complete protection against the heterologous SIVsmE660. In contrast, macaques #7 and #9, originally better protected and not subjected to transient SIVmac251 viremia, initially controlled, but later were less able to sustain SIVsmE660 control. A key difference was their lack of long term maintenance of a potent anti-envelope antibody response. The peptomer, administered without an adjuvant to all three, was not a good booster immunogen in comparison to SIV envelope itself, and induced only low-titer antibodies at the time of the first SIVmac251 challenge. Whether a vaccine regimen that provides better initial anti-envelope boosting and maintenance of high-titer, functional antibodies will preclude the necessity for continuous, cryptic antigen exposure will require further investigation. The mechanism by which macaque #7 lost viremia control is also worthy of pursuit, and should elucidate whether the initial control was primarily cellular or humoral.
Overall, study of the three elite controller macaques has attributed the potent viremia control to multifunctional cellular, humoral, and mucosal immune responses. This extraordinary level of control appropriately matches the complex nature of SIV infection and pathology.