Distinctive TLR7 Signaling, Type I IFN Production, and Attenuated Innate and Adaptive Immune Responses to in a Primate Reservoir Host This information is current as of September 24, 2021. Judith N. Mandl, Rama Akondy, Benton Lawson, Natalia Kozyr, Silvija I. Staprans, Rafi Ahmed and Mark B. Feinberg J Immunol 2011; 186:6406-6416; Prepublished online 22 April 2011; Downloaded from doi: 10.4049/jimmunol.1001191 http://www.jimmunol.org/content/186/11/6406 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2011/04/22/jimmunol.100119 Material 1.DC1 References This article cites 76 articles, 28 of which you can access for free at: http://www.jimmunol.org/content/186/11/6406.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Distinctive TLR7 Signaling, Type I IFN Production, and Attenuated Innate and Adaptive Immune Responses to Yellow Fever Virus in a Primate Reservoir Host

Judith N. Mandl,*,†,1 Rama Akondy,† Benton Lawson,† Natalia Kozyr,† Silvija I. Staprans,†,‡,2 Rafi Ahmed,†,‡ and Mark B. Feinberg†,‡,x

Why cross-species transmissions of zoonotic viral infections to humans are frequently associated with severe disease when responsible for many zoonotic diseases appear to cause only benign infections in their reservoir hosts is unclear. Sooty mangabeys (SMs), a reservoir host for SIV, do not develop disease following SIV infection, unlike nonnatural HIV-infected human or SIV- infected rhesus macaque (RM) hosts. SIV infections of SMs are characterized by an absence of chronic immune activation, in

association with significantly reduced IFN-a production by plasmacytoid dendritic cells (pDCs) following exposure to SIV or Downloaded from other defined TLR7 or TLR9 ligands. In this study, we demonstrate that SM pDCs produce significantly less IFN-a following ex vivo exposure to the live attenuated yellow fever virus 17D strain vaccine, a virus that we show is also recognized by TLR7, than do RM or human pDCs. Furthermore, in contrast to RMs, SMs mount limited activation of innate immune responses and adaptive T cell proliferative responses, along with only transient antiviral Ab responses, following infection with yellow fever vaccine 17D strain. However, SMs do raise significant and durable cellular and humoral immune responses comparable to those seen in RMs when infected with modified vaccinia Ankara, a virus whose immunogenicity does not require TLR7/9 recognition. Hence, http://www.jimmunol.org/ differences in the pattern of TLR7 signaling and type I IFN production by pDCs between primate species play an important role in determining their ability to mount and maintain innate and adaptive immune responses to specific viruses, and they may also contribute to determining whether disease follows infection. The Journal of Immunology, 2011, 186: 6406–6416.

he majority of emerging viruses, including HIV, avian disease (7). However, elucidation of the mechanisms by which influenza, severe acute respiratory syndrome coronavirus, reservoir hosts remain healthy despite harboring viruses that cause T and virus, have zoonotic origins (1). Although these severe disease following zoonotic transmissions will be essen- viruses typically cause benign infections in their tial to better understand how host immune responses can either hosts, cross-species transmission to humans can result in severe ameliorate or contribute to infection-associated damage. Insights by guest on September 24, 2021 disease (2–6). Unfortunately, our understanding of host-specific into healthy host–virus equilibria in natural host species will also attributes that determine whether disease develops following in- provide valuable information to guide development of effective fection with zoonotic viruses is very limited. To date, mechanisms therapeutic interventions for treating zoonotic infections. Fur- enabling resistance to infection have generally been better studied thermore, improved understanding of the biology and evolution of than those that allow hosts to tolerate particular infections without natural reservoir hosts for viruses responsible for zoonotic dis- eases will illuminate whether the association of specific host species (such as and African primates) with multiple emer- *Graduate Program in Population Biology, Ecology, and Evolution, Emory Univer- ging viruses reflects common or distinctive biological features of † sity, Atlanta, GA 30329; Emory Vaccine Center, Emory University School of Med- the host (8, 9). icine, Atlanta, GA 30329; ‡Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30329; and xDepartment of Medicine, Numerous viruses have crossed into humans from African Emory University School of Medicine, Atlanta, GA 30329 primate species with devastating consequences, such as SIV,yellow 1Current address: Lymphocyte Biology Section, Laboratory of Immunology, National fever virus (YFV), and virus (8, 10). More than 30 Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, African primate species harboring closely related SIVs in the wild MD. have been described so far (11). SIV from chimpanzees has given 2Current address: Merck Vaccines and Infectious Diseases, West Point, PA. rise to HIV-1 in humans, and SIV from a second primate reservoir Received for publication April 13, 2010. Accepted for publication March 18, 2011. host, the sooty mangabey (SM), is the zoonotic origin of SIVmac This work was supported in part by National Institutes of Health Grants R01 in rhesus macaques (RMs) and HIV-2 in humans (10). Similar to HL075766, R01 AI049155, and U19 AI057266; Emory Center for AIDS Research Grant P30-AI-50409; and Yerkes National Primate Research Center Grant SIV, there is evidence that YFV also originates in Africa, infecting RR000165. many of the same primate species, including SMs, which do not Address correspondence and reprint requests to Dr. Mark B. Feinberg at the current develop disease upon YFV infection and are important in the address: Merck Vaccines and Infectious Diseases, Merck & Co., WP97-A337, 770 ecology of the sylvatic cycle of YFV transmission (12, 13). In Sumneytown Pike, P.O. Box 4, West Point, PA 19486. E-mail address: mark_feinberg@ merck.com contrast, YFV infections of RMs (Asian primates that are not The online version of this article contains supplemental material. natural hosts for YFV) are severe and often lethal (14, 15). Abbreviations used in this article: IRF, IFN regulatory factor; mDC, myeloid den- Among emerging viruses, the immune responses of reservoir dritic cell; MVA, modified vaccinia virus Ankara; ODN, oligodeoxynucleotide; pDC, hosts for SIV have been studied in the greatest detail (16). In- plasmacytoid dendritic cell; RM, rhesus macaque; SM, sooty mangabey; YF-17D, fections of nonnatural RM hosts with SIV, as for HIV infections in yellow fever vaccine 17D strain; YFV, yellow fever virus. humans, lead to the development of immunodeficiency in asso- Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 ciation with persistent virus replication, chronic aberrant acti- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001191 The Journal of Immunology 6407 vation of multiple arms of the immune system, active but ulti- signaling and IFN-a production play in the generation of innate mately ineffective host antiviral cellular and humoral immune and adaptive immune responses to viral infection in primates. responses, and progressive CD4+ T cell depletion (17). In marked contrast, SIV-infected SMs and other natural hosts do not progress Materials and Methods to AIDS despite high levels of virus replication (18). Importantly, Human and animal subjects a key feature distinguishing nonpathogenic SIV infections of 2 natural hosts is the absence of chronic generalized immune acti- Blood samples were obtained from HIV human volunteers after obtaining vation (18). Whether this is a result of the active suppression of written informed consent. Protocols were approved by the Emory Uni- versity Institutional Review Board. Animal housing, care, and research immune activation or the absence of signals that instigate or were in accordance with the Guide for the Care and Use of Laboratory maintain ongoing immune activation, or both, is still unclear (19). Animals (National Research Council). All animal studies were approved SIV and HIV have been shown to potently activate human and RM by the Emory University Institutional Animal Use and Care Committee. plasmacytoid dendritic cells (pDCs) to produce IFN-a and other Virus (e.g., IL-12 and TNF-a) via TLR7 and TLR9 (20). We have previously shown that SM pDCs produce significantly less MVA was grown on DF-1 cells (American Type Culture Collection, IFN-a, but similar levels of other proinflammatory cytokines, after Manassas, VA) and cell lysates were collected. YF-17D was grown on Vero cells (American Type Culture Collection) and infected cell media collected. engagement of TLR7 and TLR9 by SIV or other TLR7/9 ligands, Viruses were purified by pelleting through 36% sucrose. Titres were de- than do pDCs from humans or RMs. SM pDCs produce equivalent termined by plaque assays. Sucrose-purified MVA was used for all levels of IFN-a as do those from RMs and humans following experiments, whereas sucrose-purified YF-17D was used only for in vitro exposure to innate immune response agonists (e.g., live influenza experiments, not for animal infections. Downloaded from virus) that activate pDCs via TLRs other than TLR7 and TLR9 or Infection with YF-17D via cytosolic pattern recognition receptors (e.g., RIG-I–like heli- Humans and animals were s.c. given 1 3 105 PFU of the commercially cases), demonstrating that they do not have an intrinsic inability to available YF-17D vaccine (YF-VAX; Aventis Pasteur). Animals were produce IFN-a (21). This pattern of production suggests infected in three separate groups: group 1 (RM1, RM2, RM3, RM4), group impaired signaling along the IFN regulatory factor (IRF)-7 path- 2 (SM1, SM2, SM5, SM6), and group 3 (SM3, SM4, SM7, SM8, RM5, + way, but preserved NF-kB–dependent signaling following TLR7 RM6). SM5–SM8 were SIV . http://www.jimmunol.org/ or TLR9 activation in SMs. Currently, it is unknown whether the Infection with MVA qualitatively different immune response to SIV in SMs compared with RMs is unique or whether it may also affect immune re- A subgroup of animals vaccinated with YF-17D (RM1–RM4, SM1, SM2 and SM5, SM6) was given 1 3 108 PFU sucrose-purified MVA both in- sponses mounted to other viruses that, similar to SIV, are recog- tramuscularly and intradermally. nized by TLR7 or TLR9. To investigate this question, we studied both the ex vivo Determination of YF-17D plasma RNA responses of pDCs and the in vivo immune responses of RMs and YF-17D plasma viremia was quantified by ultrasensitive quantative real- SMs to two distinct viruses: YFV vaccine 17D strain (YF-17D), time RT-PCR as described (25). Limit of detection was 12 copies/ml. a flavivirus, and modified vaccinia virus Ankara (MVA), an or- by guest on September 24, 2021 Immunophenotyping and flow cytometry thopoxvirus. YF-17D has an ssRNA and in mice has been shown to be recognized by TLRs 2, 7, 8, and 9 (22). In contrast, Whole blood staining was performed with the following mAbs as described MVA has a dsDNA genome and its innate recognition, as studied (21): anti-CD3 (SP34), anti-CD4 (L200), anti-CD8 (SK1), anti-CD20 (L27 or 2H7), anti-CD16 (3G8), anti-CD11c (S-HCL-3), anti-CD123 (7G3), in murine models, has been shown not to involve TLR7 or TLR9 anti-CD14 (M5E2), and anti-HLA-DR (L243) from BD Pharmingen (San (23, 24). Both of these viruses, the attenuated live YF-17D vaccine Jose, CA) and anti-CCR7 (150503; R&D Systems, Minneapolis, MN). strain, and the host range-restricted MVA have been shown to Samples used for anti-Ki67 or anti-Bcl2 staining were permeabilized elicit significant levels of antiviral cellular and humoral immune (Perm Solution 2; BD Biosciences). Flow cytometric acquisition of at least responses in humans after a single inoculation and have proven to 100,000 lymphocytes was done on a FACSCalibur or LSRII (BD Bio- sciences) and analyzed using FlowJo (Tree Star, Ashland, OR). be valuable tools in studying primary immune responses to virus infection in humans (25–29). Indeed, contemporary systems bi- YF-17D Ab titres ology approaches have recently been applied to study the human Plasma was assayed for anti-YF neutralizing Ab by measuring protection immune response to YF-17D in great detail (28, 29). These studies conferred on Vero cells against the YF-17D cytopathic effect as described have highlighted the importance of a number of “masterswitch (29). ” involving several pathways of the innate immune response, MVA Ab titres including type I IFNs, which are significantly upregulated fol- lowing infection and are associated with the induction of a sub- Microtiter wells were coated with 2 mg/ml sucrose gradient-purified MVA. After blocking with PBS/10% FCS, animal plasma samples were added in stantial and broad Th1/Th2 profile of immune responses elicited sixteen 1:2 serial dilutions. A polyclonal HRP-conjugated goat anti-rhesus by YF-17D immunization (28, 29). Of the identified nodes of Ig was used as secondary Ab (SouthernBiotech, Birmingham, AL) that was immune response activation by YF-17D, IRF-7 was found to cross-reactive with SM IgG. Plates were developed using SureBlue TMB feature prominently as a key regulatory pathway following YF- substrate (KPL, Gaithersburg, MD), and absorbance was read at 450 nm, 17D infection of humans, consistent with its important role in after the reaction was stopped using 1 M phosphoric acid. generation of innate and adaptive immune responses in mice (30– Detection of MVA-specific T cells 32). Additionally, TLR7 and a range of type I IFN response genes PBMCs were isolated from blood using sodium citrate Vacutainer CPT were observed to be significantly induced following YF-17D in- tubes (BD Biosciences, Franklin Lakes, NJ) centrifuged at 2500 rpm for 25 fection (28, 29). Given these studies and our earlier observations min. RBCs were lysed with ACK lysing buffer (BioSource, Camarillo, CA), of the distinctive patterns of TLR7/9 signaling and type I IFN PBMCs washed, and 2 3 106 cells incubated in 10% FBS RPMI 1640 at production in association with nonpathogenic SIV infection of 37˚C for 16 h in 200 ml media with MVA (multiplicity of infection of 2), 5 mg/ml Con A (Sigma-Aldrich, St. Louis, MO), 2 mg/ml staphylococcal SMs, the present study sought to explore not only the specific enterotoxin B (Sigma-Aldrich), or media alone. Brefeldin A (Sigma- biology of the SM immune system in response to virus infections Aldrich) was then added at final concentration of 10 mg/ml and cells that they harbor as reservoir hosts but also the role that TLR7 were incubated for an additional 5 h. Cells were surface stained for CD3 6408 VIRAL IMMUNITY IN PRIMATE RESERVOIR HOSTS and CD8, permeabilized (Perm Solution 2; BD Biosciences), and stained intracellularly for IFN-g (4S.B3; BD Pharmingen). DC stimulations PBMCs were isolated from HIV/SIV-negative humans, RMs, or SMs. Cells (4 3 105) were stimulated with a multiplicity of infection of 0.4 MVA or YF-17D in 10% FBS RPMI 1640 in the presence or absence of oligo- deoxynucleotide (ODN) antagonists IRS661, IRS869, or IRS954 (Dynavax Technologies, Berkeley, CA), or chloroquine diphosphate salt (Sigma- Aldrich) dissolved in PBS, for 17 h in 96-well plates. IFN-a and IL-12 production was measured in the supernatants by ELISA (human IFN-a multi-subtype kit [PBL Biomedical Laboratories, Piscataway, NJ] and rhesus monkey IL-12 kit [BioSource International, Camarillo, CA]). For experiments using the ODN antagonists, data are shown as a percentage of IFN-a production relative to the no inhibitor controls. Statistical analyses We analyzed data using t tests, Mann–Whitney U tests, or, in cases where more than one group was compared, by Kruskal–Wallis ANOVA (GraphPad Prism). A p value ,0.05 was considered significant. Downloaded from Results Reduced IFN-a production of SM PBMCs upon stimulation with YF-17D Persistently increased levels of type I IFN have been reported to be associated with increased levels of chronic immune activation

during pathogenic SIV infections in nonhuman primates and in http://www.jimmunol.org/ HIV infections in humans (21, 33). However, it remains unclear whether this association between IFN-a production and chronic immune activation is causal. Similarly, the extent to which host- specific differences in IFN-a production response to TLR7 and FIGURE 1. Reduced IFN-a production in SMs compared with RMs and TLR9 stimulation may influence the nature and magnitude of 2 humans upon stimulation with YF-17D, but not MVA. A, HIV human antiviral immune responses generated following infection with PBMCs were stimulated with YF-17D or MVA for 17 h in the presence of viruses whose innate immune recognitions are mediated via no inhibitor, 5.6 mm ODN of TLR7 (IRS661), TLR9 (IRS869), both TLR7 TLR7/9 pathways has not been determined previously in humans and TLR9 (IRS954), a nonspecific ODN, or chloroquine. Histograms and nonhuman primates. To investigate the effect of reduced type I represent mean 6 SEM of four to six humans. Comparisons are made to by guest on September 24, 2021 IFN production on the generation of immune responses in more without inhibitor, and data are rescaled relative to the level of IFN-a detail in primates, we examined the host-specific responses of production in the absence of inhibitors. B and C, HIV– human and SIV– RMs and SMs to two distinct viruses: YF-17D and MVA. RM or SM PBMCs were stimulated with YF-17D or MVA and IFN-a (B) We first sought to determine whether TLR7 or TLR9 is required or IL-12 production above background (IL-12 production with media alone for eliciting an IFN-a response to YF-17D or MVA in humans and is subtracted) (C), measured by ELISA. Mean cytokine production is denoted by a line. *p , 0.05, **p , 0.001, ***p , 0.0001. primates. YF-17D has been reported to activate DCs in mice to produce IL-6 and IL-12 via TLRs 2, 7, and 9, and to activate NF- kB in human HEK293 cells transfected with human TLR8 (22). TLR9. Although these data do not exclude potential contributions To date, TLR pathways responsible for activation of type I IFN of other TLR pathways in the pDC response to YFV, they are production by YF-17D in mice have not been reported. Further- consistent with what is known about the nature of TLR7 agonists more, although initial host responses to YF-17D immunization (35–37) and the key role played by TLR7-mediated activation of manifest a prominent signature of type I IFN production (28, 29), IFN-a production by other ssRNA viruses, including the specific TLR signaling pathways responsible for activation of (38) and coronaviruses (39). None of the TLR inhibitors signifi- IFN-a production by pDCs have not been determined in humans cantly reduced IFN-a production upon stimulation with MVA or primates. To investigate this, we stimulated human PBMCs (Fig. 1A), suggesting that TLR7 and TLR9 signaling are largely with YF-17D or MVA in the presence or absence of inhibitors of dispensable for generation of an IFN-a response to this virus. TLR7 and/or TLR9 and measured IFN-a production (Fig. 1A). We next examined the type I IFN responses of human, RM, and Controls for these studies included chloroquine, an endosomal SM PBMCs to YF-17D or MVA. We found that SM PBMCs acidification inhibitor that blocks activity of endosomal TLRs, produced significantly lower levels of IFN-a upon stimulation with including TLR7 and TLR9; three ODN antagonists of TLR sig- YF-17D than did human or RM PBMCs, consistent with the al- naling that are specific for TLR7 (IRS661), TLR9 (IRS869), or tered TLR7/9 signaling pathway previously described (21). Con- both TLRs (IRS954); as well as a nonspecific ODN as a negative versely, MVA-induced IFN-a production by SM PBMCs is similar control (34). IFN-a production was completely blocked upon to humans and significantly higher than RMs (Fig. 1B). As we stimulation with YF-17D in the presence of chloroquine and the have previously shown that the TLR7/9 signaling pathway leading specific ODN inhibitors of TLR7 and TLR7/9, indicating that to the production of proinflammatory cytokines in an NF-kB– TLR7 signaling is necessary for the induction of IFN-a in re- dependent manner is intact in SMs (21), we investigated whether sponse to this virus (Fig. 1A). The TLR9-specific ODN inhibitor YF-17D stimulation of SM PBMCs resulted in IL-12 production. did not inhibit IFN-a production above the control ODN, in- Interestingly, SM IL-12 production in response to YF-17D stim- dicating that unlike SIV and HIV that activate pDCs via both ulation was not significantly different from that of humans and TLR7 and TLR9, YFV-induced IFN-a production does not require RMs (Fig. 1C), confirming that although the TLR7/9 signal The Journal of Immunology 6409 bifurcation leading to IFN-a production via the IRF-7–dependent vaccination, regardless of the extent of YF-17D viremia detected. pathway is altered in SMs, the NF-kB–dependent signaling path- In the two RMs with the greatest upregulation of CCR7 on pDCs, way leading to production of other proinflammatory cytokines is an increase in CCR7+ myeloid DCs (mDCs) was also observed, similarly intact and functional as in RM and human pDCs. whereas CCR7 expression on mDCs remained unchanged in all SMs (Fig. 2B). Hence, our data suggest that differences in the No detectable DC activation in SMs upon YF-17D infection TLR-dependent ability of pDCs to recognize YF-17D may affect in vivo, despite detectable YF-17D viremia early in vivo innate responses following virus infection. We sought to determine whether species-specific differences in pDC-derived IFN-a production upon YF-17D exposure are asso- Attenuated immune responses in SMs upon infection with ciated with differences in innate and adaptive immune antiviral YF-17D + responses in vivo. A total of eight SMs, of which four were SIV , To investigate whether the lack of detectable DC activation in SMs 2 and six SIV RMs were infected with the YF-17D vaccine leads to differences in downstream innate responses elicited, we (YF-VAX). The kinetics of YF-17D viremia was measured by followed the magnitude and kinetics of the NK cell proliferative quantifying levels of YFV viral RNA in plasma following infection response following YF-17D immunization in the blood by staining (Fig. 2A). YF-17D was able to replicate in both primate species, for nuclear expression of Ki67 to identify recently divided cells. although to a lesser extent in SMs. All RMs and five of eight Although high levels of NK cell proliferation were seen in RMs by SMs exhibited detectable YF-17D viremia, with viral replication day 7 after YF-17D infection, there was little increase in NK cell peaking around day 4 (Fig. 2A). (By way of reference, in recent proliferation in YF-17D–immunized SMs (Fig. 3A). studies of YF-17D immunization of humans using the same assay Similarly, we observed substantial differences between SMs and Downloaded from for quantification of YFV RNA levels, between 58 and 75% of RMs in the magnitude of T cell proliferative responses following immunized individuals had peak detected levels of YFV of ,1000 YF-17D challenge. To identify effector T cell responses, we copies/ml and 17.5–30% of individuals had peak levels of ,100 measured Ki67 expression and expression of the anti-apoptotic copies/ml (27, 28).) To determine whether YF-17D was recog- protein Bcl-2, which is downregulated upon activation. The nized in vivo, we assessed whether DCs were activated by YF- combination of these markers has been shown to identify Ag-

17D in SMs and RMs postinfection. As DCs mature, they up- specific T cell responses upon YF-17D and vaccinia virus im- http://www.jimmunol.org/ regulate CCR7, a chemokine receptor that binds CCL19 and munization in humans, with effector T cells being Ki67+Bcl-2lo CCL21 and directs migration to lymphoid organs, facilitating Ag (25, 31). As in humans, there was an expansion of Ki67+Bcl-2lo presentation and cross-talk between DCs and T or B cells (40). In CD4+ and CD8+ T cells in RMs, whereas in SMs the percentage RMs there was a rapid upregulation of CCR7 on pDCs in the Ki67+Bcl-2lo CD4+ and CD8+ T cells remained unchanged fol- blood peaking at day 4 postinfection (Fig. 2B). In contrast, there lowing YF-17D inoculation (Fig. 3B). Because not all Ki67+ was little increase in pDC CCR7 expression in SMs after YF-17D T cells were Bcl-2lo in RMs, we also examined the kinetics of total proliferating T cells. There were no detectable increases in the percentage Ki67-expressing CD4+ or CD8+ T cells after YF-17D infection in either SIV+ or SIV2 SMs (Fig. 3C), nor did we ob- by guest on September 24, 2021 serve increases in CD69 or HLA-DR expression on T cells in SMs following YF-17D inoculation (data not shown). Given the sub- stantially higher baseline percentage of Ki67+ T cells in RMs than in SMs, as is commonly observed (M.B. Feinberg, unpublished data and Refs. 21, 41), we assessed the fold increase in pro- liferation of CD4 and CD8 T cells from baseline postinfection in both species. Peak fold changes in proliferation of T cells were significantly higher in RMs than in SMs (Fig. 3D). This was also true for CD4+ and CD8+ T cells in lymph nodes of these animals (data not shown). Thus, even in SMs with peak YF-17D viremia at 200–900 RNA copies/ml, there were no detectable NK or T cell proliferative responses following vaccination. Our efforts, using the limited cryopreserved cellular samples available from these studies, to quantify levels of YFV-specific CD4 and CD8 cells in YF-17D–vaccinated RMs (using the same intracellular cytokine assay system we have successfully employed, using freshly pre- pared PBMCs, to measure YFV-specific T cell responses in YF- 17D–vaccinated human volunteers; see Refs. 22, 25, 27) were unsuccessful (data not shown). Without a reliable positive control, we could neither quantify nor exclude a YFV-specific T cell re- sponse in SMs. FIGURE 2. Increases in CCR7 expression on RM, but not SM, DCs To determine whether the absence of detectable effector T cell following YF-17D infection, despite detectable YF-17D viremia. A, Plasma responses in SMs, as assessed by levels of Ki67+Bcl-2lo CD8+ YF-17D RNA copies/ml in eight SMs and six RMs. Symbols used for in- T cells quantified by flow cytometric analyses, was due to the dividual animals are as follows: SM1 (n), SM2 (d), SM3 (s), SM4 (N), ♦ : 4 ) n ♦ d reduced level of YF-17D viremia in these animals, we specifically SM5 ( ), SM6 ( ), SM7 ( ), SM8 ( ), RM1 ( ), RM2 ( ), RM3 ( ), + RM4 (:), RM5 (N), RM6 (4). B, Percentage CCR7+ DCs in RMs and SMs investigated the extent of CD8 T proliferation in humans vacci- prior to (day 0) and 4 d after YF-17D infection. pDCs are gated on CD123+ nated with YF-17D that had levels of plasma YF-17D viremia Lineage2HLA-DR+ cells, and mDCs are gated on CD11c+Lineage2HLA- ,500 RNA copies/ml (Fig. 4). From previous studies of YF-17D– DR+ cells. CCR7 expression was determined by comparison with a fluo- infected humans, it is clear that low YF-17D viremia after im- rescence minus one control. Mean 6 SEM is shown. *p , 0.05. munization is frequently seen, but that robust Ab responses are 6410 VIRAL IMMUNITY IN PRIMATE RESERVOIR HOSTS Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021 FIGURE 3. Kinetics and magnitude of T cell, NK cell, and Ab responses in YF-17D–vaccinated animals. A, Percentage Ki67+ NK (CD16+CD8+) cells in SMs and RMs. B, Ki67 and Bcl-2 expression on T cells in a representative RM (RM5) and SM (SM8) 0, 7, 11, 14, and 42 d following YF-17D infection. FACS plots were gated on CD3+CD8+ cells (upper panel) or CD3+CD4+ cells (lower panel), and numbers on plots indicate percentage Ki67+Bcl-2lo cells. C, Percentage Ki67+ CD4+ or CD8+ T cells in SMs and RMs. D, Maximum fold increases in proliferation were obtained by comparing day 0 to peak (day 7 or 11) Ki67 expression. Mean fold increases are denoted by a line. E, YF-17D neutralizing Ab titers. Data shown were obtained in two independent experiments. Symbols used for individual animals in A, C, and E are indicated in the Fig. 2A legend. nonetheless elicited (27, 28). Depending on the days where plasma maintained at high levels (Fig. 3E). The lower levels of YF-17D was sampled, 36 (27) to 60% (28) of YF-17D–vaccinated humans replication in SMs are unlikely to be responsible for the lower and had a peak viremia of ,500 RNA copies/ml. In four representa- only transient anti-YFV Ab responses seen in SMs since humans tive humans with such low YF-17D replication (peak viremia of who display low-level YF-17D viremia following immunization 52–435 RNA copies/ml), there was nevertheless a substantial in- mount significant and durable anti-YFVAb responses (Fig. 4). The crease in percentage Ki67+Bcl-2lo CD8+ T cells by day 14 post- induction and persistence of significant anti-YFV Ab responses vaccination (Fig. 4). From this comparison with vaccinated in humans who manifest low peak levels of viremia following humans, we would expect to see detectable increases in T cell YF-17D immunization, including those with undetectable YFV proliferation in SMs with measurable, albeit lower, YF-17D vi- viremia postvaccination, has also been reported by others (28). remia. Therefore, it is unlikely that the absence of any increases in Interestingly, although transient, anti–YF-17D Abs were never- T cell proliferation following YF-17D infection in SMs are due theless measured in all animals, even in SMs where YF-17D vi- only to lower levels of YF-17D replication. remia was undetectable. No differences in Ab titers were observed Ab responses play a critical role in protective immune responses between SIV+ and SIV2 SMs, further attesting to preserved im- induced by YF-17D and persist for decades following a single mune system integrity of SIV-infected SMs, unlike HIV-infected inoculation in humans (42). Thus, we next assessed the magnitude humans (43). and duration of neutralizing anti–YF-17D Ab titers in both pri- HIV viremia in infected humans has been reported to transiently mate species (Fig. 3E). In RMs and SMs the timing of appearance increase following inoculation with specific vaccines (44, 45). This of neutralizing Abs coincided with the diminution of detectable is likely due to the activation and preferential infection of Ag- YF-17D viremia. However, peak anti–YF-17D Ab titers were specific CD4+ T cells, as well as the potential reactivation of la- significantly lower in SMs than in RMs (p = 0.0013, Mann– tently infected cells, upon exposure to the vaccine (46). Hence, Whitney U test). Furthermore, YF-17D–specific Ab levels de- perturbations from the steady-state viremia during chronic SIV clined to levels below detection by 20 wk postvaccination in all infection is a potentially sensitive indicator of CD4+ T cell acti- but two SMs (Fig. 3E). In contrast, in RMs Ab levels were stably vation. We therefore determined whether there were increases in The Journal of Immunology 6411

liferating T and NK cells (Fig. 5). In contrast to YF-17D infection, there was an expansion of Ki67+Bcl-2lo CD4+ and CD8+ T cells in both species after MVA infection. Fold increases in Ki67+ T and NK cells were not significantly different between RMs and SMs (Fig. 5). An intracellular cytokine assay for MVA-specific CD8+ T cell responses confirmed that by day 29 postinfection there were increases in MVA-specific IFN-g+CD8+ T cells in all infected animals (Fig. 5D). Unlike the lower magnitude and impaired persistence of Ab responses seen in SMs following YF-17D in- fection, high anti-MVA Ab titers were measured in both species and were sustained up to 16 wk postinfection (Fig. 5E). Thus, in stark contrast to what we observed with YF-17D, SMs raised a detectable T cell response to MVA and maintained stable MVA- specific Ab responses following virus inoculation. In contrast to the transient decreased levels of SIV viremia following YF-17D infection, we observed increased SIV replica- tion in association with increased CD4+ T cell activation in SMs following MVA infection, a result consistent with reports of

vaccine-elicited increases in viremia in HIV-infected humans. Downloaded from There was a 1.4 log increase in the SIV viral load at day 11 postinfection compared with day 0 in the SM (SM5) that had a detectable CD4+ T cell proliferative response following MVA infection (Supplemental Fig. 1C). This increase in SIV replication occurred concomitantly with increased CD4+ T cell counts + (Supplemental Fig. 1D). In comparison, the other SIV SM that http://www.jimmunol.org/ + + FIGURE 4. Magnitude of CD8+ T cell and Ab responses in YF-17D– showed little increase in Ki67 CD4 T cells following MVA in- vaccinated humans with low YF-17D viremia. A, Plasma YF-17D RNA fection showed little change in SIV viremia. copies per milliliter in four humans were determined by ultrasensitive quantitative RT-PCR. B, Ki67 and Bcl-2 expression on CD8 T cells 0 and Discussion 14 d following YF-17D infection. FACS contour plots were gated on CD3+ + + lo The African primates that are natural hosts for AIDS viruses have, CD8 cells, and numbers on plots indicate percentage Ki67 Bcl-2 cells. to date, been the most intensively studied only with respect to the C, YF-17D neutralizing Ab titers. Symbols used in A and C are as follows: nature of the nonpathogenic host–SIV equilibrium. Interestingly, HU1 (♦), HU2 (:), HU3 (d), HU4 (N). these primates also harbor, without evidence of disease, other viruses that are responsible for serious zoonotic diseases in by guest on September 24, 2021 levels of plasma SIV RNA in SIV+ SMs following YF-17D vac- humans, among them YFV. Until now, little was known about cination, which might identify activation of Ag-specific CD4+ immune responses to these and other viruses in natural reservoir T cell responses in these animals that we may not have detected hosts. In particular, although we have previously shown that SM by monitoring levels of Ki67+ CD4+ T cells in the peripheral pDCs have a reduced ability to produce large amounts of IFN-a bloodstream alone. Surprisingly, rather than seeing increased lev- following stimulation with SIV and other TLR7/9 ligands (21), els of SIV production in SMs following inoculation of YF-17D, it has been unclear how the altered TLR7/9 signaling pathway there was a transient diminution of SIV replication, with a decline affects their ability to mount and maintain innate and adaptive of, on average, a half log in SIV viral loads during the first immune responses to other viruses that are recognized by TLR7/9 14 d after vaccination (Supplemental Fig. 1A). Although, due to innate pathways. In this study, to address these questions, we in- the small sample size, this decline is not statistically significant, it vestigated the immune responses of SMs to two viruses, YFV and nevertheless represents a greater change than is generally ob- MVA. served in such a short time frame during chronic SIV infection of Studies in YF-17D–immunized humans have shown that the SMs (S.I. Staprans and M.B. Feinberg, unpublished observation) activation of type I IFN-response genes represents an early and and is striking in that it occurs in all animals during the period of prominent component of anti-YFV immune responses (28, 29). active YF-17D replication. The decline in SIV viral load was not In this study, we provide evidence elucidating the underlying attributable to measurable changes in overall CD4+ T cell levels in mechanism of activation of this early innate response to YF-17D the blood or lymph nodes (Supplemental Fig. 1B and data not infection by showing that YFV requires TLR7 to induce a type I shown) or in levels of proliferating (Ki67+) CD4+ T cells in these IFN response in pDCs from humans and RMs. Conversely, we compartments (Fig. 3C and data not shown). show that, as has been previously described in mice (23, 24), in humans and primates MVA elicits IFN-a by non-TLR7/9 innate Equivalent immune responses in RMs and SMs following MVA pathways. As for SIV and other TLR7/9 ligands, we observed that infection YF-17D stimulation of SM PBMCs ex vivo results in significantly To determine whether the reduced adaptive immune responses of reduced IFN-a production compared with RMs and humans, SMs raised to YF-17D is due to how this virus is recognized by the whereas MVA-induced IFN-a production by PBMCs is similar. , we next infected SMs and RMs with MVA, Interestingly, SM PBMCs are nevertheless able to produce IL-12 a virus recognized by non-TLR7/9 innate pathways. To rule out in response to YF-17D stimulation, suggesting that proximal animal-specific differences, we used a subset of animals previously TLR7/9 signaling events are intact. This is consistent with pre- infected with YF-17D. A total of four SMs, of which two were vious studies indicating that NF-kB–dependent signaling path- 2 chronically SIV+, and four SIV RMs were infected with MVA. ways induced by TLR7/9 ligand binding are unaltered, whereas As before, we measured Ki67+Bcl-2lo T cells and total pro- activation of the IRF-7–dependent signaling pathway following 6412 VIRAL IMMUNITY IN PRIMATE RESERVOIR HOSTS Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 5. Kinetics and magnitude of T cell, NK cell, and Ab responses in MVA-vaccinated animals. A, Ki67 and Bcl-2 expression on CD8 T cells in a representative RM (RM1) and SM (SM5) 0, 7, 11, 14, and 39 d following MVA infection. FACS plots were gated on CD3+CD8+ cells (upper panel)or CD3+CD4+ cells (lower panel), and numbers on plots indicate percentage Ki67+Bcl-2lo cells. B, Kinetics of percentage Ki67+CD4+ T cells, CD8+ T cells, and NK cells in SMs and RMs. C, Maximum fold increases in proliferation were obtained by comparing day 0 to peak (day 7) Ki67 expression. Mean fold increases are denoted by a line. D, PBMCs from SMs and RMs were stimulated with MVA and stained for IFN-g on days 0 and 29 following MVA infection. Numbers on FACS plots indicate the percentage IFN-g+CD8+ T cells (gated on live CD3+CD8+ cells). E, MVA Abs (IgG) were measured by ELISA and endpoint titers calculated. Symbols used for individual animals in B and E are as follows: SM1 (n), SM2 (d), SM5 (♦), SM6 (:), RM1 (n), RM2 (♦), RM3 (d), RM4 (:).

TLR7/9 ligand engagement is markedly attenuated in SMs pDCs, of NK and T cells observed in RMs. Furthermore, although all SMs as compared with pDCs from RMs and humans (21). The precise mounted detectable neutralizing Ab responses to YF-17D, this changes in the IRF-7–mediated signaling pathway in SMs, as well response was significantly lower than in RMs and, in notable as the potential role for additional variations in post-TLR sig- distinction to humans and RMs immunized with YF-17D, it was naling molecules, that result in lower IFN-a production following generally not maintained (47). Although YF-17D replication was TLR7/9 stimulation remain to be determined. lower in SMs than in RMs, we show that humans with similarly Differences in ex vivo SM pDC type I IFN responses to YF-17D low peak YF-17D viremia nonetheless have detectable CD8+ and MVAwere mirrored by striking differences between SM in vivo T cell proliferation and robust Ab responses, suggesting that the immune responses to YF-17D and MVA immunization. In SMs, lower YF-17D viremia in SMs is not sufficient to explain the there was no detectable DC activation or proliferative NK and transient Ab responses or absence of detectable T cell responses in T cell responses following YF-17D infection, in contrast to in- this primate species. Furthermore, both SMs and RMs raised in- creased pDC activation and the significant increases in proliferation nate and adaptive cellular and humoral immune responses to MVA The Journal of Immunology 6413 that were of similar magnitude and persistence. Hence, the ab- the ex vivo PBMC stimulation data suggest, there is a reduced sence of any detectable effector T cell increases even in SMs with type I IFN response in vivo in SMs following YF-17D immuni- substantial YF-17D replication is a notable and significant result. zation. A recent study of experimental SIV infection of SMs ob- It is still unclear why YF-17D replicates to lower levels in SMs served a gene expression profile consistent with a transient type I than in RMs. It has been shown that DCs play a role in flavivirus IFN response during the acute infection period (61). Given the replication by disseminating virus from the site of infection (48, 49). exquisite sensitivity of expression levels of IFN response genes to Hence, the reduced activation and trafficking of DCs observed even very low levels of IFN-a and IFN-b (62), these data are not in SMs might also contribute to the lower viral replication in this inconsistent with lower level, but still readily detectable, levels of species. Alternatively, because YF-17D is able to replicate in IFN-a produced by SM pDCs following exposure to SIV in ex activated T cells (Ref. 51 and data not shown), the substantial vivo assays (21). Of note, these studies did not investigate the T cell activation in RMs and humans following immunization may source of the in vivo type I IFN or ascertain the innate pathways amplify YF-17D replication. If so, reduced T cell activation in by which its production was being stimulated. The observed ab- SMs might contribute to the more limited YF-17D viremia ob- sence of a type I IFN response pattern during chronic infection served. However, further experiments will be required to specifi- when levels of SIV viremia are very high (61) is consistent with cally address this question. our earlier report (21) and reinforces that important differences Importantly, the substantially reduced innate and adaptive im- exist in the innate immune responses to SIV in SMs and RMs. mune responses of SMs to YF-17D observed is consistent with the Induction of immunosuppressive mediators could, as has been key role of type I IFNs in linking innate and adaptive immunity hypothesized following SIV infection (19), also play a role in the

(28, 29, 51). Studies conducted in mice have shown that IFN-a has active inhibition of damaging immune responses to YFV in SMs, Downloaded from numerous effects on innate immune cells that indirectly affect for instance, by induction of IDO following exposure to HIV and adaptive immune responses, such as stimulating the maturation of other TLR7 and TLR9 agonists (63), an NF-kB–mediated re- DCs, inducing the upregulation of MHC class I, promoting NK sponse that we have found to be preserved in SMs (data not cell activation, as well as resulting in production of IL-15 and shown). In light of evidence indicating that potentially immuno- IFN-g, which themselves enhance adaptive immune responses suppressive mediators, such as IDO, are produced by pDCs in

(51). Moreover, type I IFN also has direct effects on the induction response to activation of the TLR7 signaling pathway, it is http://www.jimmunol.org/ of T cell responses. Clonal expansion and survival of CD4+ and tempting to speculate that such mediators might contribute to the CD8+ T cells in response to specific viruses are severely di- transient diminution of SIV viremia we observed in SMs follow- minished upon infection in mice lacking the type I IFN receptor or ing vaccination with YF-17D (but not the non-TLR7/9-active vi- when IFN-a production is blocked (30, 52, 53). Interestingly, the rus, MVA) (64, 65). Conceivably, both the limited CD4+ T cell dependency of T cells on IFN-a–mediated survival signals is activation and induction of immunosuppressive pathways might determined by the pathogen (52, 54). In IRF72/2 mice, which do be involved in protecting natural hosts from disease. An attenuated not produce IFN-a/b upon TLR7/9 stimulation, impairment of innate or compromised early CD4+ T cell response may result in T cell responses is seen only when TLR7/9 signaling is required induction of nonresponsive or readily exhausted T cell responses for DC activation (31). More recent support documenting the (66), the promotion of a state of active tolerance, and result in by guest on September 24, 2021 impact of depressed pDC number or function on the induction and impaired Ab responses. Importantly, both following YF-17D and maintenance of CD8+ T cell numbers and function have been SIV infection of SMs, little change in CD4+ T cell proliferation is obtained from studies of YF-17D infection in rapamycin-treated observed (data from this study and Refs. 21, 41, 67–69). The mice (which results in inhibition of IRF-7–dependent IFN-a extent of early CD8+ T cell proliferation has been found to vary production) and VSV-infected mice that had been conditionally between different animals and in different published reports depleted of pDCs (30, 55). Although less is known about the role (ranging from low to modest levels as compared with acute of TLRs, DCs, and IFN-a in the generation of long-lived memory pathogenic SIV and HIV infections), but in all cases when seen, B cell responses (56), IFN-a has been shown to enhance primary increased levels of CD8+ T cell proliferation in SIV-infected SMs Ab responses, promote isotype switching, influence relative po- following SIV infection have been only transient (in contrast to larization of the Ab isotype profile, and, together with IL-6, pathogenic SIV infections in RMs and HIV infections of humans) stimulate plasma cell differentiation in mice (57–60). However, (21, 41, 67–69). Notably, in the present study, we could detect detailed mechanistic studies to better understand the role and little or no increase in effector CD8+ T cell proliferative responses mechanisms of potentially important effects of TLR7/9 signaling after YF-17D infection. Clearly, pursuit of understanding the spe- and IFN-a production on the levels, isotype composition, gener- cific determinants of the differences as well as the similarities in ation of CD4+ T cell and B cell memory, and in the overall per- SM host responses to YF-17D and SIV in much greater detail, and sistence of Ab production following specific infections and how these differ from those of RMs and humans, represent im- immunizations in both humans and nonhuman primates (as well as portant topics for future studies. in other vertebrate species that represent natural hosts for zoonotic Of note, in contrast to SIV, YF-17D does not establish persistent infections) are clearly needed. infection in SMs despite their dampened immune responses to the Together with these data, our results suggest that the ability of virus. Ab responses to YF-17D in SMs, albeit transient, are likely pDCs to produce IFN-a following TLR7/9 activation is important responsible for virus clearance. In contrast to HIV or SIV, YF-17D in generating multilineage innate and adaptive immune responses is extremely sensitive to neutralizing Ab (42). In primates studied to YFV in humans and primates (21, 28, 29). It is likely that the herein, as in immunized humans (25), clearance of YF-17D vi- diminished NK and T cell responses to YF-17D in SMs are related remia coincides with the appearance of Ab responses. The ability to, and perhaps explained by, the reduced IFN-a response to this of SMs to produce Ab responses even to viruses whose recogni- virus. Additionally, lower magnitude and limited persistence of tion by the innate immune system involves TLR7/9 may enable anti–YF-17D Ab levels in vaccinated SMs may result from the them to resist the range of potentially pathogenic virus infections reduced production of IFN-a by their pDCs and the limited CD4+ they encounter in the wild. Indeed, experimental infection of SMs T cell activation they exhibit following YF-17D infection. How- with pathogenic YFV has been reported to result in transient vi- ever, further studies will be required to directly ascertain that, as remia that is cleared coincident with the production of YFV- 6414 VIRAL IMMUNITY IN PRIMATE RESERVOIR HOSTS neutralizing Abs (13). This feature, in concert with the discrete infecting novel hosts (5), the TLR7/9 pathway likely represents an nature of the alteration in TLR7/9 signaling in SMs, as well as the important candidate for possible host adaptations to tolerate these apparent redundancy in innate immune activation inferred from zoonotic viruses without disease. Interestingly, adaptations to par- studies of individuals with specific inherited polymorphisms (70), ticular viruses by their natural hosts may enable them to harbor may explain why SMs are nevertheless able to control most additional viruses recognized by the same innate immune path- infections. Additional immunization studies in SMs and RMs may ways, such that some species act as reservoir hosts for multiple provide a valuable experimental model to understand how di- zoonotic agents. If so, the observation that specific animal species, vergent TLR signaling patterns can influence host responses to including bats and African primates, serve as reservoir hosts for specific infectious agents, thereby complementing findings from multiple zoonotic viruses, along with the fact that so many emerg- genetically defined mouse models, which are increasingly recog- ing infectious diseases are caused by RNA viruses, may not be nized as being imperfect models of human TLR biology (70–72). coincidental (5, 78). The interaction of the innate immune system with a virus greatly It will be important for future studies to elucidate the immu- affects the balance between an immune response capable of nologic mechanisms that allow reservoir hosts to tolerate infection clearing infection or suppressing virus replication and a response with single or multiple emerging viruses and to determine whether that contributes to disease. For most emerging zoonotic infections, the underlying mechanisms of apathogenic infection are similar for the severe consequences of acute infection are responsible for the distinct viruses and reservoir species. Such studies promise to greatest morbidity and mortality (2–4, 6). Although acute con- provide novel insights into evolutionary solutions to host–pathogen sequences of these infections can be lethal, hosts surviving the struggles that have allowed natural hosts to avoid disease. These primary infection clear the virus and develop specific immunity. In insights should, in turn, inform efforts to better detect potential Downloaded from contrast, in HIV infection host immune responses are fundamen- reservoir hosts, anticipate the potential emergence of zoonotic tally unable to mediate clearance, and it is the long-term con- infections, and to specifically design therapies to ameliorate the sequences of chronic virus replication and generalized immune immunopathologic consequences of emerging virus infections. activation that lead to disease progression (17). In each instance, however, unintended immunopathologic consequences of the Acknowledgments virus–host immune system interactions likely influence disease We thank G. Silvestri, S. Garg, E. Farnon, T. Monath, B. Pulendran, and http://www.jimmunol.org/ severity following infection. For instance, higher levels of proin- A. Yates for very helpful discussions. We also acknowledge the Yerkes Na- flammatory mediators are seen in people with fatal hemorrhagic tional Primate Research Center veterinarians S. Ehnert, C. Souder, and YFV infections compared with people experiencing less severe E. Strobert for care of the study animals; A. Barry, K. Berger, S. Gordon, infections (15). Indeed, a more exuberant early immune response S. Klucking, J. Miller, and B. Sumpter for expert technical advice; F. Barrat is often associated with more severe disease following zoonotic and R. Coffman at Dynavax Technologies for providing the oligonucleotide infections, such as SARS coronavirus, dengue virus, hantavirus, antagonists and for very helpful advice; D. Garber for providing an initial virus, and the 1918 influenza A virus strain (73–77). stock of MVA; and anonymous human volunteers at Emory University for The precise mediators of immunopathologic responses seen in providing blood samples for these studies. nonnatural hosts following zoonotic viral infections may differ in by guest on September 24, 2021 different hosts and different infections, and many cytokine path- Disclosures ways (e.g., type I IFN) may well play both beneficial and detri- The authors have no financial conflicts of interest. mental roles in the host response to infection. However, ex- plication of the role that TLR-mediated signaling of the innate References immune response plays in determining the absence of disease in 1. Jones, K. E., N. G. Patel, M. A. Levy, A. Storeygard, D. Balk, J. L. Gittleman, natural reservoir hosts, as well as the pathogenesis of zoonotic and P. Daszak. 2008. Global trends in emerging infectious diseases. 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