Replication of Simian Immunodeficiency Virus (SIV) in ex Vivo Lymph Nodes as a Means to Assess Susceptibility of Macaques in Vivo

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1 Virology 275, (2000) doi: /viro , available online at on Replication of Simian Immunodeficiency Virus (SIV) in ex Vivo Lymph Nodes as a Means to Assess Susceptibility of Macaques in Vivo L. Margolis,* S. Glushakova,* C. Chougnet, G. Shearer, P. Markham, M. Robert-Guroff, R. Benveniste, C. J. Miller, M. Cranage,** V. Hirsch, and G. Franchini,1 *Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; Experimental Immunology Branch, Basic Research Laboratory, and Frederick Cancer Research and Development Center, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892; Advanced BioScience Laboratories, Inc., Kensington, Maryland 20895; California Regional Primate Research Center, University of California Davis, Davis, California 95616; **Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire, United Kingdom; and National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland Received December 15, 1999; returned to author for revision May 8, 2000; accepted July 9, 2000 Six macaques, apparently uninfected, following low-dose exposure to the pathogenic SIV mac251 and SIV SME660 by the mucosal route, were used in a pilot study to investigate whether infectability of ex vivo lymph nodes could predict resistance and/or susceptibility to SIV infection in vivo. Of six macaques exposed to the less-pathogenic virus SIV MNE, four resisted viral infection. Analysis of the susceptibility of the PBMC of these four animals before SIV MNE challenge indicated that all of them were resistant to infection by the SIV BK28 isolate and, in three of them, this resistance was dependent on CD8 T cells. Blocks of lymph nodes of these four macaques were resistant to SIV MNE infection ex vivo following SIV MNE viral challenge exposure. However, the same blocks from the same animals were permissive to the more virulent SIV 251(32H). Accordingly, three of these macaques were readily infected following challenge exposure with SIV 251(32H). Lymphoproliferative responses in blood or lymph nodes, local C-C chemokine production in the lymph-node explants, and cytotoxic T-cell activity measured throughout the study did not correlate with ex vivo resistance or susceptibility to in vivo infection. In conclusion, PBMC and lymph-node resistance or susceptibility to infection ex vivo appeared to correlate with in vivo infectivity and, thus, these approaches should be further tested for their predictive value for in vivo infection Academic Press INTRODUCTION SIV infection of rhesus macaques has been developed as a model to study HIV disease. In the SIV macaque model, particularly following mucosal exposure, low doses of SIV may result in latent or occult infection and, despite knowledge on the time and dose of exposure, the assessment of the mechanism(s) underlying resistance to viral infection has proven difficult (Clerici et al., 1994b; Cranage et al., 1998; Dittmer et al., 1996; Lohman et al., 1994; McChesney et al., 1998; Neildez et al., 1998; Petry et al., 1997; Trivedi et al., 1994). In humans, where exposure could only be inferred by risk behavior, the natural or acquired correlates of resistance are also unknown (Clerici et al., 1994a; Mazzoli et al., 1997; Pinto et al., 1995; Shearer and Clerici, 1996). Recent evidence in HIV-1-exposed (presumably uninfected) Gambian women and uninfected infants born to HIV-1-infected mothers suggests that virus-specific CD8 responses may be associated with viral resistance (Cheynier et al., 1 To whom correspondence and reprint requests should be addressed at National Institutes of Health, Basic Research Laboratory, National Cancer Institute, 41 Library Drive, Building 41, Room D804, Bethesda, MD Fax: (301) veffa@helix.nih.gov. 1992; De Maria et al., 1994; Rowland-Jones et al., 1993, 1995). However, multiple mechanisms may underlie the resistance of some individuals to HIV-1 (Fauci, 1996). Here we have attempted to address the issue by studying a cohort of six macaques previously exposed to low doses of SIV mac251 (Miller et al., 1994) or SIV SME660 (Goldstein et al., 1994) and studying immunological and virological parameters in blood and tissues of these macaques following sequential exposure to SIV isolates that differed in their virulence. All the viral isolates in this study were genetically and immunologically related and were able to induce AIDS in macaques. However, their levels of viral replication in vivo differed (Benson et al., 1998; Benveniste et al., 1990; Cranage et al., 1992; Hu, 1994; Miller et al., 1994). The six macaques used in this study resisted a low dose of either SIV 251 or SIV SME660 and analysis of the immune responses in the blood did not offer clues to the nature of this resistance. Thus, experiments were designed to address whether the in vitro susceptibility of the PBMC of these macaques to SIV correlated with resistance to a subsequent SIV MNE challenge exposure. In addition, we investigated whether the resistance of the PBMC to SIV replication was dependent on CD8 T cells to assess whether protection from /00 $35.00 Copyright 2000 by Academic Press All rights of reproduction in any form reserved.

2 392 MARGOLIS ET AL. TABLE 1 Stimulation Index in Tissue and Blood of the SIV SME660 - and SIV 251 -Exposed s a Lymph node PBMC PHA cona Gag Env Nef Flu Ag PHA cona gag 1 env 2 nef 3 Flu Ag 290 ND b ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND a negative animals had a stimulation index of less than 3; stimulation index was derived from the IL-2 production as previously described (Benson et al., 1998). b ND not determined. infection was innate or the result of adaptive immunity following exposure to a low dose of virus. Finally, we assessed whether the susceptibility or resistance of macaques to a given viral strain correlated with the susceptibility or resistance of their ex vivo lymph nodes. The lymph nodes of the animals that resisted SIV MNE challenge exposure remained resistant to SIV MNE in vitro. However, the same lymph nodes replicated the more virulent SIV 251(32H) and subsequent exposure of these macaques to SIV 251(32H) resulted in overt infection and disease progression. RESULTS AND DISCUSSION In an attempt to make full use of nonhuman primate resources and investigate a possible novel approach to predict resistance and susceptibility to SIV infection in vivo, we used six macaques exposed to low doses (Buge et al., 1997) of either SIV 251 (Miller et al., 1994) or SIV SME660 (Goldstein et al., 1994). All macaques were apparently uninfected, as judged by negative virus isolation, consistently negative DNA PCR, and the absence of sustained lymphoproliferative responses to gag, env, and nef in both lymph nodes and PBMC (Table 1). Virus isolation was consistently negative (eight subsequent attempts of virus isolation from PBMC of these animals within 8 months postexposure), and the sensitivity of our isolation assay was assessed by a limiting-dilution method using total PBMC from SIV-infected animals and CEMX174 as a target cell. With this assay, virus isolation is usually positive with 450 PBMC and occasionally with as few as 50 PBMC cells. The DNA PCR assay was described previously (Buge et al., 1997). To investigate possible immune correlates and assess indirect evidence of viral infection (McChesney et al., 1998; Shearer and Clerici, 1996) we measured CD4 T-cell-proliferative responses against SIV peptides derived from the gag, env, and nef proteins (Clerici et al., 1994b) simultaneously in the blood and in the lymphnode cells of these animals. Albeit all animals PBMC proliferated in response to phytohemagglutinin (PHA) or concanavalin A (ConA), proliferative responses to env and nef were transient and found in one out of three independent measurements in the blood, but not in the lymph nodes, of animals 292 and 294 (Table 1). The remaining seven animals did not proliferate in response to viral antigens. Thus, the lack of sustained proliferative response in the blood as well as in lymphoid tissues combined with the inability to detect virus suggests that these animals may have cleared the low-dose infection by the pathogenic SIV strains, rather than have an occult infection (McChesney et al., 1998). Since innate protective response mediated by the CD8 T cells has been described as another correlate in humans as well as macaques and since CD8 T-cellindependent resistance of PBMC to viral infection has been observed in vitro (Vanessa Hirsch, unpublished data), the susceptibility of PBMC in the presence and in the absence of CD8 T cells was studied. As demonstrated in Table 2, some differences were observed in the susceptibility of macaque PBMC to replicate SIV BK28 ; animals 291, 292, 295, and 352 replicated SIV BK28 at a lower level than animals 290 and 294 in the presence of CD8 T cells. However, when CD8 T cells were removed, viral replication was rescued at levels observed in animals 290 and 294 only in animal 292, suggesting that in this animal the in vitro resistance to viral replication was dependent on CD8 T cells. In animals 291 and 352, the in vitro resistance to SIV BK28 replication was observed also with a tenfold increase of virus input and was partly mediated by CD8 T cells, since their removal moderately increased viral replication. Thus, reduced susceptibility of PBMC to infection was observed in vitro in four out of six animals and, in one of the four animals, the decreased susceptibility was clearly dependent on CD8 T cells. Since all of these six animals resisted the first low-dose challenge of highly virulent SIV strains, these data indicate that none of the parameters studied correlated tightly with in vivo resistance.

3 LYMPH-NODE SUSCEPTIBILITY TO SIV INFECTION 393 TABLE 2 In Vitro Susceptibility of Macaques PBMC to SIV BK28 Infection Total PBMC 6, ; 260 a 1,625 5,400 1,288 Limit; 920 a CD8 -depleted PBMC 4, ; 520 a 10,000 5,400 1,040 Limit; 1,770 a % CD8 suppression 0 0; 50 a ND b ;94 a a 100 TCID 50. b ND not determined. Next we tested whether these animals were susceptible to the less-virulent SIV MNE strain. Since the SIV MNE stock had been previously titered only in Macaca nemestrina, we first assessed its infectiousness in Indian rhesus macaques. This virus stock (Benveniste et al., 1990) readily infected two naive rhesus macaques following intrarectal exposure either to 1:5 or to 1:10 dilutions of the SIV MNE stock, and both animals were virusisolation-positive in four out of five or in six out of six attempts, respectively. Therefore, we chose the 1:10 dilution for further experimentation. In the experimental challenge, virus from this stock infected intrarectally both the control naive animals 469 and 470, as demonstrated by the SIV MNE isolation on several occasions from their PBMC. In contrast, among animals 290, 291, 292, and 295, previously exposed to SIV SME660, and animals 294 and 352, previously exposed to SIV 251, animals 290, 291, 294, and 352 apparently resisted infection. Their blood scored negative for virus isolation in 15 of 15 attempts within 12 months postchallenge (Table 2), and the inability to isolate virus was paralleled by the negative finding of plasma viral RNA by NASBA (Romano et al., 1997). The latter assay is quantitative for more than 10 3 viral copies per ml of plasma. The two virus-isolation-positive animals, 290 and 294, also became seropositive for SIV antigen, whereas seroconversion did not occur in the four animals that were consistently negative for detection of virus (data not shown). In the search for additional correlates for resistance to the SIV MNE challenge, lymphoproliferative responses were studied as described in Table 1, and a transient nef-specific proliferative response was found only in animal 291 (Table 3). In addition, attempts to measure CTL responses in the blood of the six animals, either before or after SIV MNE challenge, did not reveal significant cytotoxic activity (less than 10% killing; data not shown). Interestingly, all of the four animals that resisted SIV MNE challenge exposure in vivo demonstrated a decreased susceptibility to SIV BK28 replication of their PBMC prior to challenge (Table 2), and only in one out of four animals this decreased susceptibility appeared to be dependent on CD8 T cells (Tables 2 and 3). Considered together, these data could suggest that a mucosal preexposure to low doses of pathogenic SIV strains may have conferred resistance to subsequent mucosal challenge with a lessvirulent virus, since both naive controls became infected and four out of the six experimental animals did not. However, one alternative interpretation of these data is that the animals that also resisted the second challenge with SIV MNE may be naturally resistant to viral infection as demonstrated by the decreased susceptibility of their PBMC to SIV BK28 infection in vitro. Other immune corre- TABLE 3 In Vivo Susceptibility of Preexposed Macaques to SIV MNE Exposure 1 (vaginal) Virological status Exposure 2 a (intrarectal) Virological status Immune response observed In vitro PBMC susceptibility Dependence on CD8 T cells 290 SIV SM SIV MNE Yes No 291 SIV SM SIV MNE T helper nef response Decreased Partly 292 SIV SM SIV MNE Decreased Fully 294 SIV 251 SIV MNE Yes No 295 SIV SM SIV MNE Decreased No 352 SIV 251 SIV MNE Decreased Partly 469 Naive SIV MNE ND b ND ND 470 Naive SIV MNE ND ND ND a The animals were exposed to a 1:10 dilution of the SIV MNE viral stock (Benveniste et al., 1990). b ND not done.

4 394 MARGOLIS ET AL. lates could not be identified because the proliferative responses in the animals before and after challenge with SIV MNE were transient and various levels of CD8 T-cellassociated suppression activity were present in most, but not all, of the animals that resisted SIV MNE challenge (Tables 2 and 3). Similarly, bulk CTL assays were negative. Because the measurement of these immunological responses did not reveal any correlation with resistance to viral infection in vivo, we resorted to exploring a novel approach. Blocks of human lymphoid tissue ex vivo support infection of diverse HIV strains in the absence of exogenous stimulation (Glushakova et al., 1997, 1998). We therefore elected to assess the ex vivo susceptibility to SIV MNE of the lymph nodes of each animal that resisted the in vivo challenge with SIV MNE. In parallel, we also tested the susceptibility to the pathogenic SIV 251(32H) viral stock of the same ex vivo lymph nodes with the intent to expose the macaques to this viral stock at a later time. Experiments were initiated on the ex vivo tissues collected 12 months following SIV MNE challenge after the virological status of the animals was confirmed. The kinetics of ex vivo replication of SIV MNE and SIV 251(32H) was first assessed in biopsies of lymph nodes from three naive animals. The latter virus was chosen because sufficient amount of in vivo titered viral stock was available to perform ex vivo and in vivo viral challenge. Figure 1 demonstrates that both viruses replicated efficiently and that the replication kinetic mirrored that observed in human tonsils infected ex vivo with different strains of HIV-1 (Glushakova et al., 1997, 1998). Aliquots from the SIV MNE and SIV 251(32H) viral stocks were then used to infect ex vivo lymph nodes from animals 291, 292, 295, and 352 obtained at 12 and 13 months postchallenge, and animal 295 at 15 months postchallenge; their susceptibility to infection was assessed by measuring the total amount of p27 gag antigen produced per tissue block over an 18-day period. Both SIV mac strains readily replicated in the ex vivo lymph nodes of naive animals to the level of ng of p27 (Fig. 1 and Table 4), whereas the replication of the same virus in the ex vivo lymph nodes of the experimental animals was at least tenfold less. None of the ex vivo lymph nodes, except one (axillary lymph node from animal 295), replicated SIV MNE efficiently (p27 gag 1.18 ng). Moreover, 1 month later, two additional lymph nodes from animal 295 also failed to replicate SIV MNE efficiently. In contrast, SIV 251(32H) replicated in nine out of ten lymph nodes from the experimental animals at levels above 13 ng of p27 gag. On average, SIV MNE replication in lymph nodes from the challenged animals produced ng of p27 gag, whereas SIV 251(32H) replication in the same lymph nodes from the same animals produced ng of p27 gag. Thus, viral replication ex vivo appeared in lymphoid tissue ex vivo to correlate with the resistance of these animals to SIV MNE infection in vivo. FIG. 1. Kinetics of SIV MNE and SIV 251(32H) replication in lymph-node histocultures. Lymph nodes extracted under anesthesia according to an approved protocol were dissected into 2- to 3-mm blocks and placed at the air liquid interface onto the collagen sponge gels (5 blocks/gel/3 ml of RPMI1640; media was changed every 3 days) as described earlier (Glushakova et al., 1997, 1998). To infect the tissue equal doses of SIV in 5 9 l were applied on top of each tissue block. To study the molecular basis of this observation, we analyzed the production of RANTES, MIP1-, and MIP1-, the natural ligands for CCR5, since local alteration of their levels could mediate the resistance of lymph nodes to SIV infection (Benveniste et al., 1990; Kannagi et al., 1988; Lehner et al., 1996; Leno et al., 1999; Wang et al., 1998). Overall, comparable amounts of C-C chemokines were observed in different ex vivo lymph nodes from the same animal, whereas variation in chemokine production was observed among animals. The level of variation in the amounts of C-C chemokine production did not allow for an evaluation of a possible correlation between C-C chemokine production and resistance of the ex vivo lymph nodes to SIV MNE (data not shown). Since the lymph nodes of the experimental macaques appeared to be susceptible ex vivo to SIV 251(32H) infection, we investigated whether this assay reflected the animals infectability in vivo. Therefore, animals 292, 295, and 352, and two naive controls (471 and 485) were exposed to 20 MID of SIV 251(32H) by the intrarectal route as described earlier (Benson et al., 1998). Both naive and experimental macaques became infected by SIV 251(32H) as demonstrated by consecutive successful attempts of virus isolation from PBMC and detection of viral RNA in the plasma (Fig. 2). The levels of acute and setpoint viremia in the naive and the previously exposed animals were similar, and all animals seroconverted to viral antigens

5 LYMPH-NODE SUSCEPTIBILITY TO SIV INFECTION 395 TABLE 4 In Vitro Susceptibility of Lymph-Node Histocultures to the SIV MNE and SIV 251(32H) Strains Location SIV MNE a (ng of p27) SIV 251(32H) b (ng of p27 c ) 13 Months 291 Inguinal ND d Axillary ND Axillary Inguinal ND Months 291 Inguinal Mesenteric 1.1 ND 292 Inguinal Mesenteric Inguinal Mesenteric 1.7 ND 352 Inguinal Mesenteric Months 295 Mesenteric (Control) Inguinal 58 ND a SIV MNE : the viral stock had a TCID50 of on the A2-clone 5 cell line and was diluted 1:50 to a presumed TCID50 of 10 4 (Benveniste et al., 1990). b SIV 251(32H) : the viral stock had a TCID50 of 10. c Total amount of p27 gag antigen produced by five blocks in 3 ml of media over an 18-day period. d ND, not determined. and experienced a decrease in CD4 T-cell counts within the 6-month observation period (Fig. 2). Thus, efficient ex vivo lymph-node infection correlated with the susceptibility of these macaques to in vivo infection with SIV 251. Furthermore, these data indicate that the resistance of these macaques to SIV infection was not absolute and was likely dependent on the virulence and/or dose of the viral challenge. Thus, the doses and virulences of the viral strains used in conjunction with innate or, in this case, perhaps acquired resistance to viral infection greatly influence the outcome of challenge exposure (Table 5). Ex vivo susceptibility of lymphoid tissues, including blood, to viral strains of different virulences may help to define correlates of natural or acquired resistance to infection in the SIV macaque model and may further develop as an additional experimental tool to search for immune correlates in studies of HIV-1 vaccine candidates. MATERIALS AND METHODS SIV infection of simian lymphoid tissue ex vivo Inguinal, mesenteric, or axillary lymph nodes were extracted under anesthesia according to an approved protocol and immediately placed in sterile tubes with RPMI1640 medium with antibiotics. Lymph nodes were transferred to the laboratory, washed thoroughly with FIG. 2. (A) Outcome of SIV 251(32H) challenge exposure. Virus load in the plasma of exposed (top) and control (bottom) animal was measured by NASBA, as previously described (Benson et al., 1998). (B) CD4 T-cell count for mm 3 of blood following viral exposure. (C and D) Western blot analyses of the exposed animals sera. The numbers on the tops of the panels represent months following SIV 251(32H) challenge. (D) 209 serum is used as a positive control (Abimiku et al., 1997).

6 396 MARGOLIS ET AL. TABLE 5 Primary and Challenge Exposures to SIV exposure to October 1996 February 1998 Challenge Outcome Challenge Outcome SIV SME660 SIV MNE Infected Euthanized 291 SIV SME660 SIV MNE Uninfected Euthanized a 292 SIV SME660 SIV MNE Uninfected SIV 251(32H) Infected Euthanized 295 SIV SME660 SIV MNE Uninfected SIV 251(32H) Infected Euthanized 294 SIV 251 SIV MNE Infected Euthanized 352 SIV 251 SIV MNE Uninfected SIV 251(32H) Infected Euthanized 469 Naive SIV MNE Infected 470 Naive SIV MNE Infected 471 Naive SIV 251(32H) Infected Euthanized 485 Naive SIV 251(32H) Infected Euthanized a This animal was euthanized because of postsurgical complications following lymph-node biopsy. medium containing antibiotics/antimicotics, and then sectioned into 2- to 3-mm blocks. These tissue blocks were placed on top of collagen sponge gels in culture medium (RPMI1640 with 15% FCS) at the air liquid interface and infected as described earlier for human lymphoid tissue (Hu, 1994; Kannagi et al., 1988), specifically, after overnight in culture, tissue blocks were infected with SIV MNE or SIV 251(32H). To normalize the infection dose for different SIV strains, between 5 and 9 l of viruscontaining media were applied on top of each tissue block. Productive SIV infection was assessed by measuring p27 in the culture medium using an SIV p27 antigen ELISA (Advanced Bioscience Laboratories, Kensington, MD). Specifically, the concentration of p27, which accumulated in 3 ml of culture medium bathing a sponge gel with five tissue blocks during the 3 days between the successive medium changes, was used as a measure of virus replication. Immunological and virological assays Ficoll-purified PBMC from each animal were stimulated in vitro with the Gag peptide (EGCTPYDIN- QML), used at 5 M concentration; the peptides from the SIV MNE Env amino acid sequence (NYVCPCHIR- QIINTWH), (CITMKCNKSETDKWG), and (KYYNLTMKCRRPGNK), used at a 5 M final concentration; and the Nef peptides (GPRYPK- TFGWLWKLV), (SQWDDPWGEVLVWKF), and (EKGGLEGIYYSERRHK), also used at the 5 M final concentration. CD8 suppression CD8 suppression activity was measured as follows: Ficoll-purified PBMC from each animal were divided in two aliquots: one was depleted of CD8 T cells by Ab -CD8 conjugated on Dynabead (Dynal, U.K.) and the other was left untreated. Both aliquots were stimulated with PHA at 2.5 g/ml for 48 h, washed, and exposed to 10 TCID50 of BK28 virus stock. The cultures were maintained in IL-2 and the amount of p27 in the sample was assayed at Day 8. The percentage suppression is derived from the value at Day 8 of p27 gag obtained in the absence of CD8 T cells and the level of p27 gag obtained in the whole PBMC (100%). Virus isolation and detection of plasma viral RNA by NASBA and Western blot were performed as previously described (Benson et al., 1998). ACKNOWLEDGMENTS We thank Barbara Felber for helpful suggestions and Steven Snodgrass for editorial assistance. REFERENCES Abimiku, A. G., Robert-Guroff, M., Benson, J., Tartaglia, J., Paoletti, E., Gallo, R. C., Markham, P. D., and Franchini, G. (1997). Long-term survival of SIVmac251-infected macaques previously immunized with NYVAC-SIV vaccines. J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 15, S78 S85. Benson, J., Chougnet, C., Robert-Guroff, M., Montefiori, D., Markham, P. D., Shearer, G. M., Gallo, R. C., Cranage, M. P., Paoletti, E., Limbach, K., Venzon, D., Tartaglia, J., and Franchini, G. (1998). Recombinant vaccine-induced protection against the highly pathogenic SIV mac251 : Dependence on route of challenge exposure. J. Virol. 72, Benveniste, R. E., Hill, R. W., Eron, L. J., Csaikl, U. M., Knott, W. B., Henderson, L. E., Sowder, R. C., Nagashima, K., and Gonda, M. A. (1990). Characterization of clones of HIV-1 infected HuT 78 cells defective in gag gene processing and of SIV clones producing large amounts of envelope glycoprotein. J. Med. Primatol. 19, Buge, S. L., Richardson, E., Alipanah, S., Markham, P. D., Cheng, S., Kalyan, N., Miller, C. J., Lubeck, M., Udem, S., Eldridge, J., and Robert-Guroff, M. (1997). An adenovirus-simian immunodeficiency virus env vaccine elicits humoral, cellular, and mucosal immune responses in rhesus macaques and decreases viral burden following vaginal challenge. J. Virol. 71, Cheynier, R., Langlade, D. P., Marescot, M. R., Blanche, S., Blondin, G., Wain, H. S., Griscelli, C., Vilmer, E., and Plata, F. (1992). Cytotoxic T lymphocyte responses in the peripheral blood of children born to

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