Mechanisms of Protection Induced by Attenuated Simian Immunodeficiency Virus

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1 Virology 296, (2002) doi: /viro Mechanisms of Protection Induced by Attenuated Simian Immunodeficiency Virus V. No Evidence for Lymphocyte-Regulated Cytokine Responses upon Rechallenge Richard J. Stebbings,*,1 Neil M. Almond, E. Jim Stott, Neil Berry, Alison M. Wade-Evans, Robin Hull, Jenny Lines,* Peter Silvera, Rebecca Sangster, Terry Corcoran, Jane Rose, and K. Barry Walker* *Division of Immunobiology and Division of Retrovirology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, United Kingdom Received January 8, 2002; accepted January 14, 2002 To determine whether attenuated simian immunodeficiency virus (SIV) vaccines confer protection against superinfection via secondary cellular immune responses, we searched for markers of immune activation following rechallenge. Productive infection with either attenuated SIVmacC8 or wild-type SIVmacJ5 resulted in a transient increase in T-lymphocyte CD25 and Mafa-DR expression. A pronounced increase in the frequency of FAS CD8 lymphocytes was observed following SIVmacJ5 infection only. A transient increase in lymphocytes positive for intracellular IFN- and IL-4 was observed following primary infection with either virus. In contrast, lymphocytes positive for intracellular IL-2 were reduced. Following SIVmacJ5 challenge of SIVmacC8-infected vaccinees, no evidence of detectable superinfection was obtained. Rechallenge of vaccinees did not alter the frequency of activated peripheral T-lymphocytes, perturb cytokine profiles, or generate an anamnestic antibody response. These data do not support the hypothesis that protection conferred by live attenuated SIV is mediated by the induction of vigorous T-cell responses upon rechallenge Elsevier Science (USA) INTRODUCTION Simian immunodeficiency virus (SIV) is closely related to human immunodeficiency virus (HIV) in its genomic organization (Chakrabarti et al., 1987; Franchini et al., 1987; Hirsch et al., 1987), its pathogenicity (Daniel et al., 1985; Letvin, 1990), and its use of CD4 and chemokine receptors to infect target cells (Deng et al., 1997; Hill et al., 1997). Infection of macaques with wild-type SIV is characterized by persistent infection, gradual erosion of immune function, loss of CD4 cells in susceptible animals, and fatal opportunistic infections similar to those observed with HIV infection of humans (Kestler et al., 1990; Letvin et al., 1985; Sharma et al., 1992). Consequently, this animal model is widely used to study the clinical progression to immunodeficiency (Lu et al., 1998; Zink et al., 1998), mechanisms of pathogenesis (Hodge et al., 1998), and the efficacy and safety of vaccines (Almond and Heeney, 1998) and drugs against HIV (Tsai et al., 1995; Wyand, 1992). At present, the most potent protection observed using the SIV model has been achieved through immunization with live attenuated virus (Almond et al., 1995; Daniel et al., 1992; Desrosiers, 1990; Lohman et al., 1994; Stahl- Hennig et al., 1996; Wyand et al., 1996). Inoculation of juvenile macaques with live attenuated SIV generates a 1 To whom correspondence and reprint requests should be addressed. Fax: 44 (0) rstebbings@nibsc.ac.uk. low-level infection that renders recipients resistant to subsequent superinfection with pathogenic wild-type SIV (Connor et al., 1998; Cranage et al., 1997; Nilsson et al., 1998; Rud et al., 1994a). Unfortunately, attenuated SIV demonstrates a propensity for repairing attenuating deletions in vivo (Carl et al., 1999; Sawai et al., 2000; Whatmore et al., 1995) and infection of infant or susceptible adult macaques with attenuated SIV can cause disease (Baba et al., 1995, 1999; Ruprecht et al., 1996). Consequently, it is unlikely that an attenuated HIV vaccine will ever be utilized in clinical trials. Nevertheless, if the mechanism of vaccine protection were understood, then it may be possible to reproduce it by less hazardous means. Yet, it still remains to be established if protection is mediated by immunological responses or through viral interference (Rud et al., 1994b). To date, passive transfer of immune sera and lymphocyte depletion experiments have yet to establish a role for serological or cell-mediated responses alone in vaccine-induced protection (Almond et al., 1997; Stebbings et al., 1998). In order to establish a role for any combination of immune responses in vaccine-mediated control of superinfection, we established assays to measure lymphocyte activation and cytokine production. These assays were used to characterize primary cellular immune responses following infection with attenuated and wild-type SIV and then to look for secondary cellular immune responses following rechallenge of vaccinees with wild-type SIV. Any /02 $ Elsevier Science (USA) All rights reserved. 338

2 CYTOKINE RESPONSES OF SIV VACCINEES TO RECHALLENGE 339 TABLE 1 Virus Detection on Indicated Days Postchallenge with SIVmacJ5 or SIVmacC8 a Days post SIV challenge (0) 233(15) 274(56) 378(160) Animal PCR Virus PCR vrna PCR Virus PCR Virus PCR Virus PCR Virus PCR Virus PCR Virus R R R R R nd nd (C8) (C8) (C8) R nd nd (C8) (C8) R nd nd (C8) (C8) R nd nd (C8) (C8) R265 (J5) (J5) (J5) R266 (J5) (J5) (J5) R267 (J5) (J5) (J5) R268 (J5) (J5) (J5) Challenge SIV J5/C8 SIV J5 a Shown is virus detected on indicated days post SIV challenge. On day 0 macaques R33 R36 were challenged with SIVmacJ5 and macaques R37 R40 with SIVmacC8. On day 218 macaques R37 R40 and R265 R268 were challenged with SIVmacJ5. Under PCR a plus sign indicates that SIV gag proviral DNA was detected. Under PCR a minus sign indicates that no SIV proviral DNA was detected. Under PCR, (C8) or (J5) indicates that the nef proviral DNA detected was from SIVmacC8 or SIVmacJ5, respectively. Figures under vrna for day 15 refer to mean log SIV RNA copies per milliliter of plasma. All samples for days 58 and 113 were below the cut-off for the assay: 2.3 log SIV RNA copies per milliliter of plasma. Under Virus a plus sign indicates that cell-associated virus was recovered from PBMC and a minus sign indicates that no virus was recovered from PBMC. nd indicates that results were not determined. recall of previously observed cellular immune responses could then be attributed to superinfection resistance. RESULTS Infection with wild-type SIVmacJ5 and attenuated SIVmacC8 Following challenge of group A (R33 through R36) with SIVmacJ5 and group B (R37 through R40) with SIVmacC8 at day 0, all eight animals were positive by DNA PCR for SIV gag by day 58 (Table 1). At this time virus could be reisolated from the peripheral blood mononuclear cells (PBMC) of three SIVmacJ5 animals (R33, R34, and R36) but from only one SIVmacC8 animal (R40). All eight animals were positive for SIV vrna at day 15 but all samples were below the detection limit of the assay on days 58 and 113 (Table 1). Mean log SIV RNA copies per milliliter of plasma from SIVmacJ5 challenged animals were significantly higher than those for SIVmacC8 challenged animals ( and , respectively), by unpaired t test (P 0.001, Table 1). Following rechallenge of SIVmacC8 animals with SIVmacJ5 on day 218, no full-length SIVmacJ5 nef proviral DNA could be detected in PBMC. In contrast, all new naive controls R265 through 268 (group C) challenged contemporaneously were positive by virus isolation, SIV gag polymerase chain reaction (PCR), and J5 nef PCR. At 15 days post SIVmacJ5 challenge there was no significant difference between the mean DNA loads for group A and group C ( copies/10 6 PBMC and copies/10 6 PBMC, respectively), by unpaired t test (P 0.666). All eight macaques challenged with SIVmacJ5 or SIVmacC8 seroconverted to SIVgp140 by day 15 and SIVp27 by day 58 (Figs. 1A and 1B; individual data not shown). Neutralizing antibody activity against SIVmacJ5 was first detected in macaques R35, R37, and R38 at day 58. Neutralizing antibody activity was detected in all eight animals by day 113 (Fig. 1C; individual data not shown). At day 113 anti-sivgp140 end-point titers of SIVmacJ5 animals were significantly higher than those of SIVmacC8 animals, by unpaired t test (P 0.042). Mean anti-sivp27 titers for the sum of days 58, 113, 218, and 274 were significantly higher for SIVmacJ5 than SIVmacC8 animals, by unpaired t test (P 0.026), but not at single time points. By day 218 mean neutralizing antibody titers were significantly higher in SIVmacJ5 than SIVmacC8 animals, by unpaired t test (P 0.026). No anamnestic anti-sivgp140, anti-sivp27, or neutralizing antibody response was detected in SIVmacC8 animals following rechallenge with SIVmacJ5. Contemporaneously challenged naive controls, R265 through R268, seroconverted to SIVgp140 and SIVp27 by day 274. Apart from macaque R268, the proportion of CD4 PBMC in groups A, B, and C remained within reference range following SIV challenge (Figs. 2A 2C). The proportion of CD8 PBMC in groups A, B, and C exhibited a transient increase above reference range at 15 days after

3 340 STEBBINGS ET AL. FIG. 1. Antibody responses to challenge of group A (R33 R36) with wild-type SIVmacJ5 on day 0, group B (R37 R40) with attenuated SIVmacC8 on day 0, and groups A and C (R265 R268) with wild-type SIVmacJ5 on day 218. (A) Log anti-siv gp140 end-point titers. (B) Log anti-siv p27 end-point titers. (C) Log anti-siv neutralizing antibody titers. challenge (Figs. 3A 3C). Following rechallenge of SIVmacC8 animals with SIVmacJ5 on day 218, no transient increase in CD8 PBMC was observed. T-lymphocyte activation following SIV challenge Following primary challenge with either SIVmacJ5 or SIVmacC8 a transient increase in the proportion of CD3 CD25 lymphocytes was observed (Figs. 4A 4C). At 4 weeks postchallenge the proportion of CD3 CD25 lymphocytes was significantly above reference range in groups A, B, and C, by paired t test (P 0.023, P 0.004, and P 0.012, respectively). No significant increase in the proportion of group B CD3 CD25 lymphocytes was detected following rechallenge with SIVmacJ5. Similar data were obtained with the proportion of CD3 PBMC expressing Mafa-DR, which cross-reacted with anti-hla-dr antibodies (data not shown). Following challenge of group A with SIVmacJ5 the proportion of CD8 FAS lymphocytes (Fig. 5A) increased significantly above reference range at day 29, by paired t test (P 0.016), remaining elevated until day 169. For group B challenged with SIVmacC8 the proportion of CD8 FAS lymphocytes (Fig. 5B) was significantly above reference range only at day 85, by paired t test (P 0.003). Following SIVmacJ5 challenge of group C the proportion of CD8 FAS lymphocytes (Fig. 5C) increased significantly above reference range at day 233,

4 CYTOKINE RESPONSES OF SIV VACCINEES TO RECHALLENGE 341 FIG. 2. Changes in the proportion of CD4 PBMC following SIV challenge. (A) Macaques R33 R36 (group A) challenged with wild-type SIVmacJ5 on day 0. (B) Macaques R37 R40 (group B) challenged with attenuated SIVmacC8 on day 0 and rechallenged with wild-type SIVmacJ5C on day 218. (C) Macaques R265 R268 (group C) challenged with wild-type SIVmacJ5 on day 218. Upper and lower dashed lines delineate normal reference range based on 2 standard deviations mean proportion of CD4 PBMC derived from two prebleeds of each animal used in this study. by paired t test (P 0.004). No significant increase in CD8 FAS lymphocytes was detected in SIVmacC8 animals following rechallenge with SIVmacJ5. No significant change in the proportion of FAS CD4 cells in groups A, B, or C was observed following SIV challenge (data not shown). Cytokine expression by lymphocytes following SIV challenge Mitogen stimulation either had no effect or decreased the numbers of IL-4 CD3 lymphocytes before and after SIV challenge (data not shown). Only the data for un-

5 342 STEBBINGS ET AL. FIG. 3. Changes in the proportion of CD8 PBMC following SIV challenge. (A) Macaques R33 R36 (group A) challenged with wild-type SIVmacJ5 on day 0. (B) Macaques R37 R40 (group B) challenged with attenuated SIVmacC8 on day 0 and rechallenged with wild-type SIVmacJ5 on day 218. (C) Macaques R265 R268 (group C) challenged with wild-type SIVmacJ5 on day 218. Upper and lower dashed lines delineate normal reference range based on 2 standard deviations mean proportion of CD8 PBMC derived from two prebleeds of each animal used in this study. stimulated IL-4 secretion are reported here. Following challenge of group A with SIVmacJ5 and group B with SIVmacC8, the proportion of IL-4 CD3 lymphocytes (Figs. 6A and 6B) increased significantly above reference range at days 85 and 113, respectively, by paired t test (P and P 0.049, respectively). At day 113 the

6 CYTOKINE RESPONSES OF SIV VACCINEES TO RECHALLENGE 343 FIG. 4. Changes in the proportion of CD25 CD3 PBMC following SIV challenge. (A) Macaques R33 R36 (group A) challenged with wild-type SIVmacJ5 on day 0. (B) Macaques R37 R40 (group B) challenged with attenuated SIVmacC8 on day 0 and rechallenged with wild-type SIVmacJ5 on day 218. (C) Macaques R265 R268 (group C) challenged with wild-type SIVmacJ5 on day 218. Upper and lower dashed lines delineate normal reference range based on 2 standard deviations mean proportion of CD25 CD3 PBMC derived from two prebleeds of each animal used in this study. mean proportion of group A IL-4 CD3 lymphocytes was significantly greater than that of group B, by unpaired t test (P 0.006). No significant increase in the proportion of group B IL-4 CD3 lymphocytes was detected following rechallenge with SIVmacJ5. In contrast, contemporaneous challenge of group C with SIVmacJ5 resulted in a significant increase in the proportion of IL-4 CD3 lymphocytes (Fig. 6C) to above reference range at day 233, by paired t test (P 0.049). For detection of IFN- or IL-2 cells only the data following mitogen stimulation are shown here. No IFN- or IL-2 cells were detected in the absence of mitogen

7 344 STEBBINGS ET AL. FIG. 5. Changes in the proportion of FAS CD8 PBMC following SIV challenge. (A) Macaques R33 R36 (group A) challenged with wild-type SIVmacJ5 on day 0. (B) Macaques R37 R40 (group B) challenged with attenuated SIVmacC8 on day 0 and rechallenged with wild-type SIVmacJ5 on day 218. (C) Macaques R265 R268 (group C) challenged with wild-type SIVmacJ5 on day 218. Upper and lower dashed lines delineate normal reference range based on 2 standard deviations mean proportion of FAS CD8 PBMC derived from two prebleeds of each animal used in this study. stimulation before or after SIV challenge (data not shown). Following challenge of group A with SIVmacJ5 and group B with SIVmacC8 the proportion of IFN- lymphocytes (Figs. 7A and 7B) peaked significantly above reference range at day 113 by paired t test (P and P 0.046, respectively). At day 113 the mean proportion of IFN- lymphocytes from group A was nonsignificantly greater than that from group B. No significant increase in the proportion of group B IFN- lymphocytes was detected following rechallenge with

8 CYTOKINE RESPONSES OF SIV VACCINEES TO RECHALLENGE 345 FIG. 6. Changes in the proportion of CD3 IL-4 PBMC following SIV challenge. (A) Macaques R33 R36 (group A) challenged with wild-type SIVmacJ5 on day 0. (B) Macaques R37 R40 (group B) challenged with attenuated SIVmacC8 on day 0 and rechallenged with wild-type SIVmacJ5 on day 218. (C) Macaques R265 R268 (group C) challenged with wild-type SIVmacJ5 on day 218. Upper and lower dashed lines delineate normal reference range based on 2 standard deviations mean proportion of IL-4 CD3 PBMC derived from two prebleeds of each animal used in this study. SIVmacJ5. In contrast, contemporaneous challenge of group C with SIVmacJ5 resulted in a significant increase in the proportion of IFN- lymphocytes (Fig. 7C) above reference range at day 246, by paired t test (P 0.047). Following challenge of group A with SIVmacJ5 and group B with SIVmacC8, the proportion of IL-2 lymphocytes (Figs. 8A and 8B) decreased significantly below reference range at day 29, by paired t test (P and P 0.002, respectively). Thereafter, levels of IL-2 lymphocytes in SIVmacJ5 animals remained significantly below reference range except on days 85 and 246. In contrast, levels of IL-2 lymphocytes in SIVmacC8 ani-

9 346 STEBBINGS ET AL. FIG. 7. Changes in the proportion of IFN- PBMC following SIV challenge. (A) Macaques R33 R36 (group A) challenged with wild-type SIVmacJ5 on day 0. (B) Macaques R37 R40 (group B) challenged with attenuated SIVmacC8 on day 0 and rechallenged with wild-type SIVmacJ5 on day 218. (C) Macaques R265 R268 (group C) challenged with wild-type SIVmacJ5 on day 218. Upper and lower dashed lines delineate normal reference range based on 2 standard deviations mean proportion of IFN PBMC derived from two prebleeds of each animal used in this study. mals remained within reference range except on day 218. No significant decrease in the proportion of group B IL-2 lymphocytes was detected following rechallenge with SIVmacJ5. Contemporaneous challenge of group C with SIVmacJ5 resulted in a significant decrease in the proportion of IL-2 lymphocytes (Fig. 8C) to below reference range at day 308, by paired t test (P 0.009). DISCUSSION This work builds upon our previous observations that protection conferred by live attenuated SIV cannot be transferred with immune serum (Almond et al., 1997) and cannot be abrogated by depletion of cytotoxic T-cells (Stebbings et al., 1998). The aim of this investigation was

10 CYTOKINE RESPONSES OF SIV VACCINEES TO RECHALLENGE 347 FIG. 8. Changes in the proportion of IL-2 PBMC following SIV challenge. (A) Macaques R33 R36 (group A) challenged with wild-type SIVmacJ5 on day 0. (B) Macaques R37 R40 (group B) challenged with attenuated SIVmacC8 on day 0 and rechallenged with wild-type SIVmacJ5 on day 218. (C) Macaques R265 R268 (group C) challenged with wild-type SIVmacJ5 on day 218. Upper and lower dashed lines delineate normal reference range based on 2 standard deviations mean proportion of IL-2 PBMC derived from two prebleeds of each animal used in this study. to address the role of secondary cellular immune responses in the potent protection against superinfection conferred by vaccination with live attenuated SIV. This study clearly demonstrates that primary infection with both wild-type and attenuated SIV strongly stimulates host cellular immune responses. Although attenuated SIV viral loads were 100-fold lower than wild-type SIV, both challenges elicited a significant increase in the numbers of activated T-cells, CD8 lymphocytes, and cells positive for IFN- or IL-4 by intracellular cytokine staining. In addition, both attenuated and wild-type SIV challenges resulted in profound loss of cells positive for

11 348 STEBBINGS ET AL. IL-2 by intracellular cytokine staining in the absence of significant CD4 lymphocyte loss. The importance of active cellular immune responses in controlling initial levels of SIV and simian human immunodeficiency virus replication in macaques has been demonstrated in lymphocyte depletion experiments (Jin et al., 1999; Matano et al., 1998; Schmitz et al., 1999). However, similar experiments have failed to identify a role for cellular immunity in mediating the protection conferred by live attenuated SIV vaccines (Stebbings et al., 1998). Recall of cell-mediated immunity is characterized by massive proliferation of antigen-specific T-cells (Flynn et al., 1999) and expression of activation markers such as CD25 and HLA-DR by responding T-cells (Mardiney et al., 1996; Neal and Splitter, 1998). If recall of a vigorous cellular immune response to infection is the mechanism of protection conferred by live attenuated SIV vaccines, then rechallenge with wild-type SIV should evoke recall of such responses. In this study macaques vaccinated with attenuated SIVmacC8 showed no evidence of superinfection with wild-type SIVmacJ5 by virology, serology, or molecular diagnostic assays. Yet, there was no evidence for reactivation of T-cells, increased numbers of CD8 PBMC, perturbed IL-4, IL-2, or IFN- cytokine profiles following challenge. The observed alterations in cytokine and T-cell activation in the SIV model are similar to those reported by others (Estaquier et al., 2000; Smit-McBride et al., 1998; Spring et al., 1997; Xiong et al., 2001). The greater increase in the proportion of IL-4 and IFN- cells observed with wild-type SIVmacJ5 versus the nef deleted SIVmacC8 challenge mirrors anti-siv antibody responses and probably reflects a stronger immune response to the greater size and duration of the SIVmacJ5 primary viremia. However, as a significant peak in the proportion of IFN- cells was observed only at a single time point, this result should be interpreted with caution. Nevertheless, an increased capacity for IFN- secretion in the SIV model is in agreement with the findings of others (Smit-McBride et al., 1998). The reduced numbers of CD3 IL-4 cells observed following mitogen stimulation may be linked to the activity of IFN- to limit IL-4 secretion (Marcinkiewicz and Chain, 1993). Most studies of IL-4 secretion in patients following HIV infection have reported increased levels (Barcellini et al., 1994; Clerici et al., 1996; Klein et al., 1997; Meroni et al., 1996) although others have reported no change (Fan et al., 1993; Meyaard et al., 1996). The absence of a significant increase in CD3 IL-4 cells following SIVmacC8 challenge in macaques R39 and R40 may indicate a less vigorous humoral response in these animals or that the peak increase was missed. It was noted that, within the SIVmacC8 group, detection of neutralizing antibodies was delayed in both R39 and R40. No clear consensus exists over changes in IFN- secretory capacity following HIV-1 infection with contradictory reports describing both increased (Caruso et al., 1995; Fan et al., 1993; Westby et al., 1998) and decreased levels (Hagiwara et al., 1996; Clerici et al., 1996; Meroni et al., 1996; Meyaard et al., 1996) in patients. These disparate observations may be related to the stage of disease progression, AIDS being associated with general failure to secrete cytokines, or technical issues such as the type of mitogen used to stimulate PBMC. Stimulation with phytohemagglutinin (PHA) was used when decreases in the potential IFN- secretion were reported (Clerici et al., 1996; Meroni et al., 1996), whereas phorbol 12-myristate 13- acetate (PMA) ionomycin was employed in studies where increases in IFN- secretion were observed (Caruso et al., 1995; Westby et al., 1998). Production of IFN- by antigen-stimulated T-cells requires the presence of IL-2 during both the priming and the expression phases. In contrast, production of IL-4 requires IL-2 only during the priming phase (Billiau, 1996). Observations of defects in IFN- but not IL-4 production during HIV-1 (Barcellini et al., 1994; Klein et al., 1997) infection may therefore be related to the paucity of IL-2 production. In the SIV model a defect in the ability of antigen-specific CD8 lymphocytes to produce IFN- is restored by IL-2 treatment (Xiong et al., 2001). Infection with wild-type SIVmacJ5 and attenuated SIVmacC8 resulted in equivalent depletion of IL-2 lymphocytes 4 weeks postchallenge. However, between weeks 7 and 16, levels of IL-2 lymphocytes returned to reference range in all SIVmacC8-infected animals but only in two SIVmacJ5 animals. On an individual animal basis infection with wild-type SIVmacJ5 produced a sustained suppression of IL-2 cells in two of four macaques. Loss of IL-2 lymphocytes in both SIVmacC8 and SIVmacJ5 animals was again observed between weeks 18 and 31. It is unlikely that an increase in IL-2 lymphocytes at week 33 in SIVmacC8 animals is related to the SIVmacJ5 rechallenge as a similar increase was observed at this time in the two SIVmacJ5-infected animals which were not rechallenged. Observed decreases in IL-2 secretion following antigen or mitogen stimulation of PBMC from HIV-infected patients (Clerici et al., 1996; Meroni et al., 1996; Meyaard et al., 1996; Westby et al., 1998) or SIVinfected macaques (Koopman et al., 2001; Spring et al., 1997) are widely accepted. Although mitogen stimulation is not antigen specific it does reflect changes in memory cell subsets, with polarized cytokine expression, that would otherwise not be observed when looking at gross lymphocyte populations (Koopman et al., 2001). These changes can be either immune-mediated expansion of memory subsets or immunopathogenic depletion. After week 2, the peak of primary viremia, less than 10 cells for SIVmacC8 and 100 cells for SIVmacJ5 challenged animals per PBMC are infected. By 8 weeks postinfection loads are normally 20- to 100-fold lower. Therefore, it is unlikely that perturbations of cytokine and activation marker expression observed here are due to a

12 CYTOKINE RESPONSES OF SIV VACCINEES TO RECHALLENGE 349 direct effect of SIV genes on host cells as less than 1% of lymphocytes are infected. As all of these animals were cohoused together in a contained facility, it is doubtful that nonspecific factors such as other viral illnesses could have affected only one group of animals and perturbed their cytokine profiles. Analysis of lymph node cells may have revealed changes in activation marker and cytokine expression earlier. However, as lymphocytes from lymph nodes traffic through the peripheral blood, changes in these lymphoid compartments are generally reflected in PBMC. Increased FAS expression has previously been reported for the CD8 fraction of PBMC in HIV-1-infected patients (Gehri et al., 1996; Sloand et al., 1997) and SIV-infected macaques (Iida et al., 1999; Xu et al., 1997). Increased FAS expression is a marker of T-cell activation (Tucek-Szabo et al., 1996) regulated by increased IFN- secretion (Novelli et al., 1996). Higher numbers of FAS CD8 lymphocytes following SIVmacJ5 than SIVmacC8 challenge may reflect greater IFN- secretion and expansion of memory cell subsets. Alternatively, increased FAS expression by CD8 lymphocytes of SIVmacJ5 animals may be due to chronic activation in the absence of IL-2, inducing a state of anergy and susceptibility to apoptosis. Although the second SIVmacJ5 challenge used was half the TCID 50 value of the first this was still sufficient to infect all four controls and induce CD4 cell loss in R268. No significant differences between the two SIVmacJ5 challenges in terms of peak viremia, serology, and expression of T-cell activation markers were observed. However, it is interesting that differences in the kinetics of cytokine responses of these two noncontemporaneous SIVmacJ5 challenges were noted. Since C8166 cells used here for virus reisolation poorly express the major SIV coreceptor CCR5, virus reisolation results may not accurately reflect cell-associated viral loads. While there are some who believe that it is very difficult to elicit protection against immunodeficiency virus by vaccination, no evidence for superinfection was found here. A molecular assay has been developed that differentiates the closely related vaccine and rechallenge viruses. This is capable of detecting even a single copy of SIVmacC8 or SIVmacJ5 (Rose et al., 1995) yet did not detect superinfection. More impressive perhaps is the failure to detect an anamnestic antibody response. Anamnestic antibody responses have previously been reported following superinfection of attenuated SIV vaccinees (Norley et al., 1996). However, despite the marked protection observed here we could not rule out the presence of minor species of viral sequences in the presence of other SIV-related sequences. The lack of a detectable systemic secondary immune response may mean that the vaccine protection obtained was mediated through local immune responses. It has been reported that virus-specific CD8 lymphocytes induced by attenuated SIV immunization express the mucosa-homing receptor 4 7 and home to the intestinal mucosa (Cromwell et al., 2000). However, 4 7 is expressed by a subset of virus-specific CD8 cells, initially exposed to antigen at gastrointestinal sites, that are readily detectable trafficking in the periphery. Consequently, activation of even local immune responses would be reflected in the periphery during subsequent trafficking. Failure to detect recall of active cellular immune responses to rechallenge may also be explained by lack of productive infection. It is possible that resistance to rechallenge does not elicit a strong systemic immune response because the level of immunity against the established attenuated virus is sufficient. Nevertheless, strong depletion of cellular immune responses in SIVmacC8-infected macaques by Campath-1H, a treatment developed to prevent transplant rejection, is unable to abrogate protection against a SIVmacJ5 challenge (Stebbings et al., 1998). Furthermore, cytotoxic T cell responses in animals immunized with attenuated SIV do not correlate with protection against pathogenic SIV infection (Nixon et al., 2000). Superinfection resistance could be explained if it were mediated by neutralizing antibody responses that prevented initial infection. Yet, it has been demonstrated that passive transfer of immune sera from attenuated SIV-infected macaques confers no protective capacity upon naive recipients (Almond et al., 1997). Even when superinfection does occur and is efficiently controlled, this occurs in the absence of detectable secondary cellular immune responses (Khatissian et al., 2001). Another possibility is that nonimmune mechanisms of protection such as retroviral interference (Corbin and Sitbon, 1993; Corbin et al., 1994) or depletion of a critical target cell population (Mattapallil et al., 1998) may have a role to play. It has been reported that receptor interference is unlikely to be a mechanism of protection in attenuated SIV-immunized animals since they are protected against superinfection with an attenuated SIV pseudotype carrying the amphotropic envelope of murine leukemia virus (Gundlach et al., 1998) that uses Pit2 and not CD4 as a receptor. However, the authors base their conclusions on the lack of virus reisolation, anamnestic immune response, and lack of seroconversion to SIV-MLV envelope. Yet, a previous wild-type SIVmac239 superinfection of the same animals did not increase antibody titers or result in increased virus reisolation after challenge. Finally, the authors concede in their conclusions that in agreement with their own PCR data low-level SIV-MLV replication did occur in the vaccinees. In conclusion, our findings indicate that potent vaccine protection conferred by live attenuated virus can occur in the absence of an active systemic secondary cellular

13 350 STEBBINGS ET AL. immune response. Further carefully developed experiments are required to address more directly the ability of live attenuated virus vaccines to elicit protection in the absence of a systemic secondary cellular immune response. Animals and virus MATERIALS AND METHODS Twelve juvenile male or female purpose-bred cynomolgus macaques (Macaca fascicularis) were used in this study. Macaques were screened by virus isolation and serology and shown to be free from infection by SIV or simian D-type retroviruses prior to entry into this study. Macaques were housed and maintained in accordance with Home Office Guidelines for the care and maintenance of nonhuman primates. All animals were sedated with ketamine before challenge or venipuncture and were examined clinically while under anesthesia. The SIVmac32H (pc8) virus clone differs from the SIVmac32H (pj5) clone by a 12-bp deletion and two nonsynonymous nucleotide changes resulting in conservative amino acid changes in the nef open reading frame (Rud et al., 1994a). Virus stocks termed SIVmacJ5 and SIVmacC8 were prepared as follows. Cultured PBMC from cynomolgus macaque L156 stimulated with phytohemagglutinin (ICN Pharmaceuticals, Ltd., Basing-stoke, Hampshire UK) and recombinant human IL-2 (NIBSC, Potters Bar, Hertfordshire, UK) were infected with either the 9/90 pool of SIVmacC8 or the 9/90 pool of SIVmacJ5 (Cranage et al., 1997). Cell-free supernatants harvested on day 14 were used to infect spleen cells from L156 stimulated with phytohemagglutinin and recombinant human IL-2. Cultures were harvested on day 11 and the cell-free supernatants were aliquoted and stored (Silvera, 1997). On day 0 macaques R33 through R36 were inoculated intravenously with 750 TCID 50 of SIVmacJ5 in parallel with macaques R37 through R40, which received 750 TCID 50 of SIVmacC8. On day 218 R37 through R40 and four naive controls, R265 through R268, were challenged with 375 TCID 50 of SIVmacJ5. Virus detection Amplification of SIV gag sequences in genomic DNA from macaque PBMC was performed using a PCR-based assay, employing two rounds of amplification with nested primer pairs as previously described (Rose et al., 1995). In order to differentiate between the two SIV clones, a diagnostic amplification of a region of the nef gene encompassing the 12-bp deletion of SIVmacC8 was employed. The PCR product derived from SIVmacC8 and SIVmacJ5 was distinguished by differential digestion with restriction enzyme RsaI and the product was resolved by gel electrophoresis, as described previously (Rose et al., 1995). Proviral DNA loads per 10 6 PBMC were determined by an end-point dilution method, as previously described (Almond et al., 1999; Slade et al., 1995). Plasma viral RNA (vrna) levels were determined 15 days postinfection from EDTA-treated plasma using a modified RT-PCR assay, based on a previously described method (Berry et al., 1998), with Titan reagents (Roche Diagnostics, Lewes, UK). The amount of specific PCR product was quantified by hybridization with an alkaline phosphatase-coupled probe using chemiluminescence in a microtiter-based assay. The sensitivity of the assay is 2.3 log SIV RNA copies per milliliter. Virus reisolation from separated PBMC was determined by coculture with C8166 cells, as previously described (Stebbings et al., 1998). Either when syncytia were observed or after 28 days the presence of replicating SIV was confirmed in an antigen capture assay, as previously described (Rose et al., 1995). Antibody detection Titers of binding antibodies to SIV envelope gp140 (RepliGen Corp., Needham, MA) or lacz-p27 (Almond et al., 1990) were determined from heat-inactivated (56 C for 1 h) plasma samples by ELISA (Silvera et al., 1994; Stott et al., 1990). Neutralizing antibody end-point titers were taken as the dilution of serum in the serum virus mixture inhibiting p27 antigen production by at least 75%, expressed as log 10 of the reciprocal of the end-point dilution (Kent et al., 1994). Flow cytometry Anti-human monoclonal antibodies previously identified to cross-react with macaque PBMC and cytokines were used in this study. For surface staining 200 l of whole blood was incubated at 4 C for 30 min with the following antibody pairs in parallel: CD4-FITC (OKT4, Ortho-Clinical Diagnostics, Amersham, Buckinghamshire, UK) and CD8-PE (Leu-2a, Becton Dickinson, Oxford, UK); monkey CD3-FITC (FN18, Serotec, Oxford, UK) and CD25-PE (M-A251, Pharmingen, San Diego, CA); monkey CD3-FITC and HLA-DR-PE (Tu36, Pharmingen); CD4-FITC and CD95-PE (DX2, Pharmingen); and CD8- FITC and CD95-PE. Red blood cells were removed using FACS lysing solution (Becton Dickinson). For intracellular cytokine staining, PBMC isolated on a Percoll gradient (Amersham Pharmacia Biotech, Uppsala, Sweden) were resuspended at /ml in RPMI 1640 medium supplemented with 10% FCS, 50 IU/ml penicillin streptomycin (Gibco BRL, Paisley, Scotland) and 2 M/ml monensin (Sigma Aldrich Co. Ltd., Dorset, UK). Cultures were incubated overnight at 37 C in 24- well plates with and without mitogen, 10 ng/ml of PMA 1 g/ml ionomycin (Sigma Aldrich Co. Ltd.), washed once in PBS, fixed at room temperature in Permeafix (Ortho Diagnostic Systems Inc.) for 30 min; washed once

14 CYTOKINE RESPONSES OF SIV VACCINEES TO RECHALLENGE 351 with PBS containing 4% FCS 0.05% sodium azide, and incubated for 90 min at 4 C with the following antibody pairs in parallel: anti-il-2 RPE conjugate (MQ1-17H12, Pharmingen) plus anti-ifn- FITC conjugate (4S.B3, Pharmingen) and anti-il-4 RPE conjugate (8D4-8, Pharmingen) plus anti-monkey CD3 FITC conjugate. Following staining, PBMC were washed twice with PBS containing 4% FCS 0.05% sodium azide and resuspended in 2% paraformaldehyde. Additional staining with appropriate isotype controls (Serotec) was used to confirm specificity of labeling. Acquisition of 10,000 events was carried out using a FACScan cytometer and analyzed using Lysis II software. ACKNOWLEDGMENTS We thank Dr. H. Holmes, the U.K. MRC AIDS Reagent Programme, and the European Union Programme EVA for essential materials. We thank Mr. A. Heath for assistance with statistical analysis of data. 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