CD8+ Lymphocytes Do Not Mediate Protection against
http://www.100md.com
病菌学杂志 2005年第19期
Division of Immunology Division of Retrovirology Division of Virology, National Institute for Biological Standards and Control, Potters Bar, United Kingdom
Sir William Dunn School of Pathology Oxford Therapeutic Antibody Centre, Oxford University, Oxford, United Kingdom
ABSTRACT
In order to test the hypothesis that CD8+ cytotoxic T lymphocytes mediate protection against acute superinfection, we depleted >99% of CD8+ lymphocytes in live attenuated simian immunodeficiency virus macC8 (SIVmacC8) vaccinees from the onset of vaccination, maintained that depletion for 20 days, and then challenged with pathogenic, wild-type SIVmacJ5. Vaccinees received 5 mg per kg of humanized anti-CD8 monoclonal antibody (MAb) 1 h before inoculation, followed by the same dose again on days 3, 7, 10, 13, and 17. On day 13, peripheral CD8+ T lymphocytes were >99% depleted in three out of four anti-CD8 MAb-treated vaccinees. At this time attenuated SIVmacC8 viral RNA loads in anti-CD8 MAb-treated vaccinees were significantly higher than control vaccinees treated contemporaneously with nonspecific human immunoglobulin. Lymphoid tissue CD8+ T lymphocyte depletion was >99% in three out of four anti-CD8 MAb-treated vaccinees on the day of wild-type SIVmacJ5 challenge. All four control vaccinees and three out of four anti-CD8 MAb-treated vaccinees were protected against detectable superinfection with wild-type SIVmacJ5. Although superinfection with wild-type SIVmacJ5 was detected at postmortem in a single anti-CD8 MAb-treated vaccinee, this did not correlate with the degree of preceding CD8+ T lymphocyte depletion. Clearance of attenuated SIVmacC8 viremia coincided with recovery of normal CD8+ T lymphocyte counts between days 48 and 76. These results support the view that cytotoxic T lymphocytes are important for host-mediated control of SIV primary viremia but do not indicate a central role in protection against acute superinfection conferred by inoculation with live attenuated SIV.
INTRODUCTION
Vaccination of macaques with live attenuated simian immunodeficiency virus (SIV) provides a valuable model to study the correlate(s) of immunity that an effective human immunodeficiency virus (HIV)/AIDS vaccine will need to emulate. Live attenuated SIV vaccines can confer effective protection against detectable superinfection with pathogenic, wild-type SIV (3, 13, 14, 29, 58, 59) and SIV/HIV-1 chimeric virus (7, 18, 46). Yet there are limits to the breadth of this protection, and live attenuated SIV vaccines have failed to protect against certain heterologous challenge viruses or failed to protect against a challenge performed several years postinoculation (22, 31, 58). Furthermore, the demonstrated potential for reversion to pathogenicity in live attenuated SIV precludes clinical evaluation of a live attenuated HIV vaccine (6, 38, 39, 57). Nevertheless, an understanding of the mechanism(s) of protection against superinfection conferred by inoculation with live attenuated SIV would further the development of a safe and effective HIV vaccine. An unambiguous correlate of protection against superinfection has so far evaded identification. Cytotoxic T lymphocytes (CTL), virus neutralizing antibodies, innate immunity, and retroviral interference have all been reported as potential mechanisms of protection against superinfection conferred by inoculation with live attenuated SIV (2, 24, 32, 37, 50, 54, 59). Here we have evaluated the role of CD8+ lymphocytes and, thereby, CD8+ CTL in mediating protection against acute superinfection conferred by inoculation with live attenuated SIV.
Inoculation with live attenuated SIV generates significant SIV-specific CD8+ CTL responses (16, 25, 32, 56). The appearance of SIV-specific CD8+ CTL responses during primary SIV infection coincides with clearance of plasma viremia and suppression of viral replication (41). Furthermore, the importance of CD8+ lymphocytes for control of pathogenic or attenuated SIV infection has been demonstrated in several studies that report a dramatic rise in plasma viremia following anti-CD8 monoclonal antibody (MAb) treatment to deplete CD8+ CTL, with control of virus replication being temporally associated with recovery of CD8+ lymphocytes (23, 28, 30, 41). In addition, an inverse correlation has been reported between the precursor frequency of SIV-specific CD8+ CTL responses elicited by certain vaccine approaches and virus load following challenge (20, 55). Although several groups have reported a correlation between SIV-specific CD8+ CTL responses in live attenuated SIV vaccinees and protection against superinfection with wild-type SIV (24-26, 56), other groups have failed to corroborate such observations and dispute a role for SIV-specific CD8+ CTL in mediating protection (1, 32, 44, 50, 52).
In a previous study we attempted to address the role of SIV-specific CD8+ CTL responses in mediating protection against superinfection by administering a pair of rat anti-human CD8 MAbs to live attenuated SIVmacC8 vaccinees 24 h prior to challenge with wild-type SIVmacJ5 (52). All CD8+ lymphocyte-depleted vaccinees resisted superinfection with wild-type SIVmacJ5, which suggests that SIV-specific CD8+ CTL responses are not central to protection against superinfection observed at 35 weeks postinoculation. However, it remained a possibility that the degree of depletion obtained in lymphoid tissues may have been insufficient and that the temporal removal of CD8+ lymphocytes would not have prevented the establishment of effective CD4+ T cell memory, which would then rapidly drive the reappearance of SIV-specific CD8+ CTL (15).
We have now established a model of superinfection resistance to wild-type SIVmacJ5 challenge that is reproducibly achieved within 21 days of inoculation with live attenuated SIVmacC8 (51). Moreover, protection against superinfection at 21 days postinoculation with live attenuated SIVmacC8 occurs before the development of detectable SIV-specific serological responses but coincides with the development of detectable SIV-specific CD8+ CTL responses (51). The development of a therapeutic humanized anti-human CD8 MAb which can be administered repeatedly has extended the period of effective CD8+ lymphocyte depletion (>99%) in blood and lymphoid tissues for periods greater than 3 weeks. We have combined these two advances to address more effectively the role of CD8+ CTL responses in mediating protection against acute superinfection conferred by inoculation with live attenuated SIVmacC8. To ensure >99% depletion of peripheral and lymphoid tissue CD8+ lymphocytes on the day of challenge and to prevent the expansion and dissemination of SIV-specific CD8+ CTL during vaccination, we employed profound depletion of CD8+ lymphocytes from the day of inoculation through the day of challenge and beyond.
MATERIALS AND METHODS
Animals and viruses. A total of 16 na?ve, D-type retrovirus-free, juvenile purpose-bred cynomolgus macaques (Macaca fascicularis) were used in this study. Macaques were housed and maintained in accordance with United Kingdom Home Office guidelines for the care and maintenance of nonhuman primates. The attenuated SIVmac32H (SIVmacC8) virus clone differs from the wild-type SIVmac32H (SIVmacJ5) clone by a 12-bp deletion and two nonsynonymous nucleotide changes, resulting in conservative amino acid changes in the nef open reading frame (36). For all live attenuated SIV inoculations, macaques were intravenously administered 5,000 50% tissue culture infective doses of the 9/90 pool of SIVmacC8 on day 0 (11, 36). For all wild-type SIV challenges, macaques were intravenously administered 20 50% median infective doses of the 3/92 stock of SIVmacJ5 on day 20 (12, 36).
Humanized anti-CD8 MAb treatment. In two preliminary experiments to determine efficacy, the ability of the humanized anti-CD8 MAb TRX2 (Oxford Therapeutic Antibody Centre, Oxford University, Oxford, United Kingdom) to deplete peripheral and lymphoid tissue CD8+ T lymphocytes in na?ve macaques was evaluated. In an initial study, macaques Z98 and Z99 were intravenously administered 3 mg per kg of body weight of anti-CD8 MAb on days 0, 1, and 3; 6 mg per kg on days 6, 10, and 13; and 9 mg per kg on days 17 and 20. In a follow-up study, macaques Z274 and Z275 were intravenously administered 3 mg per kg of humanized anti-CD8 MAb on days 0, 1, and 4.
To address the role of SIV-specific CD8+ CTL responses in mediating protection against acute superinfection, we have combined a previously described 21-day vaccination model (51) with sustained depletion of CD8+ lymphocytes. On day 0, macaques A55, A56, A66, and A125 (group A) were administered intravenously 5 mg per kg of body weight of humanized anti-CD8 MAb. Macaques A59, A60, A61, and A62 (group B) were administered intravenously 5 mg per kg of pooled nonspecific human immunoglobulin contemporaneously (Grifols, Barcelona, Spain). One hour after treatment, all eight macaques (groups A and B) were each inoculated with 5,000 50% tissue culture infective doses of live attenuated SIVmacC8 intravenously. Anti-CD8 MAb treatment of group A and nonspecific human immunoglobulin treatment of group B, both at 5 mg per kg, were repeated on days 3, 7, 10, 13, and 17. Group C consisted of four na?ve controls, macaques A58, A63, A64, and A65. On day 20, groups A, B, and C were all challenged with 20 50% median infective doses of wild-type SIVmacJ5 intravenously.
Quantification of CD8+ T lymphocyte counts. All anti-human MAbs used were previously identified by flow cytometry to cross-react with macaque peripheral blood mononuclear cells (PBMC). For measurement of CD8+ T lymphocyte depletion, whole blood was immunostained with mouse anti-monkey CD3 fluorescein isothiocyanate conjugate (clone FN-18; Serotec, Oxford, United Kingdom), mouse anti-human CD4 phycoerythrin conjugate (clone L120; BD Biosciences, San Jose, CA), and mouse anti-human CD8 allophycocyanin conjugate (clone 3B5; Caltag Laboratories, Burlingame, CA), as previously described (50). Positive staining for the T lymphocyte marker CD3 was used to distinguish CD8+ T lymphocytes from CD8+ natural killer (NK) cells. All blood samples and lymph node biopsies were taken before administration of each dose of anti-CD8 MAb or nonspecific human immunoglobulin. The addition of humanized anti-CD8 MAb to macaque whole blood did not block subsequent immunostaining with anti-human CD8 allophycocyanin conjugate. Absolute counts of CD8+ T lymphocytes were made using the BD TruCOUNT system (San Jose, CA). The detection limit for CD8+ T lymphocytes by flow cytometry was 1 cell per μl of blood or resuspended lymph node biopsy. Lymph node biopsies were trimmed of fat and weighed prior to disaggregation using the Medimachine system (DakoCytomation Ltd., Ely, United Kingdom) and then immunostained as per whole blood. A FACSCalibur cytometer was used for acquisition, and data were analyzed using CellQuest Pro software (BD Biosciences, San Jose, CA). Greater than 99% depletion of CD8+ T lymphocytes compared to pretreatment levels was deemed effective depletion.
Virus detection, quantification, and serology. The presence of SIV in PBMC or tissue samples was determined using a SIV gag DNA PCR assay, as previously described (35). The two molecular clones, SIVmacC8 and SIVmacJ5, were differentiated using a nef-specific nested PCR protocol which encompasses the attenuating 12-bp deletion in SIVmacC8 nef. RsaI restriction endonuclease digestion of the PCR product was used to discriminate between the presence of full-length SIVmacJ5 nef containing this site and SIVmacC8 nef, which does not (35). Intermediate revertants lacking the RsaI restriction site are not distinguished from SIVmacC8 by this protocol. Within a background of SIVmacC8, the lower detection limit of this assay is 10 copies of SIVmacJ5. The overall kinetics of plasma SIV RNA loads were determined as previously described (8). The sensitivity of the assay is 200 SIV RNA copies per ml of plasma. Virus isolation and titration from PBMC were determined by coculture with C8166 cells; the presence of replicating virus was confirmed by syncytia identification or by antigen capture at 28 days, as previously described (52). Titers of binding antibodies to SIV envelope gp130 (EVA670; CFAR/NIBSC, Potters Bar, United Kingdom) or recombinant SIV p27 (EVA643; CFAR/NIBSC, Potters Bar, United Kingdom) were determined in heat-inactivated (56°C for 1 h) plasma samples by enzyme-linked immunosorbent assay, as previously described (4, 47, 53). Neutralizing antibody end-point titers were determined as the dilution of serum in the serum and virus mixture inhibiting p27 antigen production by at least 75%, expressed as the log10 of the reciprocal of the end-point dilution (27).
RESULTS
Anti-CD8 MAb treatment depletes CD8+ T lymphocytes. We conducted two preliminary studies in na?ve macaques to determine a suitable protocol for anti-CD8 MAb treatment that could maintain effective (>99%) depletion of CD8+ T lymphocytes over a 3-week period. Intravenous administration of anti-CD8 MAb to na?ve macaques Z98, Z99, Z274, and Z275 resulted in >99.7% depletion of peripheral CD8+ T lymphocytes within 24 h of treatment (Fig. 1A). Three anti-CD8 MAb treatments, given to macaques Z274 and Z275 on days 0, 1, and 4, were sufficient to maintain >99.9% depletion of peripheral CD8+ T lymphocytes for at least 2 weeks (Fig. 1A). Eight anti-CD8 MAb treatments, given to macaques Z98 and Z99 on days 0, 1, 3, 6, 10, 13, 16, and 20, were sufficient to maintain >99.7% depletion of peripheral CD8+ T lymphocytes for at least 7 weeks (Fig. 1A). When compared to the rebound in peripheral CD8+ T lymphocyte counts observed in macaques that received three anti-CD8 MAb treatments, the recovery of peripheral CD8+ T lymphocyte counts was very slow in macaques that received eight anti-CD8 MAb treatments (Fig. 1A). Where all peripheral CD8+ T lymphocyte counts were below the detection limits of the flow cytometer (1 cell per μl blood), lymphoid tissue CD8+ T lymphocyte counts provided a better means of ranking the degree of depletion between individuals. On day 20, following three anti-CD8 MAb treatments, depletion of lymphoid tissue CD8+ T lymphocytes in macaques Z274 and Z275 was substantial at 92.9% and 97.4%, respectively. However, administration of seven anti-CD8 MAb treatments to macaques Z98 and Z99 by day 20 achieved >99.9% and 99.8% depletion of lymphoid tissue CD8+ T lymphocytes, respectively (Fig. 1B). Recovery of lymphoid tissue CD8+ T lymphocyte counts following cessation of anti-CD8 MAb treatment was less pronounced than that observed in the periphery. In particular, macaques Z98 and Z99 were still greater than 99.7% depleted of lymphoid tissue CD8+ T lymphocytes on day 147, in contrast to partial recovery of their peripheral CD8+ T lymphocyte counts (Fig. 1A and B). This profound depletion of CD8+ lymphocyte depletion was mirrored in mesenteric lymph nodes and spleen at termination (data not shown). CD4+ T lymphocyte and CD4– CD8– T lymphocyte counts remained stable for the duration of these studies (data not shown).
Since administration of anti-CD8 MAb antibody on seven occasions had proven sufficient to attain the desired level and duration of CD8+ T lymphocyte depletion and because of concerns over the safety of long-term CD8+ T lymphocyte depletion in SIV-infected macaques, the total number of treatments was reduced from eight to six, given over 3 weeks. Anti-CD8 MAb treatment of group A vaccinees (animals A55, A56, A66, and A125) resulted in profound depletion of peripheral CD8+ T lymphocytes (Fig. 2A). No depletion of peripheral or lymphoid tissue CD8+ T lymphocytes was evident following contemporaneous treatment of group B vaccinees (A59, A60, A61, and A62) with nonspecific human immunoglobulin (Fig. 2B and D, respectively). On day 13, following four anti-CD8 MAb treatments, peripheral CD8+ T lymphocytes were >99.8% depleted in macaques A55, A56, and A66 but were only reduced by 87.7% in macaque A125 (Fig. 2A). Peripheral CD8+ T lymphocyte counts in group A were significantly lower than group B on day 13 (P = 0.0209, Mann-Whitney U test). On day 20, after six anti-CD8 MAb treatments, CD8+ T lymphocytes recovered from lymphoid tissue were >99.4% depleted in macaques A55, A56, and A66 but were only reduced by 95.8% in macaque A125 (Fig. 2C). On day 20, the day of wild-type SIVmacJ5 challenge, lymphoid tissue CD8+ T lymphocyte counts in group A were significantly lower than group B (P = 0.0209, Mann-Whitney U test). Within group A, CD8+ T lymphocytes recovered from lymph node biopsies were most profoundly depleted in macaques A55 and A56, at >99.9% depletion (Fig. 2C). Following cessation of treatment with anti-CD8 MAb and 14 days after challenge with wild-type SIVmacJ5 (day 34), peripheral CD8+ T lymphocyte numbers had increased, but in three out of four vaccinees, CD8+ T lymphocyte depletion remained >92.5% (Fig. 2A). In particular, CD8+ T lymphocyte depletion in macaque A56 was >99.8% on day 34 (Fig. 2A). At both days 34 and 48, CD8+ T lymphocyte counts in group A vaccinees remained significantly lower than group B vaccinees, but CD8+ T lymphocyte counts increased thereafter (P = 0.0209 at both times, Mann-Whitney U test) between days 48 and 76 (Fig. 2A). By day 169, recovery of lymphoid tissue CD8+ T lymphocytes was evident in group A vaccinees, and counts did not differ significantly from group B vaccinees (Fig. 2C and D) (P = 0.2482, Mann-Whitney U test).
Inoculation with attenuated SIVmacC8 promotes resurgence of CD8+ T lymphocytes. In comparison to na?ve macaques, the recovery of CD8+ T lymphocytes in attenuated SIVmacC8 vaccinees was much more rapid and absolute (Fig. 1A and 2A). In attenuated SIVmacC8 vaccinees, this recovery was partly driven by the expansion of CD4+ T lymphocytes expressing the -chain of CD8 (data not shown). Expansion of CD4+ CD8+ T lymphocytes was not observed in anti-CD8 MAb-treated na?ve macaques during recovery. CD8+ NK cells were also profoundly depleted by anti-CD8 MAb treatment (data not shown). No significant difference in CD4+ T lymphocyte counts between vaccinees that received anti-CD8 MAb or nonspecific human immunoglobulin treatment was observed during this study (data not shown).
CD8+ lymphocyte depletion is associated with increased SIVmacC8 virus load. The kinetics of the primary viremia of attenuated SIVmacC8 in cynomolgus macaques has been reported previously (8). Depletion of CD8+ lymphocytes throughout the primary viremia of attenuated SIVmacC8 (Fig. 3A) provoked a significant increase in peak plasma viral RNA (vRNA) loads measured at day 13, compared to vaccine controls (Fig. 3B) treated with nonspecific human immunoglobulin (Fig. 3D) (for group A compared to group B, P = 0.0339, Mann-Whitney U test). This observation was corroborated by significantly higher cell-associated virus loads in group A vaccinees at day 13, compared with group B vaccinees (data not shown; P = 0.0339, Mann-Whitney U test). Plasma vRNA loads among group A vaccinees remained significantly higher than group B vaccinees until at least day 34 but not thereafter (Fig. 3D) (P = 0.0433, Mann-Whitney U test). Nonetheless, plasma virus loads in group B fell below the lower detection limit of our assay by day 34 (Fig. 3B), whereas in group A this suppression of primary viremia was delayed until at least day 48 (Fig. 3A). Following CD8+ lymphocyte depletion of group A vaccinees, the increase in peak plasma viremia of attenuated SIVmacC8 at day 13 (Fig. 3A) is more comparable to the peak in wild-type SIVmacJ5 viremia of group C on day 34 of the study (Fig. 3C) (day 14 after challenge with SIVmacJ5) than to that of group B vaccinees on day 13 (Fig. 3B).
CD8+ lymphocyte depletion increases anti-SIV envelope antibody titers. Antibodies against SIV gp130 were detected in all vaccinees (groups A and B) by day 34 (Fig. 4A). Anti-SIV gp130 antibody titers in group A vaccinees were significantly higher than in group B vaccinees at day 34 and remained elevated at all time points thereafter (Fig. 4A) (P = 0.0209 at all time-points, Mann-Whitney U test). Following wild-type SIVmacJ5 challenge on day 20, anti-SIV gp130 antibodies were detected in all group C na?ve controls on day 48 (Fig. 4A). Anti-SIV gp130 antibody titers in group A vaccinees were significantly higher than group C challenge controls at days 34, 48, and 76 (Fig. 4A) (P = 0.0209, P = 0.0433, and P = 0.0209, respectively; Mann-Whitney U test). Thereafter, anti-SIV gp130 titers in group C challenge controls increased to similar levels as group A (Fig. 4A). Anti-SIV neutralizing antibodies were first detected in vaccinees at day 76 (Table 1). There was no significant difference in neutralizing antibody titers between vaccinee groups A and B on days 76 or 104 (P = 0.3865 and P = 0.2482, respectively; Mann-Whitney U test). Antibodies against SIV p27 were detected in all group A vaccinees on day 34 but only in two out of four group B vaccinees (Fig. 4B). Antibodies against SIV p27 were not detected in all group B vaccinees until day 76 (Fig. 4B). At day 34 anti-SIV p27 titers were significantly higher in group A vaccinees compared to group B vaccinees but not thereafter (Fig. 4B) (P = 0.0209, Mann-Whitney U test).
CD8+ lymphocyte responses are not required for protection against acute superinfection. Successful inoculation of all group A and B macaques with live attenuated SIVmacC8 on day 0 was confirmed by SIV gag DNA PCR assays on day 13 (data not shown). Following intravenous challenge of all groups with wild-type SIVmacJ5, all group C macaques were confirmed to be infected with SIVmacJ5 by nef-specific DNA PCR (Table 2). All group B vaccinees were negative for detectable wild-type SIVmacJ5 nef at all time points following challenge and were therefore deemed to have resisted superinfection (Table 2). Within group A, three out of four vaccinees were negative for detectable wild-type SIVmacJ5 nef at all time points and were deemed to have resisted superinfection (Table 2). Although wild-type SIVmacJ5 nef could not initially be detected in group A, a low level of wild-type SIVmacJ5 nef signal (approximately 10% of the total signal) was detected at postmortem (day 169) in the blood and lymphoid tissues of vaccinee A66, indicating superinfection and vaccine failure in that individual (Table 2).
DISCUSSION
This study is part of a series that has characterized the early protection against superinfection conferred by inoculation with live nef-attenuated SIVmacC8 (3, 5, 48, 50-52). Previously, we showed that protection against acute superinfection with pathogenic, wild-type SIVmacJ5 can be obtained within 21 days of inoculation with live attenuated SIVmacC8, at a time when neutralizing antibodies were not present and limited CD8+ T lymphocyte responses were detected (51). The present study demonstrates that profound depletion of CD8+ lymphocytes for up to 34 days, encompassing the period macaques are vaccinated with live attenuated SIVmacC8, does not abrogate protection against challenge with pathogenic, wild-type SIVmacJ5. Our observations are consistent with those of others that CD8+ lymphocyte responses are important for control of SIV replication since we observed a dramatic increase in plasma viremia associated with the depletion of CD8+ lymphocytes in macaques inoculated with attenuated SIVmacC8 (23, 28, 30, 41). However, the ultimate conclusion of this study is that CD8+ lymphocyte responses alone are not central to the protection against acute superinfection conferred 20 days after vaccination with attenuated SIVmacC8. This conclusion is consistent with the findings of other investigators who were unable to identify a correlation between SIV-specific CD8+ CTL responses elicited by inoculation with live attenuated SIV and protection against superinfection (31, 32, 44).
Results for the depletion of CD8+ T lymphocytes in na?ve macaques given three anti-CD8 MAb treatments are comparable to those obtained by Schmitz and colleagues (43). Although peripheral CD8+ T lymphocytes are effectively (>99%) depleted within 1 h of anti-CD8 MAb treatment, the majority of CD8+ lymphocytes are found within lymphoid tissues and have been found to be resistant to effective depletion (23, 28, 30, 52). By increasing the number of anti-CD8 MAb treatments to eight, given over a 3-week period, we were able to effectively deplete lymphoid tissue CD8+ T lymphocytes in na?ve macaques and extend greatly the duration of depletion. After effective depletion of lymphoid tissue CD8+ T lymphocytes, the recovery observed in vaccinees was much faster and more complete than that observed in na?ve macaques depleted to a similar degree. Thus, inoculation with live attenuated SIVmacC8 appears to be a strong stimulus for the production of new CD8+ T lymphocytes as a mechanism to control viremia. Anti-CD8 MAb treatment also depleted CD8+ NK cells in attenuated SIVmacC8 vaccinees. NK cells are important effectors of the innate immune response against viral infections and also secrete cytokines and chemokines that play a role in recruitment and activation of the adaptive immune response (10, 34). It is possible that the depletion of these CD8+ NK cells contributed to the significant increase in plasma viremia observed in anti-CD8 MAb-treated individuals infected with attenuated SIVmacC8. Moreover, the depletion of CD8+ NK cells by anti-CD8 MAb treatment would also exclude a role for these cells in mediating early protection against acute superinfection.
The significant increase in anti-SIVgp130 titers elicited by depletion of CD8+ lymphocytes from attenuated SIVmacC8 vaccinees may be related to the accompanying dramatic increase in virus load. Challenge controls infected with wild-type SIVmacJ5 had comparable virus loads and generated comparable anti-SIVgp130 titers. It is possible that in group A the more marked anti-SIVgp130 serological response has resulted from a homeostatic compensation for the lack of CD8+ lymphocyte responses or that strong Th1 CD8+ cellular immune responses normally limit Th2 humoral responses. An inverse correlation between neutralizing antibody titers and plasma virus levels in SIV-infected and B-cell depleted macaques has been reported (42). Similarly, postchallenge immunotherapy with purified immunoglobulin from SIV-infected long-term nonprogressors is reported to reduce virus burden (21). It could be argued that the more marked anti-SIVgp130 serological response contributed to protection against superinfection and impaired the ability to determine the mechanism of protection. However, the more marked anti-SIVgp130 serological response did not prevent the significant increase in plasma viremia associated with depletion of CD8+ lymphocytes or prevent superinfection of vaccinee A66. Moreover, anti-SIV-specific neutralizing antibodies were not detected until day 76 in either vaccine group and do not correlate with early protection against acute superinfection (51). That detection of high anti-SIV p27 titers occurs later than is observed with anti-SIVgp130 antibodies and that significant CD8+ T lymphocyte recovery in anti-CD8 MAb-treated vaccinees occurs before this time may explain why significantly higher titers of anti-p27 antibodies are only observed at day 34. Alternatively, the extended viremia may have absorbed anti-SIV p27 antibody production.
Previously, where superinfection has been observed following challenge of attenuated SIVmacC8 vaccinees, one of two patterns has been apparent: either superinfection is clearly detected in the blood at 2 weeks postchallenge or not until postmortem, when the presence of virus can be investigated in a range of lymphoid tissues (51). Here, the latter pattern is applicable to superinfection of vaccinee A66. Thus, even in the presence of marked suppression of CD8+ T lymphocytes, wild-type SIVmacJ5 has not been able to establish itself readily, implying at least partial protection. It is interesting that the degree of preceding CD8+ T lymphocyte depletion in A66 did not correlate with its susceptibility to superinfection. Depletion of CD8+ lymphocytes in attenuated SIVmacC8 vaccinees A55 and A56 was more profound than in A66, but these macaques were not superinfected. It is possible that protection against superinfection in macaque A66 may not have been complete at the time of challenge, day 20. Previously, the earliest time we have observed reproducible protection against detectable superinfection has been 21 days after inoculation with live attenuated SIVmacC8 and only partial protection at earlier times (51). Although vaccinee A125 resisted superinfection, depletion of CD8+ lymphocytes was less marked than in subject A66. Nevertheless, the degree of CD8+ T lymphocyte depletion in A125 was substantial enough to produce a dramatic increase in plasma viremia and clearly sufficient to compromise anti-SIV CD8+ T lymphocyte mediated responses.
The model of protection against acute superinfection at 20 days after vaccination described here differs from models used by other groups, in which more highly attenuated vaccine virus is employed and full protection is not achieved until 10 or 20 weeks after inoculation (9, 59). Therefore, there may be significant differences in the mechanism of protection between these models (40). However, when superinfection occurs before the onset of full protection, significant control of primary viremia is still observed (9, 49, 51). Nevertheless, the results reported here do not support the hypothesis of a central role for CD8+ CTL responses in mediating protection against acute superinfection conferred by inoculation with live attenuated SIV. If cell-mediated immune responses are the mechanism of protection in live attenuated SIV vaccinees, then a characteristic expansion of memory CD8+ CTL and immune activation should be detected following virus challenge (17, 19). Yet evidence for immune activation or expansion of SIV-specific CTL responses following rechallenge has not been reported (44, 50). Even where protection against superinfection is incomplete, vaccine-mediated control of virus replication occurs before detection or expansion of CD8+ SIV-specific CTL responses (1, 51). However, lack of expansion of SIV-specific T cells preceding initial immune control does not necessarily rule out a role for SIV-specific CTL responses postchallenge. It should be noted that several previous studies have shown the correlation of CTL responses with protection (2, 26, 33).
Following inoculation with attenuated SIVmacC8, primary viremia peaks at day 10 and rapidly declines thereafter to undetectable levels (8). Nevertheless, the pathogenic SIVmacJ5 challenge at day 20 occurred in concert with detectable levels of vaccine virus in CD8+ lymphocyte-depleted macaques. This is in contrast to other attenuated SIV vaccine scenarios where challenge occurs when attenuated SIV replication is low or undetectable. Therefore, it is possible that the resistance to acute superinfection is not due to immune protection but, rather, is a result of a combination of target cell depletion and retroviral interference.
Since we have reported that vaccine protection conferred by attenuated SIVmacC8 cannot be transferred with immune serum alone (5) and demonstrate here that CD8+ T cell responses do not correlate with resistance to acute superinfection, it follows that other candidates for mediating protection need to be explored. Innate immunity and nonimmune mechanisms such as retroviral interference or target cell depletion need to be investigated to unravel this potential novel mechanism of antiviral immunity (37, 45, 51, 54). Armed with this information, we can then develop novel prophylactic vaccine approaches to reproduce this potential vaccine protection in a safe and effective HIV/AIDS vaccine.
ACKNOWLEDGMENTS
We thank Harvey Holmes, the Centralised Facility for AIDS Reagents, and the Oxford Therapeutic Antibody Centre for essential materials.
This work was funded in part by grants from the United Kingdom Medical Research Council (G9025730 and G9419998).
Deceased.
REFERENCES
Abdel-Motal, U. M., J. Gillis, K. Manson, M. Wyand, D. Montefiori, K. Stefano-Cole, R. C. Montelaro, J. D. Altman, and R. P. Johnson. 2005. Kinetics of expansion of SIV Gag-specific CD8+ T lymphocytes following challenge of vaccinated macaques. Virology 333:226-238.
Abel, K., L. Compton, T. Rourke, D. Montefiori, D. Lu, K. Rothaeusler, L. Fritts, K. Bost, and C. J. Miller. 2003. Simian-human immunodeficiency virus SHIV89.6-induced protection against intravaginal challenge with pathogenic SIVmac239 is independent of the route of immunization and is associated with a combination of cytotoxic T-lymphocyte and alpha interferon responses. J. Virol. 77:3099-3118.
Almond, N., K. Kent, M. Cranage, E. Rud, B. Clarke, and E. J. Stott. 1995. Protection by attenuated simian immunodeficiency virus in macaques against challenge with virus-infected cells. Lancet 345:1342-1344.
Almond, N., M. Page, K. Mills, A. Jenkins, C. Ling, R. Thorpe, P. Kitchin, and M. Williams. 1990. The production and purification of PCR-derived recombinant simian immunodeficiency virus p27 gag protein; its use in detecting serological and T-cell responses in macaques. J. Virol. Methods 28:305-319.
Almond, N., J. Rose, R. Sangster, P. Silvera, R. Stebbings, B. Walker, and E. J. Stott. 1997. Mechanisms of protection induced by attenuated simian immunodeficiency virus. I. Protection cannot be transferred with immune serum. J. Gen. Virol. 78:1919-1922.
Baba, T. W., Y. S. Jeong, D. Pennick, R. Bronson, M. F. Greene, and R. M. Ruprecht. 1995. Pathogenicity of live, attenuated SIV after mucosal infection of neonatal macaques. Science 267:1820-1825.
Bogers, W. M., H. Niphuis, P. ten Haaft, J. D. Laman, W. Koornstra, and J. L. Heeney. 1995. Protection from HIV-1 envelope-bearing chimeric simian immunodeficiency virus (SHIV) in rhesus macaques infected with attenuated SIV: consequences of challenge. AIDS 9:F13-F8.
Clarke, S., N. Almond, and N. Berry. 2003. Simian immunodeficiency virus Nef gene regulates the production of 2-LTR circles in vivo. Virology 306:100-108.
Connor, R. I., D. C. Montefiori, J. M. Binley, J. P. Moore, S. Bonhoeffer, A. Gettie, E. A. Fenamore, K. E. Sheridan, D. D. Ho, P. J. Dailey, and P. A. Marx. 1998. Temporal analyses of virus replication, immune responses, and efficacy in rhesus macaques immunized with a live, attenuated simian immunodeficiency virus vaccine. J. Virol. 72:7501-7509.
Cooper, M. A., T. A. Fehniger, and M. A. Caligiuri. 2001. The biology of human natural killer-cell subsets. Trends Immunol. 22:633-640.
Cranage, M. P., S. A. Sharpe, A. M. Whatmore, N. Polyanskaya, S. Norley, N. Cook, S. Leech, M. J. Dennis, and G. A. Hall. 1998. In vivo resistance to simian immunodeficiency virus superinfection depends on attenuated virus dose. J Gen. Virol. 79:1935-1944.
Cranage, M. P., A. M. Whatmore, S. A. Sharpe, N. Cook, N. Polyanskaya, S. Leech, J. D. Smith, E. W. Rud, M. J. Dennis, and G. A. Hall. 1997. Macaques infected with live attenuated SIVmac are protected against superinfection via the rectal mucosa. Virology 229:143-154.
Daniel, M. D., F. Kirchhoff, S. C. Czajak, P. K. Sehgal, and R. C. Desrosiers. 1992. Protective effects of a live attenuated SIV vaccine with a deletion in the nef gene. Science 258:1938-1941.
Desrosiers, R. C. 1990. The simian immunodeficiency viruses. Annu. Rev. Immunol. 8:557-578.
Dittmer, U., and K. J. Hasenkrug. 1999. Alternative interpretation of lymphocyte depletion studies using monoclonal antibodies in animals previously vaccinated with attenuated retroviral vaccines. AIDS Res. Hum. Retrovir. 15:785.
Dittmer, U., T. Nisslein, W. Bodemer, H. Petry, U. Sauermann, C. Stahl-Hennig, and G. Hunsmann. 1995. Cellular immune response of rhesus monkeys infected with a partially attenuated nef deletion mutant of the simian immunodeficiency virus. Virology 212:392-397.
Doherty, P. C., and J. P. Christensen. 2000. Accessing complexity: the dynamics of virus-specific T cell responses. Annu. Rev. Immunol. 18:561-592.
Dunn, C. S., B. Hurtrel, C. Beyer, L. Gloeckler, T. N. Ledger, C. Moog, M. P. Kieny, M. Mehtali, D. Schmitt, J. P. Gut, A. Kirn, and A. M. Aubertin. 1997. Protection of SIVmac-infected macaque monkeys against superinfection by a simian immunodeficiency virus expressing envelope glycoproteins of HIV type 1. AIDS Res. Hum. Retrovir. 13:913-922.
Flynn, K. J., J. M. Riberdy, J. P. Christensen, J. D. Altman, and P. C. Doherty. 1999. In vivo proliferation of naive and memory influenza-specific CD8(+) T cells. Proc. Natl. Acad. Sci. USA 96:8597-8602.
Gallimore, A., M. Cranage, N. Cook, N. Almond, J. Bootman, E. Rud, P. Silvera, M. Dennis, T. Corcoran, J. Stott, and et al. 1995. Early suppression of SIV replication by CD8+ nef-specific cytotoxic T cells in vaccinated macaques. Nat. Med. 1:1167-1173.
Haigwood, N. L., A. Watson, W. F. Sutton, J. McClure, A. Lewis, J. Ranchalis, B. Travis, G. Voss, N. L. Letvin, S. L. Hu, V. M. Hirsch, and P. R. Johnson. 1996. Passive immune globulin therapy in the SIV/macaque model: early intervention can alter disease profile. Immunol. Lett. 51:107-114.
Hofmann-Lehmann, R., J. Vlasak, A. L. Williams, A. L. Chenine, H. M. McClure, D. C. Anderson, S. O'Neil, and R. M. Ruprecht. 2003. Live attenuated, nef-deleted SIV is pathogenic in most adult macaques after prolonged observation. AIDS 17:157-166.
Jin, X., D. E. Bauer, S. E. Tuttleton, S. Lewin, A. Gettie, J. Blanchard, C. E. Irwin, J. T. Safrit, J. Mittler, L. Weinberger, L. G. Kostrikis, L. Zhang, A. S. Perelson, and D. D. Ho. 1999. Dramatic rise in plasma viremia after CD8(+) T cell depletion in simian immunodeficiency virus-infected macaques. J. Exp. Med. 189:991-998.
Johnson, R. P. 2002. Mechanisms of protection against simian immunodeficiency virus infection. Vaccine 20:1985-1987.
Johnson, R. P., R. L. Glickman, J. Q. Yang, A. Kaur, J. T. Dion, M. J. Mulligan, and R. C. Desrosiers. 1997. Induction of vigorous cytotoxic T-lymphocyte responses by live attenuated simian immunodeficiency virus. J. Virol. 71:7711-7718.
Johnson, R. P., J. D. Lifson, S. C. Czajak, K. S. Cole, K. H. Manson, R. Glickman, J. Yang, D. C. Montefiori, R. Montelaro, M. S. Wyand, and R. C. Desrosiers. 1999. Highly attenuated vaccine strains of simian immunodeficiency virus protect against vaginal challenge: inverse relationship of degree of protection with level of attenuation. J. Virol. 73:4952-4961.
Kent, K. A., P. Kitchin, K. H. Mills, M. Page, F. Taffs, T. Corcoran, P. Silvera, B. Flanagan, C. Powell, J. Rose, et al. 1994. Passive immunization of cynomolgus macaques with immune sera or a pool of neutralizing monoclonal antibodies failed to protect against challenge with SIVmac251. AIDS Res. Hum. Retrovir. 10:189-194.
Lifson, J. D., J. L. Rossio, M. Piatak, Jr., T. Parks, L. Li, R. Kiser, V. Coalter, B. Fisher, B. M. Flynn, S. Czajak, V. M. Hirsch, K. A. Reimann, J. E. Schmitz, J. Ghrayeb, N. Bischofberger, M. A. Nowak, R. C. Desrosiers, and D. Wodarz. 2001. Role of CD8+ lymphocytes in control of simian immunodeficiency virus infection and resistance to rechallenge after transient early antiretroviral treatment. J. Virol. 75:10187-10199.
Lohman, B. L., M. B. McChesney, C. J. Miller, E. McGowan, S. M. Joye, K. K. Van Rompay, E. Reay, L. Antipa, N. C. Pedersen, and M. L. Marthas. 1994. A partially attenuated simian immunodeficiency virus induces host immunity that correlates with resistance to pathogenic virus challenge. J. Virol. 68:7021-7029.
Metzner, K. J., X. Jin, F. V. Lee, A. Gettie, D. E. Bauer, M. Di Mascio, A. S. Perelson, P. A. Marx, D. D. Ho, L. G. Kostrikis, and R. I. Connor. 2000. Effects of in vivo CD8(+) T cell depletion on virus replication in rhesus macaques immunized with a live, attenuated simian immunodeficiency virus vaccine. J. Exp. Med. 191:1921-1931.
Nilsson, C., B. Makitalo, R. Thorstensson, S. Norley, D. Binninger-Schinzel, M. Cranage, E. Rud, G. Biberfeld, and P. Putkonen. 1998. Live attenuated simian immunodeficiency virus (SIV)mac in macaques can induce protection against mucosal infection with SIVsm. AIDS 12:2261-2270.
Nixon, D. F., S. M. Donahoe, W. M. Kakimoto, R. V. Samuel, K. J. Metzner, A. Gettie, T. Hanke, P. A. Marx, and R. I. Connor. 2000. Simian immunodeficiency virus-specific cytotoxic T lymphocytes and protection against challenge in rhesus macaques immunized with a live attenuated simian immunodeficiency virus vaccine. Virology 266:203-210.
Putkonen, P., B. Makitalo, D. Bottiger, G. Biberfeld, and R. Thorstensson. 1997. Protection of human immunodeficiency virus type 2-exposed seronegative macaques from mucosal simian immunodeficiency virus transmission. J. Virol. 71:4981-4984.
Robertson, M. J., and J. Ritz. 1990. Biology and clinical relevance of human natural killer cells. Blood 76:2421-2438.
Rose, J., P. Silvera, B. Flanagan, P. Kitchin, and N. Almond. 1995. The development of PCR based assays for the detection and differentiation of simian immunodeficiency virus in vivo. J. Virol. Methods 51:229-239.
Rud, E. W., M. Cranage, J. Yon, J. Quirk, L. Ogilvie, N. Cook, S. Webster, M. Dennis, and B. E. Clarke. 1994. Molecular and biological characterization of simian immunodeficiency virus macaque strain 32H proviral clones containing nef size variants. J. Gen. Virol. 75:529-543.
Rud, E. W., L. Ogilvie, B. E. Clarke, N. Almond, K. Kent, L. Chan, M. Page, P. Kitchin, E. J. Stott, N. Cook, S. Sharpe, T. Ashwork, M. Dennis, G. Hall, and M. Cranage. 1994. A naturally attenuated SIVmac32H vaccine or viral interference? p. 217-223. In F. Brown, R. M. Chanock, H. S. Ginsberg, and E. Norrby (ed.), Vaccines 94: modern approaches to new vaccines including prevention of AIDS. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Ruprecht, R. M. 1999. Live attenuated AIDS viruses as vaccines: promise or peril? Immunol. Rev. 170:135-149.
Sawai, E. T., M. S. Hamza, M. Ye, K. E. Shaw, and P. A. Luciw. 2000. Pathogenic conversion of live attenuated simian immunodeficiency virus vaccines is associated with expression of truncated Nef. J. Virol. 74:2038-2045.
Schmitz, J. E., R. P. Johnson, H. M. McClure, K. H. Manson, M. S. Wyand, M. J. Kuroda, M. A. Lifton, R. S. Khunkhun, K. J. McEvers, J. Gillis, M. Piatak, J. D. Lifson, G. Grosschupff, P. Racz, K. Tenner-Racz, E. P. Rieber, K. Kuus-Reichel, R. S. Gelman, N. L. Letvin, D. C. Montefiori, R. M. Ruprecht, R. C. Desrosiers, and K. A. Reimann. 2005. Effect of CD8+ lymphocyte depletion on virus containment after simian immunodeficiency virus SIVmac251 challenge of live attenuated SIVmac2393-vaccinated rhesus macaques. J. Virol. 79:8131-8141.
Schmitz, J. E., M. J. Kuroda, S. Santra, V. G. Sasseville, M. A. Simon, M. A. Lifton, P. Racz, K. Tenner-Racz, M. Dalesandro, B. J. Scallon, J. Ghrayeb, M. A. Forman, D. C. Montefiori, E. P. Rieber, N. L. Letvin, and K. A. Reimann. 1999. Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes. Science 283:857-860.
Schmitz, J. E., M. J. Kuroda, S. Santra, M. A. Simon, M. A. Lifton, W. Lin, R. Khunkhun, M. Piatak, J. D. Lifson, G. Grosschupff, R. S. Gelman, P. Racz, K. Tenner-Racz, K. A. Mansfield, N. L. Letvin, D. C. Montefiori, and K. A. Reimann. 2003. Effect of humoral immune responses on controlling viremia during primary infection of rhesus monkeys with simian immunodeficiency virus. J. Virol. 77:2165-2173.
Schmitz, J. E., M. A. Simon, M. J. Kuroda, M. A. Lifton, M. W. Ollert, C. W. Vogel, P. Racz, K. Tenner-Racz, B. J. Scallon, M. Dalesandro, J. Ghrayeb, E. P. Rieber, V. G. Sasseville, and K. A. Reimann. 1999. A nonhuman primate model for the selective elimination of CD8+ lymphocytes using a mouse-human chimeric monoclonal antibody. Am. J. Pathol. 154:1923-1932.
Sharpe, S. A., A. Cope, S. Dowall, N. Berry, C. Ham, J. L. Heeney, D. Hopkins, L. Easterbrook, M. Dennis, N. Almond, and M. Cranage. 2004. Macaques infected long-term with attenuated simian immunodeficiency virus (SIVmac) remain resistant to wild-type challenge, despite declining cytotoxic T lymphocyte responses to an immunodominant epitope. J. Gen. Virol. 85:2591-2602.
Sharpe, S. A., A. M. Whatmore, G. A. Hall, and M. P. Cranage. 1997. Macaques infected with attenuated simian immunodeficiency virus resist superinfection with virulence-revertant virus. J. Gen. Virol. 78:1923-1927.
Shibata, R., C. Siemon, S. C. Czajak, R. C. Desrosiers, and M. A. Martin. 1997. Live, attenuated simian immunodeficiency virus vaccines elicit potent resistance against a challenge with a human immunodeficiency virus type 1 chimeric virus. J. Virol. 71:8141-8148.
Silvera, P., B. Flanagan, K. Kent, E. Rud, C. Powell, T. Corcoran, C. Bruck, C. Thiriart, N. L. Haigwood, and E. J. Stott. 1994. Fine analysis of humoral antibody response to envelope glycoprotein of SIV in infected and vaccinated macaques. AIDS Res. Hum. Retrovir. 10:1295-1304.
Silvera, P., A. Wade-Evans, E. Rud, R. Hull, K. Silvera, R. Sangster, N. Almond, and J. Stott. 2001. Mechanisms of protection induced by live attenuated simian immunodeficiency virus. III. Viral interference and the role of CD8+ T-cells and beta-chemokines in the inhibition of virus infection of PBMCs in vitro. J. Med. Primatol. 30:1-13.
Stahl-Hennig, C., U. Dittmer, T. Nisslein, H. Petry, E. Jurkiewicz, D. Fuchs, H. Wachter, K. Matz-Rensing, E. M. Kuhn, F. J. Kaup, E. W. Rud, and G. Hunsmann. 1996. Rapid development of vaccine protection in macaques by live-attenuated simian immunodeficiency virus. J. Gen. Virol. 77:2969-2981.
Stebbings, R., N. M. Almond, E. J. Stott, N. Berry, A. M. Wade-Evans, R. Hull, J. Lines, P. Silvera, R. Sangster, T. Corcoran, J. Rose, and K. B. Walker. 2002. Mechanisms of protection induced by attenuated simian immunodeficiency virus. V. No evidence for lymphocyte-regulated cytokine responses upon rechallenge. Virology 296:338-353.
Stebbings, R., N. Berry, J. Stott, R. Hull, B. Walker, J. Lines, W. Elsley, S. Brown, A. Wade-Evans, G. Davis, J. Cowie, M. Sethi, and N. Almond. 2004. Vaccination with live attenuated simian immunodeficiency virus for 21 days protects against superinfection. Virology 330:249-260.
Stebbings, R., J. Stott, N. Almond, R. Hull, J. Lines, P. Silvera, R. Sangster, T. Corcoran, J. Rose, S. Cobbold, F. Gotch, A. McMichael, and B. Walker. 1998. Mechanisms of protection induced by attenuated simian immunodeficiency virus. II. Lymphocyte depletion does not abrogate protection. AIDS Res. Hum. Retrovir. 14:1187-1198.
Stott, E. J., W. L. Chan, K. H. Mills, M. Page, F. Taffs, M. Cranage, P. Greenaway, and P. Kitchin. 1990. Preliminary report: protection of cynomolgus macaques against simian immunodeficiency virus by fixed infected-cell vaccine. Lancet 336:1538-1541.
Tenner-Racz, K., C. S. Hennig, K. Uberla, H. Stoiber, R. Ignatius, J. Heeney, R. M. Steinman, and P. Racz. 2004. Early protection against pathogenic virus infection at a mucosal challenge site after vaccination with attenuated simian immunodeficiency virus. Proc. Natl. Acad. Sci. USA 101:3017-3022.
Vogel, T. U., B. E. Beer, J. zur Megede, H. G. Ihlenfeldt, G. Jung, S. Holzammer, D. I. Watkins, J. D. Altman, R. Kurth, and S. Norley. 2002. Induction of anti-simian immunodeficiency virus cellular and humoral immune responses in rhesus macaques by peptide immunogens: correlation of CTL activity and reduction of cell-associated but not plasma virus load following challenge. J. Gen. Virol. 83:81-91.
Vogel, T. U., J. Fournier, A. Sherring, D. Ko, M. Parenteau, D. Bogdanovic, J. Mihowich, and E. W. Rud. 1998. Presence of circulating CTL induced by infection with wild-type or attenuated SIV and their correlation with protection from pathogenic SHIV challenge. J. Med. Primatol. 27:65-72.
Whatmore, A. M., N. Cook, G. A. Hall, S. Sharpe, E. W. Rud, and M. P. Cranage. 1995. Repair and evolution of nef in vivo modulates simian immunodeficiency virus virulence. J. Virol. 69:5117-5123.
Wyand, M. S., K. Manson, D. C. Montefiori, J. D. Lifson, R. P. Johnson, and R. C. Desrosiers. 1999. Protection by live, attenuated simian immunodeficiency virus against heterologous challenge. J. Virol. 73:8356-8363.
Wyand, M. S., K. H. Manson, M. Garcia-Moll, D. Montefiori, and R. C. Desrosiers. 1996. Vaccine protection by a triple deletion mutant of simian immunodeficiency virus. J. Virol. 70:3724-3733.(Richard Stebbings, Neil B)
Sir William Dunn School of Pathology Oxford Therapeutic Antibody Centre, Oxford University, Oxford, United Kingdom
ABSTRACT
In order to test the hypothesis that CD8+ cytotoxic T lymphocytes mediate protection against acute superinfection, we depleted >99% of CD8+ lymphocytes in live attenuated simian immunodeficiency virus macC8 (SIVmacC8) vaccinees from the onset of vaccination, maintained that depletion for 20 days, and then challenged with pathogenic, wild-type SIVmacJ5. Vaccinees received 5 mg per kg of humanized anti-CD8 monoclonal antibody (MAb) 1 h before inoculation, followed by the same dose again on days 3, 7, 10, 13, and 17. On day 13, peripheral CD8+ T lymphocytes were >99% depleted in three out of four anti-CD8 MAb-treated vaccinees. At this time attenuated SIVmacC8 viral RNA loads in anti-CD8 MAb-treated vaccinees were significantly higher than control vaccinees treated contemporaneously with nonspecific human immunoglobulin. Lymphoid tissue CD8+ T lymphocyte depletion was >99% in three out of four anti-CD8 MAb-treated vaccinees on the day of wild-type SIVmacJ5 challenge. All four control vaccinees and three out of four anti-CD8 MAb-treated vaccinees were protected against detectable superinfection with wild-type SIVmacJ5. Although superinfection with wild-type SIVmacJ5 was detected at postmortem in a single anti-CD8 MAb-treated vaccinee, this did not correlate with the degree of preceding CD8+ T lymphocyte depletion. Clearance of attenuated SIVmacC8 viremia coincided with recovery of normal CD8+ T lymphocyte counts between days 48 and 76. These results support the view that cytotoxic T lymphocytes are important for host-mediated control of SIV primary viremia but do not indicate a central role in protection against acute superinfection conferred by inoculation with live attenuated SIV.
INTRODUCTION
Vaccination of macaques with live attenuated simian immunodeficiency virus (SIV) provides a valuable model to study the correlate(s) of immunity that an effective human immunodeficiency virus (HIV)/AIDS vaccine will need to emulate. Live attenuated SIV vaccines can confer effective protection against detectable superinfection with pathogenic, wild-type SIV (3, 13, 14, 29, 58, 59) and SIV/HIV-1 chimeric virus (7, 18, 46). Yet there are limits to the breadth of this protection, and live attenuated SIV vaccines have failed to protect against certain heterologous challenge viruses or failed to protect against a challenge performed several years postinoculation (22, 31, 58). Furthermore, the demonstrated potential for reversion to pathogenicity in live attenuated SIV precludes clinical evaluation of a live attenuated HIV vaccine (6, 38, 39, 57). Nevertheless, an understanding of the mechanism(s) of protection against superinfection conferred by inoculation with live attenuated SIV would further the development of a safe and effective HIV vaccine. An unambiguous correlate of protection against superinfection has so far evaded identification. Cytotoxic T lymphocytes (CTL), virus neutralizing antibodies, innate immunity, and retroviral interference have all been reported as potential mechanisms of protection against superinfection conferred by inoculation with live attenuated SIV (2, 24, 32, 37, 50, 54, 59). Here we have evaluated the role of CD8+ lymphocytes and, thereby, CD8+ CTL in mediating protection against acute superinfection conferred by inoculation with live attenuated SIV.
Inoculation with live attenuated SIV generates significant SIV-specific CD8+ CTL responses (16, 25, 32, 56). The appearance of SIV-specific CD8+ CTL responses during primary SIV infection coincides with clearance of plasma viremia and suppression of viral replication (41). Furthermore, the importance of CD8+ lymphocytes for control of pathogenic or attenuated SIV infection has been demonstrated in several studies that report a dramatic rise in plasma viremia following anti-CD8 monoclonal antibody (MAb) treatment to deplete CD8+ CTL, with control of virus replication being temporally associated with recovery of CD8+ lymphocytes (23, 28, 30, 41). In addition, an inverse correlation has been reported between the precursor frequency of SIV-specific CD8+ CTL responses elicited by certain vaccine approaches and virus load following challenge (20, 55). Although several groups have reported a correlation between SIV-specific CD8+ CTL responses in live attenuated SIV vaccinees and protection against superinfection with wild-type SIV (24-26, 56), other groups have failed to corroborate such observations and dispute a role for SIV-specific CD8+ CTL in mediating protection (1, 32, 44, 50, 52).
In a previous study we attempted to address the role of SIV-specific CD8+ CTL responses in mediating protection against superinfection by administering a pair of rat anti-human CD8 MAbs to live attenuated SIVmacC8 vaccinees 24 h prior to challenge with wild-type SIVmacJ5 (52). All CD8+ lymphocyte-depleted vaccinees resisted superinfection with wild-type SIVmacJ5, which suggests that SIV-specific CD8+ CTL responses are not central to protection against superinfection observed at 35 weeks postinoculation. However, it remained a possibility that the degree of depletion obtained in lymphoid tissues may have been insufficient and that the temporal removal of CD8+ lymphocytes would not have prevented the establishment of effective CD4+ T cell memory, which would then rapidly drive the reappearance of SIV-specific CD8+ CTL (15).
We have now established a model of superinfection resistance to wild-type SIVmacJ5 challenge that is reproducibly achieved within 21 days of inoculation with live attenuated SIVmacC8 (51). Moreover, protection against superinfection at 21 days postinoculation with live attenuated SIVmacC8 occurs before the development of detectable SIV-specific serological responses but coincides with the development of detectable SIV-specific CD8+ CTL responses (51). The development of a therapeutic humanized anti-human CD8 MAb which can be administered repeatedly has extended the period of effective CD8+ lymphocyte depletion (>99%) in blood and lymphoid tissues for periods greater than 3 weeks. We have combined these two advances to address more effectively the role of CD8+ CTL responses in mediating protection against acute superinfection conferred by inoculation with live attenuated SIVmacC8. To ensure >99% depletion of peripheral and lymphoid tissue CD8+ lymphocytes on the day of challenge and to prevent the expansion and dissemination of SIV-specific CD8+ CTL during vaccination, we employed profound depletion of CD8+ lymphocytes from the day of inoculation through the day of challenge and beyond.
MATERIALS AND METHODS
Animals and viruses. A total of 16 na?ve, D-type retrovirus-free, juvenile purpose-bred cynomolgus macaques (Macaca fascicularis) were used in this study. Macaques were housed and maintained in accordance with United Kingdom Home Office guidelines for the care and maintenance of nonhuman primates. The attenuated SIVmac32H (SIVmacC8) virus clone differs from the wild-type SIVmac32H (SIVmacJ5) clone by a 12-bp deletion and two nonsynonymous nucleotide changes, resulting in conservative amino acid changes in the nef open reading frame (36). For all live attenuated SIV inoculations, macaques were intravenously administered 5,000 50% tissue culture infective doses of the 9/90 pool of SIVmacC8 on day 0 (11, 36). For all wild-type SIV challenges, macaques were intravenously administered 20 50% median infective doses of the 3/92 stock of SIVmacJ5 on day 20 (12, 36).
Humanized anti-CD8 MAb treatment. In two preliminary experiments to determine efficacy, the ability of the humanized anti-CD8 MAb TRX2 (Oxford Therapeutic Antibody Centre, Oxford University, Oxford, United Kingdom) to deplete peripheral and lymphoid tissue CD8+ T lymphocytes in na?ve macaques was evaluated. In an initial study, macaques Z98 and Z99 were intravenously administered 3 mg per kg of body weight of anti-CD8 MAb on days 0, 1, and 3; 6 mg per kg on days 6, 10, and 13; and 9 mg per kg on days 17 and 20. In a follow-up study, macaques Z274 and Z275 were intravenously administered 3 mg per kg of humanized anti-CD8 MAb on days 0, 1, and 4.
To address the role of SIV-specific CD8+ CTL responses in mediating protection against acute superinfection, we have combined a previously described 21-day vaccination model (51) with sustained depletion of CD8+ lymphocytes. On day 0, macaques A55, A56, A66, and A125 (group A) were administered intravenously 5 mg per kg of body weight of humanized anti-CD8 MAb. Macaques A59, A60, A61, and A62 (group B) were administered intravenously 5 mg per kg of pooled nonspecific human immunoglobulin contemporaneously (Grifols, Barcelona, Spain). One hour after treatment, all eight macaques (groups A and B) were each inoculated with 5,000 50% tissue culture infective doses of live attenuated SIVmacC8 intravenously. Anti-CD8 MAb treatment of group A and nonspecific human immunoglobulin treatment of group B, both at 5 mg per kg, were repeated on days 3, 7, 10, 13, and 17. Group C consisted of four na?ve controls, macaques A58, A63, A64, and A65. On day 20, groups A, B, and C were all challenged with 20 50% median infective doses of wild-type SIVmacJ5 intravenously.
Quantification of CD8+ T lymphocyte counts. All anti-human MAbs used were previously identified by flow cytometry to cross-react with macaque peripheral blood mononuclear cells (PBMC). For measurement of CD8+ T lymphocyte depletion, whole blood was immunostained with mouse anti-monkey CD3 fluorescein isothiocyanate conjugate (clone FN-18; Serotec, Oxford, United Kingdom), mouse anti-human CD4 phycoerythrin conjugate (clone L120; BD Biosciences, San Jose, CA), and mouse anti-human CD8 allophycocyanin conjugate (clone 3B5; Caltag Laboratories, Burlingame, CA), as previously described (50). Positive staining for the T lymphocyte marker CD3 was used to distinguish CD8+ T lymphocytes from CD8+ natural killer (NK) cells. All blood samples and lymph node biopsies were taken before administration of each dose of anti-CD8 MAb or nonspecific human immunoglobulin. The addition of humanized anti-CD8 MAb to macaque whole blood did not block subsequent immunostaining with anti-human CD8 allophycocyanin conjugate. Absolute counts of CD8+ T lymphocytes were made using the BD TruCOUNT system (San Jose, CA). The detection limit for CD8+ T lymphocytes by flow cytometry was 1 cell per μl of blood or resuspended lymph node biopsy. Lymph node biopsies were trimmed of fat and weighed prior to disaggregation using the Medimachine system (DakoCytomation Ltd., Ely, United Kingdom) and then immunostained as per whole blood. A FACSCalibur cytometer was used for acquisition, and data were analyzed using CellQuest Pro software (BD Biosciences, San Jose, CA). Greater than 99% depletion of CD8+ T lymphocytes compared to pretreatment levels was deemed effective depletion.
Virus detection, quantification, and serology. The presence of SIV in PBMC or tissue samples was determined using a SIV gag DNA PCR assay, as previously described (35). The two molecular clones, SIVmacC8 and SIVmacJ5, were differentiated using a nef-specific nested PCR protocol which encompasses the attenuating 12-bp deletion in SIVmacC8 nef. RsaI restriction endonuclease digestion of the PCR product was used to discriminate between the presence of full-length SIVmacJ5 nef containing this site and SIVmacC8 nef, which does not (35). Intermediate revertants lacking the RsaI restriction site are not distinguished from SIVmacC8 by this protocol. Within a background of SIVmacC8, the lower detection limit of this assay is 10 copies of SIVmacJ5. The overall kinetics of plasma SIV RNA loads were determined as previously described (8). The sensitivity of the assay is 200 SIV RNA copies per ml of plasma. Virus isolation and titration from PBMC were determined by coculture with C8166 cells; the presence of replicating virus was confirmed by syncytia identification or by antigen capture at 28 days, as previously described (52). Titers of binding antibodies to SIV envelope gp130 (EVA670; CFAR/NIBSC, Potters Bar, United Kingdom) or recombinant SIV p27 (EVA643; CFAR/NIBSC, Potters Bar, United Kingdom) were determined in heat-inactivated (56°C for 1 h) plasma samples by enzyme-linked immunosorbent assay, as previously described (4, 47, 53). Neutralizing antibody end-point titers were determined as the dilution of serum in the serum and virus mixture inhibiting p27 antigen production by at least 75%, expressed as the log10 of the reciprocal of the end-point dilution (27).
RESULTS
Anti-CD8 MAb treatment depletes CD8+ T lymphocytes. We conducted two preliminary studies in na?ve macaques to determine a suitable protocol for anti-CD8 MAb treatment that could maintain effective (>99%) depletion of CD8+ T lymphocytes over a 3-week period. Intravenous administration of anti-CD8 MAb to na?ve macaques Z98, Z99, Z274, and Z275 resulted in >99.7% depletion of peripheral CD8+ T lymphocytes within 24 h of treatment (Fig. 1A). Three anti-CD8 MAb treatments, given to macaques Z274 and Z275 on days 0, 1, and 4, were sufficient to maintain >99.9% depletion of peripheral CD8+ T lymphocytes for at least 2 weeks (Fig. 1A). Eight anti-CD8 MAb treatments, given to macaques Z98 and Z99 on days 0, 1, 3, 6, 10, 13, 16, and 20, were sufficient to maintain >99.7% depletion of peripheral CD8+ T lymphocytes for at least 7 weeks (Fig. 1A). When compared to the rebound in peripheral CD8+ T lymphocyte counts observed in macaques that received three anti-CD8 MAb treatments, the recovery of peripheral CD8+ T lymphocyte counts was very slow in macaques that received eight anti-CD8 MAb treatments (Fig. 1A). Where all peripheral CD8+ T lymphocyte counts were below the detection limits of the flow cytometer (1 cell per μl blood), lymphoid tissue CD8+ T lymphocyte counts provided a better means of ranking the degree of depletion between individuals. On day 20, following three anti-CD8 MAb treatments, depletion of lymphoid tissue CD8+ T lymphocytes in macaques Z274 and Z275 was substantial at 92.9% and 97.4%, respectively. However, administration of seven anti-CD8 MAb treatments to macaques Z98 and Z99 by day 20 achieved >99.9% and 99.8% depletion of lymphoid tissue CD8+ T lymphocytes, respectively (Fig. 1B). Recovery of lymphoid tissue CD8+ T lymphocyte counts following cessation of anti-CD8 MAb treatment was less pronounced than that observed in the periphery. In particular, macaques Z98 and Z99 were still greater than 99.7% depleted of lymphoid tissue CD8+ T lymphocytes on day 147, in contrast to partial recovery of their peripheral CD8+ T lymphocyte counts (Fig. 1A and B). This profound depletion of CD8+ lymphocyte depletion was mirrored in mesenteric lymph nodes and spleen at termination (data not shown). CD4+ T lymphocyte and CD4– CD8– T lymphocyte counts remained stable for the duration of these studies (data not shown).
Since administration of anti-CD8 MAb antibody on seven occasions had proven sufficient to attain the desired level and duration of CD8+ T lymphocyte depletion and because of concerns over the safety of long-term CD8+ T lymphocyte depletion in SIV-infected macaques, the total number of treatments was reduced from eight to six, given over 3 weeks. Anti-CD8 MAb treatment of group A vaccinees (animals A55, A56, A66, and A125) resulted in profound depletion of peripheral CD8+ T lymphocytes (Fig. 2A). No depletion of peripheral or lymphoid tissue CD8+ T lymphocytes was evident following contemporaneous treatment of group B vaccinees (A59, A60, A61, and A62) with nonspecific human immunoglobulin (Fig. 2B and D, respectively). On day 13, following four anti-CD8 MAb treatments, peripheral CD8+ T lymphocytes were >99.8% depleted in macaques A55, A56, and A66 but were only reduced by 87.7% in macaque A125 (Fig. 2A). Peripheral CD8+ T lymphocyte counts in group A were significantly lower than group B on day 13 (P = 0.0209, Mann-Whitney U test). On day 20, after six anti-CD8 MAb treatments, CD8+ T lymphocytes recovered from lymphoid tissue were >99.4% depleted in macaques A55, A56, and A66 but were only reduced by 95.8% in macaque A125 (Fig. 2C). On day 20, the day of wild-type SIVmacJ5 challenge, lymphoid tissue CD8+ T lymphocyte counts in group A were significantly lower than group B (P = 0.0209, Mann-Whitney U test). Within group A, CD8+ T lymphocytes recovered from lymph node biopsies were most profoundly depleted in macaques A55 and A56, at >99.9% depletion (Fig. 2C). Following cessation of treatment with anti-CD8 MAb and 14 days after challenge with wild-type SIVmacJ5 (day 34), peripheral CD8+ T lymphocyte numbers had increased, but in three out of four vaccinees, CD8+ T lymphocyte depletion remained >92.5% (Fig. 2A). In particular, CD8+ T lymphocyte depletion in macaque A56 was >99.8% on day 34 (Fig. 2A). At both days 34 and 48, CD8+ T lymphocyte counts in group A vaccinees remained significantly lower than group B vaccinees, but CD8+ T lymphocyte counts increased thereafter (P = 0.0209 at both times, Mann-Whitney U test) between days 48 and 76 (Fig. 2A). By day 169, recovery of lymphoid tissue CD8+ T lymphocytes was evident in group A vaccinees, and counts did not differ significantly from group B vaccinees (Fig. 2C and D) (P = 0.2482, Mann-Whitney U test).
Inoculation with attenuated SIVmacC8 promotes resurgence of CD8+ T lymphocytes. In comparison to na?ve macaques, the recovery of CD8+ T lymphocytes in attenuated SIVmacC8 vaccinees was much more rapid and absolute (Fig. 1A and 2A). In attenuated SIVmacC8 vaccinees, this recovery was partly driven by the expansion of CD4+ T lymphocytes expressing the -chain of CD8 (data not shown). Expansion of CD4+ CD8+ T lymphocytes was not observed in anti-CD8 MAb-treated na?ve macaques during recovery. CD8+ NK cells were also profoundly depleted by anti-CD8 MAb treatment (data not shown). No significant difference in CD4+ T lymphocyte counts between vaccinees that received anti-CD8 MAb or nonspecific human immunoglobulin treatment was observed during this study (data not shown).
CD8+ lymphocyte depletion is associated with increased SIVmacC8 virus load. The kinetics of the primary viremia of attenuated SIVmacC8 in cynomolgus macaques has been reported previously (8). Depletion of CD8+ lymphocytes throughout the primary viremia of attenuated SIVmacC8 (Fig. 3A) provoked a significant increase in peak plasma viral RNA (vRNA) loads measured at day 13, compared to vaccine controls (Fig. 3B) treated with nonspecific human immunoglobulin (Fig. 3D) (for group A compared to group B, P = 0.0339, Mann-Whitney U test). This observation was corroborated by significantly higher cell-associated virus loads in group A vaccinees at day 13, compared with group B vaccinees (data not shown; P = 0.0339, Mann-Whitney U test). Plasma vRNA loads among group A vaccinees remained significantly higher than group B vaccinees until at least day 34 but not thereafter (Fig. 3D) (P = 0.0433, Mann-Whitney U test). Nonetheless, plasma virus loads in group B fell below the lower detection limit of our assay by day 34 (Fig. 3B), whereas in group A this suppression of primary viremia was delayed until at least day 48 (Fig. 3A). Following CD8+ lymphocyte depletion of group A vaccinees, the increase in peak plasma viremia of attenuated SIVmacC8 at day 13 (Fig. 3A) is more comparable to the peak in wild-type SIVmacJ5 viremia of group C on day 34 of the study (Fig. 3C) (day 14 after challenge with SIVmacJ5) than to that of group B vaccinees on day 13 (Fig. 3B).
CD8+ lymphocyte depletion increases anti-SIV envelope antibody titers. Antibodies against SIV gp130 were detected in all vaccinees (groups A and B) by day 34 (Fig. 4A). Anti-SIV gp130 antibody titers in group A vaccinees were significantly higher than in group B vaccinees at day 34 and remained elevated at all time points thereafter (Fig. 4A) (P = 0.0209 at all time-points, Mann-Whitney U test). Following wild-type SIVmacJ5 challenge on day 20, anti-SIV gp130 antibodies were detected in all group C na?ve controls on day 48 (Fig. 4A). Anti-SIV gp130 antibody titers in group A vaccinees were significantly higher than group C challenge controls at days 34, 48, and 76 (Fig. 4A) (P = 0.0209, P = 0.0433, and P = 0.0209, respectively; Mann-Whitney U test). Thereafter, anti-SIV gp130 titers in group C challenge controls increased to similar levels as group A (Fig. 4A). Anti-SIV neutralizing antibodies were first detected in vaccinees at day 76 (Table 1). There was no significant difference in neutralizing antibody titers between vaccinee groups A and B on days 76 or 104 (P = 0.3865 and P = 0.2482, respectively; Mann-Whitney U test). Antibodies against SIV p27 were detected in all group A vaccinees on day 34 but only in two out of four group B vaccinees (Fig. 4B). Antibodies against SIV p27 were not detected in all group B vaccinees until day 76 (Fig. 4B). At day 34 anti-SIV p27 titers were significantly higher in group A vaccinees compared to group B vaccinees but not thereafter (Fig. 4B) (P = 0.0209, Mann-Whitney U test).
CD8+ lymphocyte responses are not required for protection against acute superinfection. Successful inoculation of all group A and B macaques with live attenuated SIVmacC8 on day 0 was confirmed by SIV gag DNA PCR assays on day 13 (data not shown). Following intravenous challenge of all groups with wild-type SIVmacJ5, all group C macaques were confirmed to be infected with SIVmacJ5 by nef-specific DNA PCR (Table 2). All group B vaccinees were negative for detectable wild-type SIVmacJ5 nef at all time points following challenge and were therefore deemed to have resisted superinfection (Table 2). Within group A, three out of four vaccinees were negative for detectable wild-type SIVmacJ5 nef at all time points and were deemed to have resisted superinfection (Table 2). Although wild-type SIVmacJ5 nef could not initially be detected in group A, a low level of wild-type SIVmacJ5 nef signal (approximately 10% of the total signal) was detected at postmortem (day 169) in the blood and lymphoid tissues of vaccinee A66, indicating superinfection and vaccine failure in that individual (Table 2).
DISCUSSION
This study is part of a series that has characterized the early protection against superinfection conferred by inoculation with live nef-attenuated SIVmacC8 (3, 5, 48, 50-52). Previously, we showed that protection against acute superinfection with pathogenic, wild-type SIVmacJ5 can be obtained within 21 days of inoculation with live attenuated SIVmacC8, at a time when neutralizing antibodies were not present and limited CD8+ T lymphocyte responses were detected (51). The present study demonstrates that profound depletion of CD8+ lymphocytes for up to 34 days, encompassing the period macaques are vaccinated with live attenuated SIVmacC8, does not abrogate protection against challenge with pathogenic, wild-type SIVmacJ5. Our observations are consistent with those of others that CD8+ lymphocyte responses are important for control of SIV replication since we observed a dramatic increase in plasma viremia associated with the depletion of CD8+ lymphocytes in macaques inoculated with attenuated SIVmacC8 (23, 28, 30, 41). However, the ultimate conclusion of this study is that CD8+ lymphocyte responses alone are not central to the protection against acute superinfection conferred 20 days after vaccination with attenuated SIVmacC8. This conclusion is consistent with the findings of other investigators who were unable to identify a correlation between SIV-specific CD8+ CTL responses elicited by inoculation with live attenuated SIV and protection against superinfection (31, 32, 44).
Results for the depletion of CD8+ T lymphocytes in na?ve macaques given three anti-CD8 MAb treatments are comparable to those obtained by Schmitz and colleagues (43). Although peripheral CD8+ T lymphocytes are effectively (>99%) depleted within 1 h of anti-CD8 MAb treatment, the majority of CD8+ lymphocytes are found within lymphoid tissues and have been found to be resistant to effective depletion (23, 28, 30, 52). By increasing the number of anti-CD8 MAb treatments to eight, given over a 3-week period, we were able to effectively deplete lymphoid tissue CD8+ T lymphocytes in na?ve macaques and extend greatly the duration of depletion. After effective depletion of lymphoid tissue CD8+ T lymphocytes, the recovery observed in vaccinees was much faster and more complete than that observed in na?ve macaques depleted to a similar degree. Thus, inoculation with live attenuated SIVmacC8 appears to be a strong stimulus for the production of new CD8+ T lymphocytes as a mechanism to control viremia. Anti-CD8 MAb treatment also depleted CD8+ NK cells in attenuated SIVmacC8 vaccinees. NK cells are important effectors of the innate immune response against viral infections and also secrete cytokines and chemokines that play a role in recruitment and activation of the adaptive immune response (10, 34). It is possible that the depletion of these CD8+ NK cells contributed to the significant increase in plasma viremia observed in anti-CD8 MAb-treated individuals infected with attenuated SIVmacC8. Moreover, the depletion of CD8+ NK cells by anti-CD8 MAb treatment would also exclude a role for these cells in mediating early protection against acute superinfection.
The significant increase in anti-SIVgp130 titers elicited by depletion of CD8+ lymphocytes from attenuated SIVmacC8 vaccinees may be related to the accompanying dramatic increase in virus load. Challenge controls infected with wild-type SIVmacJ5 had comparable virus loads and generated comparable anti-SIVgp130 titers. It is possible that in group A the more marked anti-SIVgp130 serological response has resulted from a homeostatic compensation for the lack of CD8+ lymphocyte responses or that strong Th1 CD8+ cellular immune responses normally limit Th2 humoral responses. An inverse correlation between neutralizing antibody titers and plasma virus levels in SIV-infected and B-cell depleted macaques has been reported (42). Similarly, postchallenge immunotherapy with purified immunoglobulin from SIV-infected long-term nonprogressors is reported to reduce virus burden (21). It could be argued that the more marked anti-SIVgp130 serological response contributed to protection against superinfection and impaired the ability to determine the mechanism of protection. However, the more marked anti-SIVgp130 serological response did not prevent the significant increase in plasma viremia associated with depletion of CD8+ lymphocytes or prevent superinfection of vaccinee A66. Moreover, anti-SIV-specific neutralizing antibodies were not detected until day 76 in either vaccine group and do not correlate with early protection against acute superinfection (51). That detection of high anti-SIV p27 titers occurs later than is observed with anti-SIVgp130 antibodies and that significant CD8+ T lymphocyte recovery in anti-CD8 MAb-treated vaccinees occurs before this time may explain why significantly higher titers of anti-p27 antibodies are only observed at day 34. Alternatively, the extended viremia may have absorbed anti-SIV p27 antibody production.
Previously, where superinfection has been observed following challenge of attenuated SIVmacC8 vaccinees, one of two patterns has been apparent: either superinfection is clearly detected in the blood at 2 weeks postchallenge or not until postmortem, when the presence of virus can be investigated in a range of lymphoid tissues (51). Here, the latter pattern is applicable to superinfection of vaccinee A66. Thus, even in the presence of marked suppression of CD8+ T lymphocytes, wild-type SIVmacJ5 has not been able to establish itself readily, implying at least partial protection. It is interesting that the degree of preceding CD8+ T lymphocyte depletion in A66 did not correlate with its susceptibility to superinfection. Depletion of CD8+ lymphocytes in attenuated SIVmacC8 vaccinees A55 and A56 was more profound than in A66, but these macaques were not superinfected. It is possible that protection against superinfection in macaque A66 may not have been complete at the time of challenge, day 20. Previously, the earliest time we have observed reproducible protection against detectable superinfection has been 21 days after inoculation with live attenuated SIVmacC8 and only partial protection at earlier times (51). Although vaccinee A125 resisted superinfection, depletion of CD8+ lymphocytes was less marked than in subject A66. Nevertheless, the degree of CD8+ T lymphocyte depletion in A125 was substantial enough to produce a dramatic increase in plasma viremia and clearly sufficient to compromise anti-SIV CD8+ T lymphocyte mediated responses.
The model of protection against acute superinfection at 20 days after vaccination described here differs from models used by other groups, in which more highly attenuated vaccine virus is employed and full protection is not achieved until 10 or 20 weeks after inoculation (9, 59). Therefore, there may be significant differences in the mechanism of protection between these models (40). However, when superinfection occurs before the onset of full protection, significant control of primary viremia is still observed (9, 49, 51). Nevertheless, the results reported here do not support the hypothesis of a central role for CD8+ CTL responses in mediating protection against acute superinfection conferred by inoculation with live attenuated SIV. If cell-mediated immune responses are the mechanism of protection in live attenuated SIV vaccinees, then a characteristic expansion of memory CD8+ CTL and immune activation should be detected following virus challenge (17, 19). Yet evidence for immune activation or expansion of SIV-specific CTL responses following rechallenge has not been reported (44, 50). Even where protection against superinfection is incomplete, vaccine-mediated control of virus replication occurs before detection or expansion of CD8+ SIV-specific CTL responses (1, 51). However, lack of expansion of SIV-specific T cells preceding initial immune control does not necessarily rule out a role for SIV-specific CTL responses postchallenge. It should be noted that several previous studies have shown the correlation of CTL responses with protection (2, 26, 33).
Following inoculation with attenuated SIVmacC8, primary viremia peaks at day 10 and rapidly declines thereafter to undetectable levels (8). Nevertheless, the pathogenic SIVmacJ5 challenge at day 20 occurred in concert with detectable levels of vaccine virus in CD8+ lymphocyte-depleted macaques. This is in contrast to other attenuated SIV vaccine scenarios where challenge occurs when attenuated SIV replication is low or undetectable. Therefore, it is possible that the resistance to acute superinfection is not due to immune protection but, rather, is a result of a combination of target cell depletion and retroviral interference.
Since we have reported that vaccine protection conferred by attenuated SIVmacC8 cannot be transferred with immune serum alone (5) and demonstrate here that CD8+ T cell responses do not correlate with resistance to acute superinfection, it follows that other candidates for mediating protection need to be explored. Innate immunity and nonimmune mechanisms such as retroviral interference or target cell depletion need to be investigated to unravel this potential novel mechanism of antiviral immunity (37, 45, 51, 54). Armed with this information, we can then develop novel prophylactic vaccine approaches to reproduce this potential vaccine protection in a safe and effective HIV/AIDS vaccine.
ACKNOWLEDGMENTS
We thank Harvey Holmes, the Centralised Facility for AIDS Reagents, and the Oxford Therapeutic Antibody Centre for essential materials.
This work was funded in part by grants from the United Kingdom Medical Research Council (G9025730 and G9419998).
Deceased.
REFERENCES
Abdel-Motal, U. M., J. Gillis, K. Manson, M. Wyand, D. Montefiori, K. Stefano-Cole, R. C. Montelaro, J. D. Altman, and R. P. Johnson. 2005. Kinetics of expansion of SIV Gag-specific CD8+ T lymphocytes following challenge of vaccinated macaques. Virology 333:226-238.
Abel, K., L. Compton, T. Rourke, D. Montefiori, D. Lu, K. Rothaeusler, L. Fritts, K. Bost, and C. J. Miller. 2003. Simian-human immunodeficiency virus SHIV89.6-induced protection against intravaginal challenge with pathogenic SIVmac239 is independent of the route of immunization and is associated with a combination of cytotoxic T-lymphocyte and alpha interferon responses. J. Virol. 77:3099-3118.
Almond, N., K. Kent, M. Cranage, E. Rud, B. Clarke, and E. J. Stott. 1995. Protection by attenuated simian immunodeficiency virus in macaques against challenge with virus-infected cells. Lancet 345:1342-1344.
Almond, N., M. Page, K. Mills, A. Jenkins, C. Ling, R. Thorpe, P. Kitchin, and M. Williams. 1990. The production and purification of PCR-derived recombinant simian immunodeficiency virus p27 gag protein; its use in detecting serological and T-cell responses in macaques. J. Virol. Methods 28:305-319.
Almond, N., J. Rose, R. Sangster, P. Silvera, R. Stebbings, B. Walker, and E. J. Stott. 1997. Mechanisms of protection induced by attenuated simian immunodeficiency virus. I. Protection cannot be transferred with immune serum. J. Gen. Virol. 78:1919-1922.
Baba, T. W., Y. S. Jeong, D. Pennick, R. Bronson, M. F. Greene, and R. M. Ruprecht. 1995. Pathogenicity of live, attenuated SIV after mucosal infection of neonatal macaques. Science 267:1820-1825.
Bogers, W. M., H. Niphuis, P. ten Haaft, J. D. Laman, W. Koornstra, and J. L. Heeney. 1995. Protection from HIV-1 envelope-bearing chimeric simian immunodeficiency virus (SHIV) in rhesus macaques infected with attenuated SIV: consequences of challenge. AIDS 9:F13-F8.
Clarke, S., N. Almond, and N. Berry. 2003. Simian immunodeficiency virus Nef gene regulates the production of 2-LTR circles in vivo. Virology 306:100-108.
Connor, R. I., D. C. Montefiori, J. M. Binley, J. P. Moore, S. Bonhoeffer, A. Gettie, E. A. Fenamore, K. E. Sheridan, D. D. Ho, P. J. Dailey, and P. A. Marx. 1998. Temporal analyses of virus replication, immune responses, and efficacy in rhesus macaques immunized with a live, attenuated simian immunodeficiency virus vaccine. J. Virol. 72:7501-7509.
Cooper, M. A., T. A. Fehniger, and M. A. Caligiuri. 2001. The biology of human natural killer-cell subsets. Trends Immunol. 22:633-640.
Cranage, M. P., S. A. Sharpe, A. M. Whatmore, N. Polyanskaya, S. Norley, N. Cook, S. Leech, M. J. Dennis, and G. A. Hall. 1998. In vivo resistance to simian immunodeficiency virus superinfection depends on attenuated virus dose. J Gen. Virol. 79:1935-1944.
Cranage, M. P., A. M. Whatmore, S. A. Sharpe, N. Cook, N. Polyanskaya, S. Leech, J. D. Smith, E. W. Rud, M. J. Dennis, and G. A. Hall. 1997. Macaques infected with live attenuated SIVmac are protected against superinfection via the rectal mucosa. Virology 229:143-154.
Daniel, M. D., F. Kirchhoff, S. C. Czajak, P. K. Sehgal, and R. C. Desrosiers. 1992. Protective effects of a live attenuated SIV vaccine with a deletion in the nef gene. Science 258:1938-1941.
Desrosiers, R. C. 1990. The simian immunodeficiency viruses. Annu. Rev. Immunol. 8:557-578.
Dittmer, U., and K. J. Hasenkrug. 1999. Alternative interpretation of lymphocyte depletion studies using monoclonal antibodies in animals previously vaccinated with attenuated retroviral vaccines. AIDS Res. Hum. Retrovir. 15:785.
Dittmer, U., T. Nisslein, W. Bodemer, H. Petry, U. Sauermann, C. Stahl-Hennig, and G. Hunsmann. 1995. Cellular immune response of rhesus monkeys infected with a partially attenuated nef deletion mutant of the simian immunodeficiency virus. Virology 212:392-397.
Doherty, P. C., and J. P. Christensen. 2000. Accessing complexity: the dynamics of virus-specific T cell responses. Annu. Rev. Immunol. 18:561-592.
Dunn, C. S., B. Hurtrel, C. Beyer, L. Gloeckler, T. N. Ledger, C. Moog, M. P. Kieny, M. Mehtali, D. Schmitt, J. P. Gut, A. Kirn, and A. M. Aubertin. 1997. Protection of SIVmac-infected macaque monkeys against superinfection by a simian immunodeficiency virus expressing envelope glycoproteins of HIV type 1. AIDS Res. Hum. Retrovir. 13:913-922.
Flynn, K. J., J. M. Riberdy, J. P. Christensen, J. D. Altman, and P. C. Doherty. 1999. In vivo proliferation of naive and memory influenza-specific CD8(+) T cells. Proc. Natl. Acad. Sci. USA 96:8597-8602.
Gallimore, A., M. Cranage, N. Cook, N. Almond, J. Bootman, E. Rud, P. Silvera, M. Dennis, T. Corcoran, J. Stott, and et al. 1995. Early suppression of SIV replication by CD8+ nef-specific cytotoxic T cells in vaccinated macaques. Nat. Med. 1:1167-1173.
Haigwood, N. L., A. Watson, W. F. Sutton, J. McClure, A. Lewis, J. Ranchalis, B. Travis, G. Voss, N. L. Letvin, S. L. Hu, V. M. Hirsch, and P. R. Johnson. 1996. Passive immune globulin therapy in the SIV/macaque model: early intervention can alter disease profile. Immunol. Lett. 51:107-114.
Hofmann-Lehmann, R., J. Vlasak, A. L. Williams, A. L. Chenine, H. M. McClure, D. C. Anderson, S. O'Neil, and R. M. Ruprecht. 2003. Live attenuated, nef-deleted SIV is pathogenic in most adult macaques after prolonged observation. AIDS 17:157-166.
Jin, X., D. E. Bauer, S. E. Tuttleton, S. Lewin, A. Gettie, J. Blanchard, C. E. Irwin, J. T. Safrit, J. Mittler, L. Weinberger, L. G. Kostrikis, L. Zhang, A. S. Perelson, and D. D. Ho. 1999. Dramatic rise in plasma viremia after CD8(+) T cell depletion in simian immunodeficiency virus-infected macaques. J. Exp. Med. 189:991-998.
Johnson, R. P. 2002. Mechanisms of protection against simian immunodeficiency virus infection. Vaccine 20:1985-1987.
Johnson, R. P., R. L. Glickman, J. Q. Yang, A. Kaur, J. T. Dion, M. J. Mulligan, and R. C. Desrosiers. 1997. Induction of vigorous cytotoxic T-lymphocyte responses by live attenuated simian immunodeficiency virus. J. Virol. 71:7711-7718.
Johnson, R. P., J. D. Lifson, S. C. Czajak, K. S. Cole, K. H. Manson, R. Glickman, J. Yang, D. C. Montefiori, R. Montelaro, M. S. Wyand, and R. C. Desrosiers. 1999. Highly attenuated vaccine strains of simian immunodeficiency virus protect against vaginal challenge: inverse relationship of degree of protection with level of attenuation. J. Virol. 73:4952-4961.
Kent, K. A., P. Kitchin, K. H. Mills, M. Page, F. Taffs, T. Corcoran, P. Silvera, B. Flanagan, C. Powell, J. Rose, et al. 1994. Passive immunization of cynomolgus macaques with immune sera or a pool of neutralizing monoclonal antibodies failed to protect against challenge with SIVmac251. AIDS Res. Hum. Retrovir. 10:189-194.
Lifson, J. D., J. L. Rossio, M. Piatak, Jr., T. Parks, L. Li, R. Kiser, V. Coalter, B. Fisher, B. M. Flynn, S. Czajak, V. M. Hirsch, K. A. Reimann, J. E. Schmitz, J. Ghrayeb, N. Bischofberger, M. A. Nowak, R. C. Desrosiers, and D. Wodarz. 2001. Role of CD8+ lymphocytes in control of simian immunodeficiency virus infection and resistance to rechallenge after transient early antiretroviral treatment. J. Virol. 75:10187-10199.
Lohman, B. L., M. B. McChesney, C. J. Miller, E. McGowan, S. M. Joye, K. K. Van Rompay, E. Reay, L. Antipa, N. C. Pedersen, and M. L. Marthas. 1994. A partially attenuated simian immunodeficiency virus induces host immunity that correlates with resistance to pathogenic virus challenge. J. Virol. 68:7021-7029.
Metzner, K. J., X. Jin, F. V. Lee, A. Gettie, D. E. Bauer, M. Di Mascio, A. S. Perelson, P. A. Marx, D. D. Ho, L. G. Kostrikis, and R. I. Connor. 2000. Effects of in vivo CD8(+) T cell depletion on virus replication in rhesus macaques immunized with a live, attenuated simian immunodeficiency virus vaccine. J. Exp. Med. 191:1921-1931.
Nilsson, C., B. Makitalo, R. Thorstensson, S. Norley, D. Binninger-Schinzel, M. Cranage, E. Rud, G. Biberfeld, and P. Putkonen. 1998. Live attenuated simian immunodeficiency virus (SIV)mac in macaques can induce protection against mucosal infection with SIVsm. AIDS 12:2261-2270.
Nixon, D. F., S. M. Donahoe, W. M. Kakimoto, R. V. Samuel, K. J. Metzner, A. Gettie, T. Hanke, P. A. Marx, and R. I. Connor. 2000. Simian immunodeficiency virus-specific cytotoxic T lymphocytes and protection against challenge in rhesus macaques immunized with a live attenuated simian immunodeficiency virus vaccine. Virology 266:203-210.
Putkonen, P., B. Makitalo, D. Bottiger, G. Biberfeld, and R. Thorstensson. 1997. Protection of human immunodeficiency virus type 2-exposed seronegative macaques from mucosal simian immunodeficiency virus transmission. J. Virol. 71:4981-4984.
Robertson, M. J., and J. Ritz. 1990. Biology and clinical relevance of human natural killer cells. Blood 76:2421-2438.
Rose, J., P. Silvera, B. Flanagan, P. Kitchin, and N. Almond. 1995. The development of PCR based assays for the detection and differentiation of simian immunodeficiency virus in vivo. J. Virol. Methods 51:229-239.
Rud, E. W., M. Cranage, J. Yon, J. Quirk, L. Ogilvie, N. Cook, S. Webster, M. Dennis, and B. E. Clarke. 1994. Molecular and biological characterization of simian immunodeficiency virus macaque strain 32H proviral clones containing nef size variants. J. Gen. Virol. 75:529-543.
Rud, E. W., L. Ogilvie, B. E. Clarke, N. Almond, K. Kent, L. Chan, M. Page, P. Kitchin, E. J. Stott, N. Cook, S. Sharpe, T. Ashwork, M. Dennis, G. Hall, and M. Cranage. 1994. A naturally attenuated SIVmac32H vaccine or viral interference? p. 217-223. In F. Brown, R. M. Chanock, H. S. Ginsberg, and E. Norrby (ed.), Vaccines 94: modern approaches to new vaccines including prevention of AIDS. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Ruprecht, R. M. 1999. Live attenuated AIDS viruses as vaccines: promise or peril? Immunol. Rev. 170:135-149.
Sawai, E. T., M. S. Hamza, M. Ye, K. E. Shaw, and P. A. Luciw. 2000. Pathogenic conversion of live attenuated simian immunodeficiency virus vaccines is associated with expression of truncated Nef. J. Virol. 74:2038-2045.
Schmitz, J. E., R. P. Johnson, H. M. McClure, K. H. Manson, M. S. Wyand, M. J. Kuroda, M. A. Lifton, R. S. Khunkhun, K. J. McEvers, J. Gillis, M. Piatak, J. D. Lifson, G. Grosschupff, P. Racz, K. Tenner-Racz, E. P. Rieber, K. Kuus-Reichel, R. S. Gelman, N. L. Letvin, D. C. Montefiori, R. M. Ruprecht, R. C. Desrosiers, and K. A. Reimann. 2005. Effect of CD8+ lymphocyte depletion on virus containment after simian immunodeficiency virus SIVmac251 challenge of live attenuated SIVmac2393-vaccinated rhesus macaques. J. Virol. 79:8131-8141.
Schmitz, J. E., M. J. Kuroda, S. Santra, V. G. Sasseville, M. A. Simon, M. A. Lifton, P. Racz, K. Tenner-Racz, M. Dalesandro, B. J. Scallon, J. Ghrayeb, M. A. Forman, D. C. Montefiori, E. P. Rieber, N. L. Letvin, and K. A. Reimann. 1999. Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes. Science 283:857-860.
Schmitz, J. E., M. J. Kuroda, S. Santra, M. A. Simon, M. A. Lifton, W. Lin, R. Khunkhun, M. Piatak, J. D. Lifson, G. Grosschupff, R. S. Gelman, P. Racz, K. Tenner-Racz, K. A. Mansfield, N. L. Letvin, D. C. Montefiori, and K. A. Reimann. 2003. Effect of humoral immune responses on controlling viremia during primary infection of rhesus monkeys with simian immunodeficiency virus. J. Virol. 77:2165-2173.
Schmitz, J. E., M. A. Simon, M. J. Kuroda, M. A. Lifton, M. W. Ollert, C. W. Vogel, P. Racz, K. Tenner-Racz, B. J. Scallon, M. Dalesandro, J. Ghrayeb, E. P. Rieber, V. G. Sasseville, and K. A. Reimann. 1999. A nonhuman primate model for the selective elimination of CD8+ lymphocytes using a mouse-human chimeric monoclonal antibody. Am. J. Pathol. 154:1923-1932.
Sharpe, S. A., A. Cope, S. Dowall, N. Berry, C. Ham, J. L. Heeney, D. Hopkins, L. Easterbrook, M. Dennis, N. Almond, and M. Cranage. 2004. Macaques infected long-term with attenuated simian immunodeficiency virus (SIVmac) remain resistant to wild-type challenge, despite declining cytotoxic T lymphocyte responses to an immunodominant epitope. J. Gen. Virol. 85:2591-2602.
Sharpe, S. A., A. M. Whatmore, G. A. Hall, and M. P. Cranage. 1997. Macaques infected with attenuated simian immunodeficiency virus resist superinfection with virulence-revertant virus. J. Gen. Virol. 78:1923-1927.
Shibata, R., C. Siemon, S. C. Czajak, R. C. Desrosiers, and M. A. Martin. 1997. Live, attenuated simian immunodeficiency virus vaccines elicit potent resistance against a challenge with a human immunodeficiency virus type 1 chimeric virus. J. Virol. 71:8141-8148.
Silvera, P., B. Flanagan, K. Kent, E. Rud, C. Powell, T. Corcoran, C. Bruck, C. Thiriart, N. L. Haigwood, and E. J. Stott. 1994. Fine analysis of humoral antibody response to envelope glycoprotein of SIV in infected and vaccinated macaques. AIDS Res. Hum. Retrovir. 10:1295-1304.
Silvera, P., A. Wade-Evans, E. Rud, R. Hull, K. Silvera, R. Sangster, N. Almond, and J. Stott. 2001. Mechanisms of protection induced by live attenuated simian immunodeficiency virus. III. Viral interference and the role of CD8+ T-cells and beta-chemokines in the inhibition of virus infection of PBMCs in vitro. J. Med. Primatol. 30:1-13.
Stahl-Hennig, C., U. Dittmer, T. Nisslein, H. Petry, E. Jurkiewicz, D. Fuchs, H. Wachter, K. Matz-Rensing, E. M. Kuhn, F. J. Kaup, E. W. Rud, and G. Hunsmann. 1996. Rapid development of vaccine protection in macaques by live-attenuated simian immunodeficiency virus. J. Gen. Virol. 77:2969-2981.
Stebbings, R., N. M. Almond, E. J. Stott, N. Berry, A. M. Wade-Evans, R. Hull, J. Lines, P. Silvera, R. Sangster, T. Corcoran, J. Rose, and K. B. Walker. 2002. Mechanisms of protection induced by attenuated simian immunodeficiency virus. V. No evidence for lymphocyte-regulated cytokine responses upon rechallenge. Virology 296:338-353.
Stebbings, R., N. Berry, J. Stott, R. Hull, B. Walker, J. Lines, W. Elsley, S. Brown, A. Wade-Evans, G. Davis, J. Cowie, M. Sethi, and N. Almond. 2004. Vaccination with live attenuated simian immunodeficiency virus for 21 days protects against superinfection. Virology 330:249-260.
Stebbings, R., J. Stott, N. Almond, R. Hull, J. Lines, P. Silvera, R. Sangster, T. Corcoran, J. Rose, S. Cobbold, F. Gotch, A. McMichael, and B. Walker. 1998. Mechanisms of protection induced by attenuated simian immunodeficiency virus. II. Lymphocyte depletion does not abrogate protection. AIDS Res. Hum. Retrovir. 14:1187-1198.
Stott, E. J., W. L. Chan, K. H. Mills, M. Page, F. Taffs, M. Cranage, P. Greenaway, and P. Kitchin. 1990. Preliminary report: protection of cynomolgus macaques against simian immunodeficiency virus by fixed infected-cell vaccine. Lancet 336:1538-1541.
Tenner-Racz, K., C. S. Hennig, K. Uberla, H. Stoiber, R. Ignatius, J. Heeney, R. M. Steinman, and P. Racz. 2004. Early protection against pathogenic virus infection at a mucosal challenge site after vaccination with attenuated simian immunodeficiency virus. Proc. Natl. Acad. Sci. USA 101:3017-3022.
Vogel, T. U., B. E. Beer, J. zur Megede, H. G. Ihlenfeldt, G. Jung, S. Holzammer, D. I. Watkins, J. D. Altman, R. Kurth, and S. Norley. 2002. Induction of anti-simian immunodeficiency virus cellular and humoral immune responses in rhesus macaques by peptide immunogens: correlation of CTL activity and reduction of cell-associated but not plasma virus load following challenge. J. Gen. Virol. 83:81-91.
Vogel, T. U., J. Fournier, A. Sherring, D. Ko, M. Parenteau, D. Bogdanovic, J. Mihowich, and E. W. Rud. 1998. Presence of circulating CTL induced by infection with wild-type or attenuated SIV and their correlation with protection from pathogenic SHIV challenge. J. Med. Primatol. 27:65-72.
Whatmore, A. M., N. Cook, G. A. Hall, S. Sharpe, E. W. Rud, and M. P. Cranage. 1995. Repair and evolution of nef in vivo modulates simian immunodeficiency virus virulence. J. Virol. 69:5117-5123.
Wyand, M. S., K. Manson, D. C. Montefiori, J. D. Lifson, R. P. Johnson, and R. C. Desrosiers. 1999. Protection by live, attenuated simian immunodeficiency virus against heterologous challenge. J. Virol. 73:8356-8363.
Wyand, M. S., K. H. Manson, M. Garcia-Moll, D. Montefiori, and R. C. Desrosiers. 1996. Vaccine protection by a triple deletion mutant of simian immunodeficiency virus. J. Virol. 70:3724-3733.(Richard Stebbings, Neil B)