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

Sally A. Sharpe1, Alethea Cope1,{dagger}, Stuart Dowall1, Neil Berry2, Claire Ham2, Jonathan L. Heeney3, Donna Hopkins1, Linda Easterbrook1, Mike Dennis1, Neil Almond2 and Martin Cranage4

1 Health Protection Agency, Porton Down, Salisbury SP4 0JG, UK
2 Division of Retrovirology, National Institute for Biological Standards and Control, South Mimms, Herts EN6 3QG, UK
3 Department of Virology, Biomedical Primate Research Centre, Rijswijk, the Netherlands
4 Department of Cellular and Molecular Medicine, St George's Hospital Medical School, London SW17 0RE, UK

Correspondence
Sally A. Sharpe
sally.sharpe{at}hpa.org.uk


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
To further investigate mechanisms of protective immunity that are induced by live, attenuated simian immunodeficiency virus (SIV), three macaques were infected with SIVmacGX2, a nef-disrupted molecular clone. In two of these animals, which expressed the MamuA*01 major histocompatibility complex class I allele, loss of functional activity against an SIV-Gag-encoded immunodominant cytotoxic T lymphocyte (CTL) epitope was observed following prolonged infection. Nonetheless, all three animals were resistant to challenge with an uncloned pool of wild-type SIVmac, whereas four naïve controls became infected. Tetramer staining revealed the rapid generation of CD8+ T-cell responses against gag- and tat-encoded immunodominant epitopes in MamuA*01+ challenge controls. The dynamics of these T-cell responses to the wild-type virus were similar to those observed following primary infection of the vaccine group with attenuated virus. In contrast, neither tetramer staining nor gamma interferon ELISpot assay revealed an immediate, systemic, anamnestic response in the wild-type-challenged, attenuated SIV-infected animals. Functional CTL capacity had not been lost in this group, as lytic activity was still evident 17 weeks after challenge. Both attenuated and wild-type viruses induced a disseminated CD8+ T-cell response, which was of a higher magnitude in lymphoid tissues than in the periphery. These results suggest that, at least as measured in the periphery, protection against wild-type infection that is induced by live, attenuated SIV is not dependent on a rechallenge-driven expansion of immunodominant epitope-specific CD8+ T cells and, therefore, pre-existing activity may be sufficient to prevent superinfection.

{dagger}Present address: Department of Cellular and Molecular Medicine, St George's Hospital Medical School, London SW17 0RE, UK.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Live, attenuated virus vaccines confer potent and durable protection against infection with a range of pathogenic viruses by the induction of broad-based immune responses. It has been shown that macaques infected with strains of simian immunodeficiency virus (SIV) that has been attenuated through deletions in the nef gene are resistant to challenge with wild-type virus (Daniel et al., 1992; Lohman et al., 1994; Wyand et al., 1996), enabling this system to be used as a model for the development of vaccines for the prevention of human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS). It is unlikely, however, that live, attenuated strains of HIV will be used as vaccines in humans, due to safety concerns. There is an incomplete understanding of immunodeficiency lentivirus pathogenicity and these viruses have the capacity to revert to a virulent phenotype (Whatmore et al., 1995; Baba et al., 1995, 1999; Sawai et al., 2000; Hofmann-Lehmann et al., 2003). Nonetheless, the simian model offers the potential to reveal insights into mechanisms of protection (Johnson & Desrosiers, 1998; Almond & Stott, 1999).

Despite many studies of live, attenuated SIV vaccines, correlates of protective immunity remain to be defined. It is unlikely that antibodies play a major role, as virus-neutralizing antibodies do not correlate with protection (Norley et al., 1996; Connor et al., 1998; Gundlach et al., 1998; Nilsson et al., 1998); animals that are infected with attenuated SIV can be protected against challenge with HIV-1 envelope-expressing SIV chimeric viruses (Stott et al., 1994; Bogers et al., 1995) and protection cannot be transferred with immune serum (Almond et al., 1997). More recently, attention has focused on the role of cytotoxic T lymphocytes (CTLs). Live, attenuated SIV vaccines have been shown to generate circulating (Dittmer et al., 1995; Johnson et al., 1997) and disseminated (Cranage et al., 1997) CTL responses, and depletion of CD8+ T cells in macaques that are infected with attenuated SIV has been associated with an increase in viraemia (Metzner et al., 2000). Circumstantial evidence has been used to support the notion that SIV-specific CTLs have a role in live, attenuated vaccine-induced protection from wild-type challenge, but this inference remains controversial. Stebbings et al. (1998) were unable to abrogate the protective effect by depletion of CD8+ T lymphocytes; however, depletion of cells in lymph nodes was only partial. Furthermore, depletion of specific cell populations perturbs the overall balance of T-cell populations, including targets for virus infection, further complicating the interpretation of results.

In previous studies of live, attenuated vaccine protection, we have used the SIVmac32H C8 molecular clone, which has a 12 bp deletion in the nef/3' long terminal repeat that is associated with pronounced attenuation of replication in rhesus macaques (Rud et al., 1994). This virus has been shown to induce potent protection, as evidenced by resistance against intravenous challenge with cell-free and cell-associated SIV (Almond et al., 1995), as well as against rectal mucosal challenge with both homologous and heterologous virus strains (Cranage et al., 1997; Nilsson et al., 1998). However, the small size of the attenuating lesion makes this virus susceptible to reversion to virulence by genetic repair in vivo (Whatmore et al., 1995). Therefore, in the present study, we have utilized a newly generated molecular clone, designated SIVmacGX2, which is based on an SIVmacJ5 backbone from which a 66 bp fragment in the nef unique region has been removed, a deletion that has been observed in a sequence recovered from an SIVmacJ5-infected macaque.

To further investigate the mechanism of vaccine-induced superinfection resistance, three rhesus macaques were infected with SIVmacGX2. Two of the macaques expressed the MamuA*01 class I major histocompatibility complex (MHC) allele that restricts immunodominant epitopes in the Gag (Gag181–189; CM9) (Allen et al., 1998) and Tat (Tat28–35; SL8) (Allen et al., 2000) proteins of SIVmac, allowing quantitative estimations of CTL epitope-specific CD8+ T-cell frequencies by staining with MHC-tetramer–peptide complexes and gamma interferon (IFN-{gamma}) enzyme-linked immunospot (ELISpot) assay. A decline in CTL activity against the Gag-CM9 epitope in animals that had been infected long-term with SIVmacGX2, when virus was essentially undetectable in the peripheral circulation, afforded the opportunity to determine whether the macaques remained resistant to wild-type challenge and whether this exposure induced an anamnestic CTL response. Any response detected could be compared with that induced by primary infection in previously naïve, MamuA*01+ macaques, following challenge with wild-type virus.


   METHODS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Animals and virus stocks.
UK captive-bred rhesus macaques of Indian origin were housed according to the Code of Practice of the UK Home Office (1989). Primates were sedated with ketamine hydrochloride prior to inoculation with virus and/or venepuncture and were killed humanely by an overdose of anaesthetic. Animals with the MamuA*01 MHC class I allele were selected by sequence-specific primer PCR and direct sequencing.

Derivation of the SIVmacGX2 molecular clone will be described in detail elsewhere. Briefly, an EcoRI–NdeI fragment of an SIVmacJ5 proviral clone was replaced with a PCR product that was amplified from proviral DNA isolated from an SIVmacJ5-infected macaque. This resulted in a 66 bp deletion in nef, removing the coding sequence for aa 62–83. A pool of virus, designated 8/99, was prepared from the supernatant of virus-infected CEMX174 cells. The titre in vitro was determined to be 104·2 TCID50 ml–1 on C8166 cells and the Gag p27 concentration of the virus stock was 103·5 ng ml–1, as determined by ELISA (Coulter). Primates were infected by atraumatic, intrarectal instillation of 50 000 TCID50 8/99 pool in a volume of 3 ml.

To determine superinfection-resistance status, animals that had previously been infected with SIVmacGX2 and naïve controls were challenged intravenously with 30 TCID50 SIVmac220, pool 6/94, a virus swarm that was derived directly from the spleen of an SIVmacJ5-infected macaque, as described previously (Polyanskaya et al., 1997).

Mononuclear cell (MNC) fractionation, virus isolation and cell-associated virus loads.
MNCs were obtained from peripheral blood and lymphoid tissues. In brief, for tissues, samples of approximately 2 cm2 were cut into small pieces and suspended in 1 ml RPMI medium that was supplemented with L-glutamine (2 mM), penicillin (50 U ml–1) and streptomycin (50 µg ml–1). Cell suspensions were made by placing the tissue suspension into a sterile Medicon, followed by maceration for 1 min in a Medimachine (both from Becton Dickinson). Ficoll density gradient separation was performed to obtain MNCs from tissues and heparinized whole blood. Cells were either used directly for virus isolation and IFN-{gamma} ELISpot assay or were cryo-preserved in 10 % (v/v) DMSO/FCS (fetal calf serum).

Virus isolation was performed by co-cultivation with C8166 cells, either at a single concentration of 106 MNCs or by limiting dilution to determine cell-associated virus load, as described previously (Cranage et al., 1998). Immunofluorescent staining with the monoclonal anti-Nef antibody KK75, which binds only to the intact Nef protein that is present in the wild-type challenge virus (Arnold et al., 1999), was used to distinguish challenge virus from attenuated virus.

Differential PCR analysis of SIV nef proviral DNA.
Amplification of SIV nef sequences from DNA extracted from tissues and/or peripheral blood mononuclear cells (PBMCs) was performed by using nested PCR primers. Outer primers in the forward (5'-ATGGGTGGAGCTATTTCCAGGA-3') and reverse (5'-TCAGCGAGTTTCCTTCTTGTCA-3') directions were followed with inner primers in the forward (5'-GCAATCCCTAGGAGGATT-3') and reverse (5'-GCCTGACTTGCTTCCAAA-3') directions. Initial comparisons were made according to migration on 2 % agarose gels, relating to the 66 bp size difference in PCR amplicons. To determine the specific presence of SIVmac220, secondary amplifications were performed with the primer 5'-GAATACTCCATGGAGAAACC-3', which is located in the forward direction in the corresponding region of the deletion in GX2 with the type-common reverse primer described above. The SIV nef assays were demonstrated to detect low-threshold target by limiting dilution analysis of plasmid DNA. SIV gag sequences were amplified from samples in parallel, in order to confirm the presence of SIV DNA, by using primer sequences that were described previously (Clarke et al., 2003).

Plasma vRNA.
Plasma vRNA levels were determined by quantitative competitive (qc) RT-PCR with a cut-off sensitivity of 40 RNA equivalents ml–1 (Ten Haaft et al., 1998).

Assay of humoral immune responses.
Serum-binding antibodies against SIV proteins were determined by using a standard ELISA. Wells were coated with recombinant SIV p27 or SIV gp130 (MRC Centralised Facility for AIDS Reagents, EVA643, EVA655).

Assay of SIV epitope-specific CTL activity.
CTL activity was determined essentially as described previously (Sharpe et al., 2003), following bulk culture restimulation in vitro with either Gag-CM9 (CTPYDINQM) or Tat-SL8 (TTPESANL) synthetic peptides at 20 µg ml–1. Briefly, precursor CTLs were restimulated in vitro on day 7 by the addition of mitomycin C-inactivated, peptide-pulsed, autologous B-lymphoblastoid cells (B-LCLs). At day 13, effector cells were used in triplicate at various effector : target (E : T) ratios in a standard chromium-release assay. Autologous B-LCL targets were pulsed with Gag or Tat peptides and labelled with 150 µCi 51Cr for 2 h. Chromium release was determined in the supernatants after 4 h. Spontaneous and total release was determined from wells that contained target cells and medium alone or with 5 % Triton X-100. Responses were considered to be positive if there was >=10 % specific lysis above control targets.

Analysis of specific CD8+ T-cell prevalence by staining with tetrameric MHC–peptide complexes.
Cryopreserved PBMCs and tissue MNCs were thawed rapidly at 37 °C and washed three times in FACS buffer (2 % FCS/PBS, 0·01 % sodium azide). The cell concentration was adjusted to 2x106 MNCs in 50 µl FACS buffer. Cells were pre-incubated (after initial titration of the tetramers) with 50 µl of either phycoerythrin (PE)-conjugated Gag-CM9 or Tat-SL8 tetramer (kindly provided by the NIH Tetramer Core facility, USA) at 4 °C for 30 min. Surface labelling of cells with CD3–FITC (fluorescein isothiocyanate) and CD8–PerCP (Becton Dickinson) was performed for a further 30 min at room temperature in the dark. Excess unlabelled antibodies were removed from the cells by washing three times in FACS buffer. Cells were fixed in 2 % paraformaldehyde in PBS and analysed by using a single 488 nm argon laser flow cytometer (Epics XL.MCL; Beckman Coulter). Compensation settings were made by using directly labelled IgG isotype controls (Becton Dickinson) and single-stained samples for all channels. Analysis was performed by gating on CD3–FITC-positive lymphocytes, followed by bi-variate dot-plot analysis of CD8–PerCP-positive, tetramer–PE positive cells; a minimum of 50 000 events were collected per sample.

Analysis of specific CD8+ T-cell prevalence by IFN-{gamma} ELISpot assay.
The frequency of epitope-specific CD8+ T cells was enumerated directly ex vivo by IFN-{gamma} ELISpot assay. The assay was carried out by using PVDF multiscreen plates (Millipore), GZ-4 anti-IFN-{gamma} capture antibody and biotinylated 7-B6-1 detector antibody, according to the manufacturer's instructions (MabTech). Synthetic peptides for epitopes Gag-CM9 and Tat-SL8 were used at 1 µg per well. Medium alone was used as a negative control and 50 ng phorbol 12-myristate ml–1+1 µg ionomycin ml–1 as a positive control. Cells were tested at 2x105 and 1x105 cells per well in triplicate and incubated undisturbed at 37 °C in 5 % CO2 for 16 h. Spots were counted under low magnification by using a dissecting microscope equipped with a vertical white light and are shown as mean values.


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Replication of nef-disrupted SIVmacGX2 in rhesus macaques
Three rhesus macaques (X59, 398 and 440) were inoculated intrarectally with 50 000 TCID50 SIVmacGX2 and monitored for circulating virus load by virus isolation from PBMCs, using limiting dilution and plasma vRNA determined by qc RT-PCR. Virus was recovered from the PBMCs of all animals 1–2 weeks after inoculation (Fig. 1). Cell-associated virus loads peaked at week 2 in all animals, whereas macaque 398 had a delayed peak of plasma vRNA at week 4. Cell-associated virus loads declined rapidly between weeks 2 and 6 and by week 16, virus could not be recovered from any of the animals. Likewise, plasma vRNA levels declined rapidly after the early burst of virus replication and were undetectable by week 15 in animal X59. Taken together, these data show that replication of SIVmacGX2 is attenuated in vivo.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 1. Virus load following infection of macaques X59 ({bullet}), 398 ({blacktriangledown}) and 440 ({blacksquare}) with nef-disrupted SIVmacGX2. (a) Cell-associated load as determined by limiting dilution co-cultivation of PBMCs with C8166 cells. (b) Plasma vRNA load as determined by qc RT-PCR.

 
Peripheral T-cell responses in SIVmacGX2-infected macaques before wild-type challenge
Functional lytic responses against the immunodominant, MamuA*01-restricted Gag-CM9 epitope were studied over time in animals X59 and 398, following restimulation in vitro (Fig. 2). Following the primary response, activity was maintained in animal X59 for approximately 50 weeks and thereafter showed a progressive decline, being undetectable prior to wild-type challenge. Animal 398 had a biphasic early response and also lost functional activity by the time of challenge. Results are shown for an E : T ratio of 100 : 1, but were typical of those seen over a range of cell ratios. Lytic responses to another MamuA*01-restricted immunodominant CTL epitope, Tat-SL8, were measured only following long-term infection with SIVmacGX2. Animal X59 had activity on both occasions tested [at weeks 60 and 100 (38 and 56 % specific activity, respectively, at 100 : 1)]. Animal 398 had transient peaks of reactivity (data not shown), but on three occasions tested prior to challenge (weeks 76, 80 and 82), activity was absent.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 2. Longitudinal analysis of CTL responses as measured by lytic activity against Gag-CM9 ({bullet}) in SIVmacGX2-infected, MamuA*01+ animals X59 (a) and 398 (c), compared with the frequency of CD3+ CD8+ T cells with specificity to Gag-CM9 and Tat-SL8 ({circ}) in animals X59 (b) and 398 (d), as measured by tetramer staining.

 
Staining of CD3+ CD8+ PBMCs with epitope-specific MHC tetramers showed that both animals raised an early response to both Tat-SL8 and Gag-CM9 epitopes, with a strong dominance to Tat-SL8 (Fig. 2). The SL8 tetramer response peaked at 4 weeks in both animals, concomitant with the peak and downturn in virus load (Fig. 1). Gag-CM9 tetramer staining reached a low plateau level in animal X59, which was maintained over time with only minor fluctuations, despite the loss in functional CTL activity. A peak in CM9 tetramer frequency was seen 12 weeks after infection of animal 398. Interestingly, this corresponded with the second peak of lytic activity. Furthermore, animal 398 had a delayed and extended peak of plasma vRNA, but subsequently showed good control of virus load (Fig. 1). The frequency of CM9-tetramer-staining cells predominated over SL8+ cells after the initial response, despite the decline in functional activity (Fig. 2).

Wild-type challenge of macaques infected long-term with attenuated SIVmacGX2
Following long-term infection (122 weeks for animal X59 and 89 weeks for animals 398 and 440) and at a time when CTL responses to Gag had declined in the two MamuA*01+ animals, all three animals, together with four naïve macaques (C12, C81, C25 and C48), were challenged intravenously with 30 MID50 of uncloned SIVmac220 as a representative wild-type challenge.

Prior to challenge, virus could not be recovered from the PBMCs of the SIVmacGX2-infected macaques, although proviral gag was detected regularly by PCR from animals 398 and 440 and, on one occasion only, from animal X59. PCR for nef confirmed the presence of SIVmacGX2. Furthermore, SIVmacGX2 was isolated from biopsies of inguinal lymph nodes from animals 398 and 440. Challenge-control animals were shown to be free of SIV by virus isolation and PCR (Table 1). Virus was recovered regularly from all control animals following challenge; immunofluorescent staining and PCR for proviral DNA confirmed this to be SIVmac220. In contrast, long-term SIVmacGX2-infected animals remained largely virus isolation-negative following challenge, SIVmacGX2 being isolated from one animal (440) on one occasion only. Furthermore, where proviral DNA was detected by PCR in PBMCs from these animals, amplification for nef showed specificity for SIVmacGX2.


View this table:
[in this window]
[in a new window]
 
Table 1. Detection of SIV in PBMCs before and following challenge of macaques with wild-type SIVmac220

g-PCR, Detection of proviral gag DNA; Ing ln, inguinal lymph nodes; ND, not determined; n-PCR, detection of proviral nef DNA; VI, virus isolated.

 
All naïve, challenged animals seroconverted (data not shown), but animals that had been infected long-term with SIVmacGX2 failed to demonstrate anamnestic antibody responses to SIV Gag p27 and Env gp130 following challenge (Fig. 3). A gradual increase in antibody titres was seen in animal 398; however, this was not characteristic of a secondary recall response. Interestingly, antibody titres to gp130 were maintained at high levels, before and after wild-type challenge, in all SIVmacGX2 long-term-infected animals, despite low circulating virus loads, whereas anti-p27 titres were relatively low.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3. Serum antibodies against SIV Gag p27 ({bullet}) and SIV Env gp130 ({blacktriangledown}) in macaques infected with attenuated SIVmacGX2, before and following wild-type challenge with SIVmac220. Animals: (a) X59; (b) 398; (c) 440.

 
Plasma vRNA remained below the limit of detection in animals X59 and 440 following challenge, but a transient low level of vRNA was detected in animal 398 (Fig. 4). Despite this, only SIVmacGX2 proviral DNA was detected and virus could not be isolated from PBMCs. Furthermore, analysis of day 56 plasma for nef-specific sequences following amplification by RT-PCR indicated the viraemic spike to be due to the reappearance of the vaccine strain, SIVmacGX2, and not to the breakthrough of SIVmac220 challenge. All control animals developed a rapid rise in vRNA following challenge, although in two animals (C14 and C46), loads 2 weeks after infection were no higher than those seen in SIVmacGX2-infected macaques following primary infection (Fig. 1). However, unlike SIVmacGX2-infected animals, loads in two animals persisted at a high level; in fact, rather unusually, animal C46, despite not showing an early peak in plasma vRNA, had a continuing increase over time. The profile of virus replication was not associated with expression of the MamuA*01 allele, as of the two animals with this MHC type (C33 and C45), only C45 displayed good control of circulating virus burden, and the two animals with relatively low loads at 2–4 weeks after challenge did not express this allele.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4. Plasma vRNA load as measured by qc RT-PCR immediately before and following challenge with SIVmac220. Animals X59 ({circ}), 398 ({triangledown}) and 440 ({square}) were infected long-term with SIVmacGX2 prior to challenge and animals C45 ({blacklozenge}), C33 ({blacktriangleup}), C14 ({blacksquare}) and C46 ({bullet}) were previously naïve.

 
Post-mortem analysis of spleen, inguinal, mesenteric and axillary lymph nodes and external, internal and common iliac lymph nodes failed to reveal, either by virus isolation or detection of proviral DNA, sequestration of SIVmac220 in animals that were previously infected with SIVmacGX2. Virus could not be recovered from any of the lymphoid tissues of animal X59, although SIVmac GX2-specific proviral DNA was detected in mesenteric and axillary lymph nodes. SIVmacGX2 was recovered from the inguinal and axillary lymph nodes of animals 398 and 440, from the mesenteric and common iliac lymph nodes of animal 398 and the external and internal iliac lymph nodes of animal 440.

Peripheral T-cell responses in naïve control and SIVmacGX2-infected macaques following challenge with SIVmac220
Six weeks after challenge with SIVmac220, lytic activity against Gag-CM9 was detected in both MamuA*01+ control animals. At 8 weeks after challenge, Tat-SL8-specific activity was detected in animal C33, but not in C45. By week 17, when activity against both epitopes was measured, only Tat-SL8-specific activity was detected in both animals. In contrast, lytic activity against target cells pulsed with these peptides was not detected 6 or 10 weeks after challenge of the long-term SIVmacGX2-infected MamuA*01+ macaques. Surprisingly, however, lytic activity against both epitopes was detected 18 weeks after wild-type challenge, albeit at a low level in animal 398 against Gag (30 and 55 % specific lysis for Gag and Tat in animal X59 and 11 and 32 % for Gag and Tat in animal 398, all at an E : T ratio of 100 : 1).

Quantitative determinations of circulating CTL epitope-specific responses were determined by IFN-{gamma} ELISpot assay and tetramer staining in the MamuA*01+ animals (Fig. 5). Previously naïve animals (C33 and C45) mounted rapid responses with both assays, showing dominance in the frequency of Tat-SL8 reactive cells, which peaked 4 weeks after challenge. Subsequently, Gag-CM9-reactive cells predominated in both animals. Despite the lower magnitude of response in animal C45, both animals had similar peak vRNA loads and C45 demonstrated better control of plasma vRNA load (Fig. 3). In general, the frequency of reactive T cells showed good concordance between the two assays.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 5. Longitudinal analysis of the frequency of SIV CTL epitope-specific T cells, assayed by enumeration of tetramer staining [Gag-CM9 ({bullet}) and Tat-SL8 ({circ})] and IFN-{gamma} spot-forming cells [Flu (open bars), Gag-CM9 (hatched bars) and Tat-SL8 (filled bars)] in MamuA*01+ macaques before and after challenge with wild-type SIVmac220. Animals X59 (a) and 398 (c) were infected long-term with attenuated SIVmacGX2 and animals C33 (b) and C45 (d) were naïve prior to challenge.

 
In contrast to the results seen in the previously naïve animals, SIVmacGX2-infected animals showed no significant increase in the frequencies of specific T cells during the first 4 weeks following challenge with SIVmac220. Indeed, the frequency of CM9- and SL8-specific cells detected by ELISpot assay had declined by 2 weeks after challenge in animal X59. Likewise, low frequencies of spot-forming cells were detected in animal 398 at this time; however, the significance of this is uncertain, as frequencies were already low at the time of challenge in this animal. Between 4 and 18 weeks, the percentage of CD3+ CD8+ T cells that stained with epitope-specific tetramers was perturbed in both animals, with a peak of activity 12 weeks after challenge. By the time that lytic activity was detected, 18 weeks after challenge, tetramer percentages had returned to low levels. In contrast, cell frequencies as measured by ELISpot assay showed no consistent changes during this time period. Gag-CM9-specific cells were detected at the highest frequency by ELISpot assay in both of the animals; however, by tetramer staining, Tat-SL8-specific cells were detected at a slightly higher frequency than Gag-CM9-specific cells in animal X59. Interestingly, in this animal, which appeared to control infection with the attenuated virus extremely well, as evidenced by absence of virus isolation, the proportion of Tat-specific cells as measured by ELISpot assay was significantly higher than in animal 398 (P<0·001; Fisher's exact test).

Frequency of epitope-specific T cells in lymphoid tissues
An extensive, tissue-specific analysis of the frequency and distribution of specific CD8+ T cells in MamuA*01+ animals was possible post-mortem, 18 weeks after challenge with SIVmac220. Measurement of tetramer frequency showed that levels of specific cells were generally higher in tissues than in PBMCs, regardless of the infecting virus (Fig. 6). Furthermore, Gag-CM9-specific cells were present in the highest proportion. Cell frequencies were, in general, higher in animals that were infected short-term with SIVmac220, ranging from 1·9 to 5·4 % Gag-CM9-specific and 0·2 to 0·9 % Tat-SL8-specific in SIVmac220-infected animals and from 0·8 to 4 % Gag-CM9-specific and 0·1 to 0·7 % Tat-SL8-specific in long-term SIVmacGX2-infected animals. The frequency of tetramer-positive cells in any one tissue was not associated directly with either the ability to isolate virus or the detection of proviral DNA, although in three animals (X59, 398 and C45), virus could not be recovered from the tissues with the highest frequency of tetramer-positive CD8+ T cells.



View larger version (39K):
[in this window]
[in a new window]
 
Fig. 6. Distribution of SIV CTL epitope-specific CD8+ T cells in the blood and lymphoid tissues of macaques infected long-term with attenuated SIVmacGX2: animals X59 (a) and 398 (b); or short-term with wild-type SIVmac220: animals C45 (c) and C33 (d). Tetramer frequencies for Gag-CM9 (open bars) and Tat-SL8 (filled bars) are shown. ND, Not determined.

 

   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Infection of macaques with live, attenuated SIV leads to the generation of persistent and disseminated humoral and cellular immune responses. It is often concluded that these are responsible for the protective effect that is observed in vaccine–challenge experiments. However, defining the correlates of the protection that is afforded by attenuated SIV strains has proven difficult, not least due to the complex nature of the responses generated. Doubts about the involvement of antibody and T cell-specific immune responses in protection against challenge have been raised in previous studies that used the SIVmacC8 replication-attenuated molecular clone. Passive transfer of immune serum failed to protect cynomolgus macaque recipients against infection with challenge virus (Almond et al., 1997), depletion of CD8+ T cells failed to abrogate protection (Stebbings et al., 1998) and there was no evidence for lymphocyte-regulated cytokine responses following challenge of SIVmacC8-infected cynomolgus macaques with the cognate wild-type SIVmacJ5 molecular clone (Stebbings et al., 2002). We have extended and strengthened these observations by using a related attenuated virus, SIVmacGX2, in rhesus macaques, where we were able to make use of the well-defined, MamuA*01-restricted, SIV-specific immunodominant epitopes that are recognized by CD8+ T cells to determine whether responsiveness to these epitopes correlated to protection and whether anamnestic, specific T-cell responses were evident on challenge.

SIVmacGX2 clearly demonstrated an attenuated phenotype in rhesus macaques. The profile of cell-associated virus loads was similar to that reported previously for SIVmacC8 (Cranage et al., 1998) and virus was unrecoverable from the periphery by week 16, consistent with the low set point vRNA load of <40 and approximately 1000 RNA equivalents ml–1. A similar pattern was seen in a further five macaques that were infected similarly as part of another study (data not shown). The overall dynamics of infection were also similar to those reported for another attenuated virus, SIVmac239{Delta}nef (Connor et al., 1998), although direct quantitative comparison is precluded by differences in the detection assays that were used.

Infection with SIVmacGX2 generated an early CTL response that was concomitant with reduction in virus load, as has been seen in infection of macaques with attenuated SIVmac239{Delta}nef (Johnson et al., 1997), attenuated SIVmacC8 (S. A. Sharpe & M. P. Cranage, unpublished data), virulent SIVmac251 (Yasutomi et al., 1993) and virulent SIVmac239 (Allen et al., 2000), as well as in HIV-1-infected humans (Borrow et al., 1994; Koup et al., 1994). Analysis by tetramer staining confirmed the early response to the immunodominant Gag-CM9 epitope, as seen in the lytic assay, and also demonstrated a much larger early-peak response to the Tat-SL8 immunodominant epitope, as seen in acute infection of macaques with SIVmac239 (Allen et al., 2000). Interestingly, in the present study, the pattern and magnitude of early CD8 T-cell responses, as measured by tetramer staining, were similar in macaques that were infected either with attenuated SIVmacGX2 or with wild-type SIVmac220, probably reflecting the similarity in early replication in vivo, as detected by plasma vRNA loads.

Despite persistence of Gag-specific CD8+ T cells (detected by tetramer staining) in both MamuA*01+ and SIVmacGX2 persistently infected animals, lytic activity to the CM9 epitope declined. Loss of CTL activity may be linked to the lack of antigenic stimulation that occurs in long-term infected animals that do not progress to disease (Geretti et al., 1999). Loss of CD8 functional activity has also been reported in chronic infection with HIV (Rinaldo et al., 2000; Kostense et al., 2001) and SIV (McKay et al., 2002) that is associated with clinical progression. The decreased long-term memory for CTLs with functional capacity may be linked to the levels of interleukin 2 (IL2) that are present during the priming phase. Although we did not measure IL2 capacity in the present study with SIVmacGX2, infection with attenuated SIVmacC8, a closely related virus, as well as infection with wild-type virus, have been associated with a profound acute-phase loss of IL2+ lymphocytes in the absence of significant loss of CD4+ T lymphocytes (Stebbings et al., 2002).

Although there was evidence of waning humoral and cellular immune responses in macaques that were infected long-term with SIVmacGX2, these animals appeared to be protected completely against superinfection with SIVmac220. No anamnestic antibody responses were seen, even though anti-SIVp27 antibody titres had declined to relatively low levels prior to challenge. Anti-Env antibody titres were maintained at relatively high levels before and after wild-type challenge. Likewise, CD8+ T-cell responses appeared to be unaffected in the period immediately after challenge, suggesting a lack of recall of cell-mediated immunity characterized by a massive proliferation of antigen-specific CD8+ T lymphocytes (Flynn et al., 1999). Not only was Gag-CM9-specific CTL reactivity undetectable in both MamuA*01+ animals following challenge, but so also was reactivity to Tat-SL8, even in the animal that had reactivity prior to challenge. Importantly, the quantitative assays of tetramer staining and IFN-{gamma} ELISpot also failed to reveal an anamnestic response. These results support those that show a lack of general T-cell activation following wild-type challenge (Khatissian et al., 2001; Stebbings et al., 2002) and a lack of correlation between the breadth and strength of CTL precursor frequency that is generated by infection with live, attenuated SIV and protection against subsequent infection (Nixon et al., 2000).

The perturbation in tetramer frequency, peaking 12 weeks after superinfection challenge, and the subsequent detection of lytic activity demonstrated that the earlier lack of functional activity was not due to clonal exhaustion. It is possible that the increase in activity was a delayed response to challenge. A transient increase in plasma vRNA was detected in one animal and it is possible that a similar effect occurred in the other two animals at a level below the sensitivity of the qc RT-PCR reaction. This may have provided an antigenic boost. Nevertheless, where detected, the increase in plasma vRNA was observed by 2 weeks following wild-type challenge and it is difficult to equate this to such a delayed effect on circulating T cells. The identification of SIVmacGX2, but not SIVmac220, sequences in the plasma of animal 398 suggested that there was a local perturbation of the cellular environment that was a direct consequence of the incoming virus challenge. Further analysis of the replication parameters relating to superinfection resistance is required to investigate this intriguing observation.

The higher frequency of virus isolation and detection of proviral DNA from lymphoid tissues compared to PBMCs following long-term infection with SIVmacGX2 indicated that the attenuated vaccine strain probably replicated preferentially in these sites and may account for the high levels of antigen-specific CD8+ T cells that were detected in the tissues. Also, virus antigen-positive cells have been found to persist preferentially in lymphoid tissues compared to the periphery in macaques infected with SIVmacGX2 and a number of other nef-disrupted, attenuated strains of SIV (N. Almond and D. Ferguson, unpublished data). A similarly distributed CD8+ T-cell profile has been reported for monkeys infected with wild-type SIVmac251 (Kuroda et al., 2000). However, in that study, the frequency of antigen-specific cells in the periphery and in tissues was found to be similar, whereas in the present study, animals infected with either wild-type or attenuated SIV had higher frequencies of antigen-specific CD8+ T cells in the tissues than in the periphery. Although the frequency of these cells was generally higher in animals that were infected short-term with SIVmac220, where virus could be isolated readily from tissues and PBMCs, surprisingly high frequencies were also detected in SIVmacGX2-infected animals. It is possible that the high frequency of tissue-resident cells represents an expanded population, due to local antigenic stimulation following exposure to wild-type virus. However, this is considered to be unlikely, as in one SIVmacGX2-infected animal where sufficient cells were available for analysis, the numbers of Tat-SL8-specific and Gag-CM9-specific IFN-{gamma} spot-forming cells were found to be remarkably similar in inguinal lymph nodes taken prior to challenge and at analysis post-mortem (data not shown). Furthermore, changes in lymph nodes would also be expected to induce perturbations in the circulating peripheral population. In another study, where macaques infected with live, attenuated SIV were only protected incompletely against superinfection with wild-type virus, no evidence of general T-cell activation was seen in lymph nodes taken 2 weeks after challenge (Khatissian et al., 2001).

Although, in the present study, an anamnestic CD8+ T-cell response to the two immunodominant epitopes studied was not evident immediately following rechallenge, pre-existing effector activity may have been sufficient to prevent infection with wild-type virus. Despite the decline in lytic activity against the Gag CM9 epitope, cytokine-secreting CD8+ cells were detected at the time of challenge with wild-type virus and tetramer staining revealed an abundance of specific cells in tissues. Furthermore, functional activity directed against other subdominant epitopes may have contributed to protection. MamuA*01 MHC class I binds a large number of SIV epitopes (Allen et al., 2001) and other epitopes may be presented by the co-expressed MHC class I molecule. Further work is needed to determine the role of CD8+ T cells in the protection afforded by infection with attenuated SIV. It will also be important to determine the potential contribution of other mechanisms, such as virus-enhanced or -induced innate immunity, receptor modulation and, as postulated previously, occupancy of a critical niche (Sharpe et al., 1997).


   ACKNOWLEDGEMENTS
 
We are grateful to Drs Ulrike Sauermann (German Primate Centre) and David Watkins (Wisconsin Regional Primate Research Center) for Mamu typing and to Zahra Fagrouch and Stuart Brown for assistance with PCR. This work was supported by the UK Department of Health, the Edward Jenner Institute for Vaccine Research and the Medical Research Council (N. A., N. B. and C. H., programme grant G925730). The views expressed in the publication are those of the authors and not necessarily those of the Department of Health.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Allen, T. M., Sidney, J., del Guercio, M.-F. & 8 other authors (1998). Characterization of the peptide binding motif of a rhesus MHC class I molecule (Mamu-A*01) that binds an immunodominant CTL epitope from simian immunodeficiency virus. J Immunol 160, 6062–6071.[Abstract/Free Full Text]

Allen, T. M., O'Connor, D. H., Jing, P. & 16 other authors (2000). Tat-specific cytotoxic T lymphocytes select for SIV escape variants during resolution of primary viraemia. Nature 407, 386–390.[CrossRef][Medline]

Allen, T. M., Mothé, B. R., Sidney, J. & 11 other authors (2001). CD8+ lymphocytes from simian immunodeficiency virus-infected rhesus macaques recognize 14 different epitopes bound by the major histocompatibility complex class I molecule MamuA*01: implications for vaccine design and testing. J Virol 75, 738–749.[Abstract/Free Full Text]

Almond, N. & Stott, J. (1999). Live attenuated SIV – a model of a vaccine for AIDS. Immunol Lett 66, 167–170.[CrossRef][Medline]

Almond, N., Kent, K., Cranage, M., Rud, E., Clarke, B. & Stott, E. J. (1995). Protection by attenuated simian immunodeficiency virus in macaques against challenge with virus-infected cells. Lancet 345, 1342–1344.[CrossRef][Medline]

Almond, N., Rose, J., Sangster, R., Silvera, P., Stebbings, R., Walker, B. & Stott, E. J. (1997). Mechanisms of protection induced by attenuated simian immunodeficiency virus. I. Protection cannot be transferred with immune serum. J Gen Virol 78, 1919–1922.[Abstract]

Arnold, C., Jenkins, A., Almond, N., Stott, E. J. & Kent, K. A. (1999). Monoclonal antibodies recognize at least five epitopes on the SIV Nef protein and identify an in vitro-induced mutation. AIDS Res Hum Retrovir 15, 1087–1097.[CrossRef][Medline]

Baba, T. W., Jeong, Y. S., Pennick, D., Bronson, R., Greene, M. F. & Ruprecht, R. M. (1995). Pathogenicity of live, attenuated SIV after mucosal infection of neonatal macaques. Science 267, 1820–1825.[Medline]

Baba, T. W., Liska, V., Khimani, A. H. & 8 other authors (1999). Live attenuated, multiply deleted simian immunodeficiency virus causes AIDS in infant and adult macaques. Nat Med 5, 194–203.[CrossRef][Medline]

Bogers, W. M. J. M., Niphuis, H., ten Haaft, P., Laman, J. D., Koornstra, W. & Heeney, J. L. (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–F18.[Medline]

Borrow, P., Lewicki, H., Hahn, B. H., Shaw, G. M. & Oldstone, M. B. A. (1994). Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection. J Virol 68, 6103–6110.[Abstract]

Clarke, S., Almond, N. & Berry, N. (2003). Simian immunodeficiency virus Nef gene regulates the production of 2-LTR circles in vivo. Virology 306, 100–108.[CrossRef][Medline]

Connor, R. I., Montefiori, D. C., Binley, J. M. & 8 other authors (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.[Abstract/Free Full Text]

Cranage, M. P., Whatmore, A. M., Sharpe, S. A. & 7 other authors (1997). Macaques infected with live attenuated SIVmac are protected against superinfection via the rectal mucosa. Virology 229, 143–154.[CrossRef][Medline]

Cranage, M. P., Sharpe, S. A., Whatmore, A. M., Polyanskaya, N., Norley, S., Cook, N., Leech, S., Dennis, M. J. & Hall, G. A. (1998). In vivo resistance to simian immunodeficiency virus superinfection depends on attenuated virus dose. J Gen Virol 79, 1935–1944.[Abstract]

Daniel, M. D., Kirchhoff, F., Czajak, S. C., Sehgal, P. K. & Desrosiers, R. C. (1992). Protective effects of a live attenuated SIV vaccine with a deletion in the nef gene. Science 258, 1938–1941.[Medline]

Dittmer, U., Nißlein, T., Bodemer, W., Petry, H., Sauermann, U., Stahl-Hennig, C. & Hunsmann, G. (1995). Cellular immune response of rhesus monkeys infected with a partially attenuated nef deletion mutant of the simian immunodeficiency virus. Virology 212, 392–397.[CrossRef][Medline]

Flynn, K. J., Riberdy, J. M., Christensen, J. P., Altman, J. D. & Doherty, P. C. (1999). In vivo proliferation of naïve and memory influenza-specific CD8+ T cells. Proc Natl Acad Sci U S A 96, 8597–8602.[Abstract/Free Full Text]

Geretti, A.-M., Hulskotte, E. G. J., Dings, M. E. M., van Baalen, C. A., van Amerongen, G., Norley, S. G., Boers, P., Gruters, R. & Osterhaus, A. D. M. E. (1999). Decline of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocytes in the peripheral blood of long-term nonprogressing macaques infected with SIVmac32H-J5. J Infect Dis 180, 1133–1141.[CrossRef][Medline]

Gundlach, B. R., Reiprich, S., Sopper, S., Means, R. E., Dittmer, U., Mätz-Rensing, K., Stahl-Hennig, C. & Überla, K. (1998). Env-independent protection induced by live, attenuated simian immunodeficiency virus vaccines. J Virol 72, 7846–7851.[Abstract/Free Full Text]

Hofmann-Lehmann, R., Vlasak, J., Williams, A. L., Chenine, A.-L., McClure, H. M., Anderson, D. C., O'Neil, S. & Ruprecht, R. M. (2003). Live attenuated, nef-deleted SIV is pathogenic in most adult macaques after prolonged observation. AIDS 17, 157–166.[CrossRef][Medline]

Home Office (1989). Code of Practice for the Housing and Care of Animals used in Scientific Procedures. London: HMSO.

Johnson, R. P. & Desrosiers, R. C. (1998). Protective immunity induced by live attenuated simian immunodeficiency virus. Curr Opin Immunol 10, 436–443.[CrossRef][Medline]

Johnson, R. P., Glickman, R. L., Yang, J. Q., Kaur, A., Dion, J. T., Mulligan, M. J. & Desrosiers, R. C. (1997). Induction of vigorous cytotoxic T-lymphocyte responses by live attenuated simian immunodeficiency virus. J Virol 71, 7711–7718.[Abstract]

Khatissian, E., Monceaux, V., Cumont, M.-C., Kieny, M.-P., Aubertin, A.-M. & Hurtrel, B. (2001). Persistence of pathogenic challenge virus in macaques protected by simian immunodeficiency virus SIVmac{Delta}nef. J Virol 75, 1507–1515.[Abstract/Free Full Text]

Kostense, S., Ogg, G. S., Manting, E. H. & 8 other authors (2001). High viral burden in the presence of major HIV-specific CD8+ T cell expansions: evidence for impaired CTL effector function. Eur J Immunol 31, 677–686.[CrossRef][Medline]

Koup, R. A., Safrit, J. T., Cao, Y., Andrews, C. A., McLeod, G., Borkowsky, W., Farthing, C. & Ho, D. D. (1994). Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome. J Virol 68, 4650–4655.[Abstract]

Kuroda, M. J., Schmitz, J. E., Seth, A. & 8 other authors (2000). Simian immunodeficiency virus-specific cytotoxic T lymphocytes and cell-associated viral RNA levels in distinct lymphoid compartments of SIVmac-infected rhesus monkeys. Blood 96, 1474–1479.[Abstract/Free Full Text]

Lohman, B. L., McChesney, M. B., Miller, C. J. & 7 other authors (1994). A partially attenuated simian immunodeficiency virus induces host immunity that correlates with resistance to pathogenic virus challenge. J Virol 68, 7021–7029.[Abstract]

McKay, P. F., Schmitz, J. E., Barouch, D. H., Kuroda, M. J., Lifton, M. A., Nickerson, C. E., Gorgone, D. A. & Letvin, N. L. (2002). Vaccine protection against functional CTL abnormalities in simian human immunodeficiency virus-infected rhesus monkeys. J Immunol 168, 332–337.[Abstract/Free Full Text]

Metzner, K. J., Jin, X., Lee, F. V. & 8 other authors (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.[Abstract/Free Full Text]

Nilsson, C., Mäkitalo, B., Thorstensson, R., Norley, S., Binninger-Schinzel, D., Cranage, M., Rud, E., Biberfeld, G. & Putkonen, P. (1998). Live attenuated simian immunodeficiency virus (SIV)mac in macaques can induce protection against mucosal infection with SIVsm. AIDS 12, 2261–2270.[CrossRef][Medline]

Nixon, D. F., Donahoe, S. M., Kakimoto, W. M., Samuel, R. V., Metzner, K. J., Gettie, A., Hanke, T., Marx, P. A. & Connor, R. I. (2000). Simian immunodeficiency virus-specfic cytotoxic T lymphocytes and protection against challenge in rhesus macaques immunized with a live attenuated simian immunodeficiency virus vaccine. Virology 266, 203–210.[CrossRef][Medline]

Norley, S., Beer, B., Binninger-Schinzel, D., Cosma, C. & Kurth, R. (1996). Protection from pathogenic SIVmac challenge following short-term infection with a nef-deficient attenuated virus. Virology 219, 195–205.[CrossRef][Medline]

Polyanskaya, N., Sharpe, S., Cook, N., Leech, S., Banks, J., Dennis, M., Hall, G., Stott, J. & Cranage, M. (1997). Anti-major histocompatibility complex antibody responses to simian B cells do not protect macaques against SIVmac infection. AIDS Res Hum Retrovir 13, 923–931.[Medline]

Rinaldo, C. R., Jr, Huang, X.-L., Fan, Z. & 9 other authors (2000). Anti-human immunodeficiency virus type 1 (HIV-1) CD8+ T-lymphocyte reactivity during combination antiretroviral therapy in HIV-1-infected patients with advanced immunodeficiency. J Virol 74, 4127–4138.[Abstract/Free Full Text]

Rud, E. W., Cranage, M., Yon, J., Quirk, J., Ogilvie, L., Cook, N., Webster, S., Dennis, M. & Clarke, B. E. (1994). Molecular and biological characterization of simian immunodeficiency virus macaque strain 32H proviral clones containing nef size variants. J Gen Virol 75, 529–543.[Abstract]

Sawai, E. T., Hamza, M. S., Ye, M., Shaw, K. E. & Luciw, P. A. (2000). Pathogenic conversion of live attenuated simian immunodeficiency virus vaccines is associated with expression of truncated Nef. J Virol 74, 2038–2045.[Abstract/Free Full Text]

Sharpe, S. A., Whatmore, A. M., Hall, G. A. & Cranage, M. P. (1997). Macaques infected with attenuated simian immunodeficiency virus resist superinfection with virulence-revertant virus. J Gen Virol 78, 1923–1927.[Abstract]

Sharpe, S., Hanke, T., Tinsley-Bown, A., Dennis, M., Dowall, S., McMichael, A. & Cranage, M. (2003). Mucosal immunization with PLGA-microencapsulated DNA primes a SIV-specific CTL response revealed by boosting with cognate recombinant modified vaccinia virus Ankara. Virology 313, 13–21.[CrossRef][Medline]

Stebbings, R., Stott, J., Almond, N. & 10 other authors (1998). Mechanisms of protection induced by attenuated simian immunodeficiency virus. II. Lymphocyte depletion does not abrogate protection. AIDS Res Hum Retrovir 14, 1187–1198.[Medline]

Stebbings, R. J., Almond, N. M., Stott, E. J. & 9 other authors (2002). Mechanisms of protection induced by attenuated simian immunodeficiency virus. V. No evidence for lymphocyte-regulated cytokine responses upon rechallenge. Virology 296, 338–353.[CrossRef][Medline]

Stott, E. J., Almond, N., West, W., Kent, K., Cranage, M. & Rud, E. (1994). Protection against simian immunodeficiency virus infection of macaques by cellular or viral antigens. In Neuvieme Colloque Des Cent Gardes, pp. 219–224. Edited by M. Girard & L. Vallette. Lyon, France: Fondation Marcel Merieux.

Ten Haaft, P., Verstrepen, B., Überla, K., Rosenwirth, B. & Heeney, J. (1998). A pathogenic threshold of virus load defined in simian immunodeficiency virus- or simian-human immunodeficiency virus-infected macaques. J Virol 72, 10281–10285.[Abstract/Free Full Text]

Whatmore, A. M., Cook, N., Hall, G. A., Sharpe, S., Rud, E. W. & Cranage, M. P. (1995). Repair and evolution of nef in vivo modulates simian immunodeficiency virus virulence. J Virol 69, 5117–5123.[Abstract]

Wyand, M. S., Manson, K. H., Garcia-Moll, M., Montefiori, D. & Desrosiers, R. C. (1996). Vaccine protection by a triple deletion mutant of simian immunodeficiency virus. J Virol 70, 3724–3733.[Abstract]

Yasutomi, Y., Reimann, K. A., Lord, C. I., Miller, M. D. & Letvin, N. L. (1993). Simian immunodeficiency virus-specific CD8+ lymphocyte response in acutely infected rhesus monkeys. J Virol 67, 1707–1711.[Abstract]

Received 20 February 2004; accepted 11 May 2004.