| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Division of Retrovirology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK
2 Division of Biotherapeutics, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK
3 Division of Biological Services, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK
Correspondence
Neil Berry
nberry{at}nibsc.ac.uk
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
A supplementary figure is available with the online version of this paper.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Nilsson et al., 1998) and parenteral (Almond et al., 1995; Daniel et al., 1992) routes. While live retrovirus-based vaccines raise profound safety concerns (Baba et al., 1995, 1999), studies in macaques may lead to an understanding of how such vaccines operate. The ability for live attenuated SIV to protect against wild-type virus challenge and the parameters governing this protection are of crucial importance in unravelling which responses might be required to develop a successful AIDS vaccine. A role for antiviral antibodies is unlikely, since vaccination with live attenuated SIV confers protection against challenge with chimeric simian–human immunodeficiency viruses (SHIVs) expressing human immunodeficiency virus (HIV)-1 envelope which is not cross-neutralized in vitro (Bogers et al., 1995; Dunn et al., 1997; Wyand et al., 1999) and since transfer of immune serum has not transferred protection (Almond et al., 1997). Live attenuated SIV elicits potent, long-lived CD4+ responses (Gauduin et al., 1999; Sarkar et al., 2002) and CD8+ responses (Johnson et al., 1997; Nixon et al., 2000; Sharpe et al., 2004) in macaques. Depletion of CD8+ T cells with humanized anti-CD8 monoclonal antibodies (mAbs) results in increased primary and set-point viraemias (Matano et al., 1998; Jin et al., 1999; Schmitz et al., 1999; Stebbings et al., 2005). However, as noted by McMichael (2006), the impact of depleting CD8+ lymphocytes on protection against wild-type virus challenge has been inconsistent between studies (Schmitz et al., 2005; Stebbings et al., 2005).
Different outcomes may be due to the different vaccine viruses used and/or different biological properties of the challenge virus, as well as the host species. In cynomolgus macaques, vaccination with SIVmacC8 for 3 weeks is sufficient to protect against detectable infection with the homologous wild-type virus clone SIVmacJ5 (Stebbings et al., 2004, 2005). SIVmac239
nef and SIVmac2393 vaccines in rhesus macaques, however, require significantly longer periods to elicit protection (Connor et al., 1998; Wyand et al., 1996).
In the current study, an ex vivo, uncloned stock of SIVmac251 derived for vigorous replication in cynomolgus macaques was used. The kinetics of virus replication and pathology in lymphoid tissue induced by this new stock, SIVmac32H/L28, were characterized in naïve cynomolgus macaques. Vaccination with SIVmacC8 20 weeks prior to infection with SIVmac32H/L28 prevented detection of this virus in the blood over 6 months and yielded no evidence of superinfection with the challenge virus in 2/4 vaccinates. Infection 3 weeks after vaccination conferred less potent protection, yet one macaque was protected against detectable infection with wild-type virus. Detailed analysis of the viral kinetics after SIVmac32H/L28 challenge in both protected and superinfected macaques revealed evidence of restimulation of the vaccine virus. The implications of this in explaining the mode of protection are discussed.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Rud et al., 1994) due to a 12 bp in-frame deletion in nef and two further conservative amino acid changes. Macaques were inoculated intravenously with 5000 TCID50 SIVmacC8 (9/90 pool; Almond et al., 1992), with an end-point titre of 104 TCID50 ml–1 on C8166 cells (Cranage et al., 1998). Wild-type challenge virus, SIVmac32H/L28, was prepared by inoculation of a cynomolgus macaque (L28) with 10 MID50 11/88 stock of SIVmac251/32H reisolate (Stott et al., 1990; Cranage et al., 1992); the macaque was killed humanely after 9 weeks. Disaggregated, Percoll-purified spleen cells were cultured in RPMI 1640, 9 % (v/v) fetal calf serum (FCS), 2 mM glutamine, 100 IU ml–1 penicillin/100 µg streptomycin ml–1, 5 µg phytohaemagglutinin (PHA) mitogen ml–1 and 10 IU IL-2 ml–1 at 37 °C in a CO2 incubator for 15 days. At days 4, 8 and 11, cultures were split 1 : 2 and fed with fresh medium containing IL-2 but not PHA until day 15. Cells were sedimented (800 g for 5 min) and the supernatant was aliquoted and stored in liquid nitrogen vapour. In vitro titration of the SIVmac32H/L28 7/8/92 stock determined on C8166 cells was 103.5 TCID50; this was found to be 104.5 MID50 following intravenous inoculation of cynomolgus macaques in vivo.
Experimental outline.
Eighteen naïve, D-type-retrovirus-free, juvenile cynomolgus macaques (Macaca fascicularis) were used in accordance with UK Home Office guidelines. The primary replication kinetics of SIVmac32H/L28 were determined by inoculating four naïve Mauritian-derived cynomolgus macaques (Z3–Z6) with 10 MID50 SIVmac32H/L28, sampled 0, 3, 7, 10, 21, 29, 42 and 56 days post-challenge (p.c.) and monthly until 24–26 weeks.
In the vaccine study, four cynomolgus macaques (A138–A142; group A) were inoculated by intravenous injection of 5000 TCID50 SIVmacC8; 17 weeks later, a further four (A143–A146; group B) were vaccinated identically. Three weeks later, all eight vaccinates and four naïve challenge controls (A147–A150; group C) were challenged with 10 MID50 SIVmac32H/L28. Macaques were bled at 14, 28, 56, 84, 112 and 170 days and killed humanely 
24 weeks p.c. with SIVmac32H/L28. Two additional macaques (W341/W342) were challenged intravenously with 10 MID50 SIVmac32H/L28 and exsanguinated 14 days later under terminal anaesthesia. EDTA-treated plasma was collected, split into aliquots, stored at –80 °C and used to derive SIV RNA reference materials.
Quantification of SIV RNA.
SIV RNA levels in plasma were determined by quantitative real-time RT-PCR (qRT-PCR), calibrated using the SIV RNA reference materials derived from the 14-day bulk bleed of macaque W342. Viral RNA was extracted from 140 µl plasma using viral RNA mini-kits (QIAamp; Qiagen) eluted in a total volume of 50 µl AVE buffer. RNA (5 µl) extracted from reference or experimental samples was amplified in triplicate, by using the Brilliant QRT-PCR plus Core Reagents one-step kit (Stratagene). Oligonucleotide primers and probe sequences, located in conserved regions of gag, were optimized at 300 and 100 nM respectively. qRT-PCR was performed with forward (5'-AGTGCCAACAGGCTCAGAAAA-3') and reverse (5'-TGCGTGAATGCACCAGATG-3') primers and Taqman hydrolysis probe [5'-(6'-FAM-TTAAAAAGCCTTTATAATACTGTCTGCG-BHQ1)-3']. The amplification profile was optimized at 51 °C for 30 min for the reverse transcription step, inactivation/activation of Taq polymerase (Stratascript) at 95 °C for 10 min, and 45 cycles of denaturation (95 °C for 30 s) and annealing/elongation (60 °C for 90 s) on an Mx3000P genetic analyser (Stratagene); quantitative data was determined using the Mx3000P software. The lower level of sensitivity of the qRT-PCR assay is 50 SIV RNA copies ml–1 plasma.
Detection and quantification of SIV DNA.
SIV infection and proviral SIV gag DNA levels were determined by quantitative PCR (qPCR), using the same primer/probe sequences as the qRT-PCR assay. Genomic DNA was extracted from whole blood or lymphoid samples as previously described (Clarke et al., 2003). The concentration added to each PCR assay was determined retrospectively using a fluorometic DNA quantification kit (Sigma) in a microtitre format. Aliquots of DNA (1 µl) were assayed in triplicate, by using a Taqman Universal PCR Master Mix (ABI) against a standard curve of the p2-LTR plasmid (Clarke et al., 2003) serially diluted in herring sperm DNA (60 µg ml–1). Amplifications were performed on an Mx3000P analyser (Stratagene) with a thermal profile of initial hot start at 95 °C for 10 min, followed by 95 °C for 30 s and 60 °C for 1 min for 45 cycles. Optimal primer and probe concentrations were 900 nM and 225 nM, respectively. SIV DNA levels were expressed as copies of SIV DNA per 105 peripheral blood mononuclear cells (PBMC) or mononuclear cells (MNC). The absolute limit of detection is a single SIV DNA copy, determined by limit-dilution analysis and Poisson statistics.
Serological and virological assays.
Binding antibodies to SIV envelope gp130 (EVA670 CFAR/NIBSC) were determined by enzyme immunoassay as described previously (Stebbings et al., 2004). Neutralizing antibody titres were determined by mixing serum diluted in RPMI containing 10 % FCS with virus. The serum dilution representing 75 % inhibition of p27 antigen production was recorded as the titre (Kent et al., 1994). The 11/88 virus stock of SIVmac251/32H (Almond et al., 1992) represented the challenge virus and SIVmacJ5 represented the vaccine virus (Stebbings et al., 2002). Virus isolation from Ficoll-purified PBMC was determined by co-culture with C8166 indicator cells up to 28 days, by syncytium formation and p27 antigen detection (Silvera et al., 2001; Almond et al., 1990).
Immunological and haematological analyses.
CD3+/CD4+ and CD3+/CD8+ lymphocyte populations were monitored in whole blood by flow cytometry following immunostaining with cross-reactive anti-human mAbs. Whole blood (200 µl) was incubated with 10 µl fluorescein isothiocyanate (FITC)-labelled anti-monkey CD3 monoclonal antibody FN18 (Serotec), 20 µl phycoerythrin (PE)-labelled anti-human CD4 mAb Leu-3a (BD Biosciences) and 10 µl APC-labelled anti-human CD8 monoclonal antibody 3B5 (Caltag Laboratories), for 1 h at 4 °C. FACS lysing solution (BD Biosciences) was used to remove red blood cells; cells were washed twice in PBS containing 4 % (v/v) FCS and 0.1 % sodium azide. Samples were fixed overnight in 2 % (v/v) formaldehyde in PBS, gating on the lymphocyte fraction, then analysed by FACS (FACSCalibur; BD Biosciences). Platelet counts were monitored to assess gross haematological changes as an independent marker of disease onset.
Cellular immune responses against SIV nef.
Intracellular gamma interferon (IFN-
) detection by flow cytometry was used to investigate cellular immune responses to specific peptides from SIVmacJ5 Nef protein. Percoll-purified PBMC in RPMI 1640 medium supplemented with 10 % FCS containing 50 IU penicillin or streptomycin ml–1 (Gibco-BRL) were rested overnight prior to incubation for 1.5 h with individual and pooled SIV Nef 9-mer peptides (5 µg ml–1; EVA7071.1-36; CFAR/NIBSC) and co-stimulatory antibodies anti-CD28 and anti-CD49D (1 µg ml–1; BD Biosciences). SIV Nef pool 1 consisted of seven peptides (2, 4, 9, 13, 19, 20 and 21), Nef pool 2 consisted of eight peptides (3, 7, 8, 10, 16, 18, 22 and 25), Nef pool 3 consisted of nine peptides (1, 5, 6, 12, 14, 15, 23, 24 and 30), Nef pool 4 consisted of six peptides (11, 17, 26, 27, 28 and 29) and Nef pool 5 consisted of six peptides (31–36). Brefeldin A (10 µg ml–1; Sigma-Aldrich) was added to the culture, incubated for 5 h and refrigerated overnight. Cultured PBMC were fixed and permeabilized (Fix and Perm; Caltag Laboratories), stained with anti-monkey CD3 FITC conjugate (Serotec), anti-human IFN- PE conjugate (Caltag Laboratories) and anti-human CD8 APC conjugate (Caltag Laboratories) as previously described (Stebbings et al., 1998). Data collected on a FACS Calibur cytometer (BD Biosciences) were analysed using CXP software (Beckman Coulter).
SIV nef sequence analyses.
Differential PCR analysis of nef vaccine SIVmacC8 and wild-type SIVmac32H/L28 was performed using nested SIV nef-specific PCR primers spanning the entire nef open reading frame. Outer primers (forward, 5'-ATGGGTGGAGCTATTTCCAGGA-3' and reverse, 5'-TGAGCGAGTTTCCTTCTTGTCA-3') and inner primers (forward, 5'-ACCAGTGATGCCACGAGTTCC-3' and reverse 5'-GCCATGTTAAGAAGGCCTCT-3') were used with AmpliTaq Gold reagents (Roche Diagnostics) according to the manufacturer's instructions. Undiluted and limiting template PCRs were digested using RsaI (Rose et al., 1995) to determine the relative frequencies of SIVmacC8/SIVmac32H/L28 in PBMC DNA or plasma RNA by RT-PCR using Titan kits (Roche Diagnostics). Additional nef sequence analysis was performed on PBMC and MNC DNA or plasma RNA. Amplicons were cloned into a TOPO TA cloning vector (Invitrogen) or sequenced directly using an automated capillary DNA sequencer (ABI Prism 3130).
Amplification and sequencing of SIV Env.
SIV Env sequence analysis of replication-competent virus was determined by deriving fragments by an initial RT-PCR amplification step from plasma using Titan reagents (Roche Diagnostics) by nested PCR. Env sequence was determined by direct sequence analysis of product amplified from plasma-derived virus by RT-PCR.
In situ hybridization (ISH).
ISH was performed on lymphoid tissues [spleen, mesenteric and peripheral lymph nodes (MLN and PLN, respectively)] collected post-mortem as described previously (Canto-Nogues et al., 2001). Hybridization mixes consisted of sense and anti-sense probes to SIV gag, env and nef transcripts. ISH-positive cells were quantified by determining numbers of positive cells within up to 10 random fields of view (x10 lens and x10 eyepiece magnification; equivalent to 2.2 mm2); these were counted manually and converted to mean number of positive cells mm–2 expressed in a grading key (see Table 3).
|
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
) and compared overall with similar time points described for SIVmacJ5 and SIVmacC8 in a previous study by Clarke et al. (2003), which were re-evaluated using the qRT- PCR assay (Fig. 1b). Plasma SIV RNA was detected in 3/4 macaques at day 3 p.c. and rose markedly, peaking at day 10 p.c. (log10 mean SIV RNA loads of 7.46±0.38 copies ml–1). Group mean levels for SIVmac32H/L28 at day 29 and day 56 were log10 6.23±0.31 and log10 5.65±0.51 SIV RNA copies ml–1, respectively. The same time points for SIVmacJ5 were statistically significantly lower (Mann–Whitney, P<0.03). Peak and persistent levels of SIVmac32H/L28 viraemia were consistently higher than SIVmacJ5 or SIVmacC8, persisting at log10 >4.5 in Z3, Z4 and Z6 though the levels declined in Z5. Similar trends to the viral RNA (vRNA) data were reflected by co-culture analysis of infected cells at days 7 and 10 (log10 3.12±0.43 and log10 4.12±0.24 infected cells per million PBMC).
|
Vaccination with SIVmacC8
All eight vaccinates (groups A and B) seroconverted; anti-SIV rgp130 was detectable in 7/8 vaccinates by 3 weeks and in all vaccinates by 8 weeks (Fig. 2). In group A, 20 weeks after SIVmacC8 vaccination, binding anti-rgp130 antibody titres were log10 2.6–3.7. Cellular immune responses to SIV Nef peptides were assessed for groups A and B 3 weeks after vaccination with SIVmacC8; this indicated that there were high levels of effector cells in peripheral blood. Anti-Nef cellular responses were detected by intracellular IFN- staining (Fig. 3) in A141/A142 (group A) and A143/A144/A145 (group B). All vaccinates (groups A and B) were SIV DNA PCR-positive 21 days after inoculation. SIV RNA levels at day 14 were log10 3.36±0.14 and 2.96±2.31 copies ml–1 for groups A and B, respectively, which, in group A vaccinates, became undetectable from day 56 until challenge with SIVmac32H/L28. On the day of SIVmac32H/L28 challenge, neutralizing antibody titres against SIVmacJ5 (representing the vaccine virus SIVmacC8) were less than 1/10 in A139–A141 (group A), 1/60 in A142 and 1/30 in A143–A146 (group B). Neutralization titres against SIVmac251/32H were less than 1/10 in all vaccinated macaques.
|
|
. In challenge controls (group C), SIV RNA levels rose rapidly, peaking at 14 days (log10 8.24±0.39 SIV RNA copies ml–1). By contrast, vRNA levels in group A remained below detection (<50 SIV RNA copies ml–1), a statistically highly significant observation even at a 99 % confidence interval (Mann–Whitney P<0.001). In challenge controls, SIV RNA levels decreased to log10 5.51±0.19 copies ml–1 by day 112, but they remained undetectable in A139/A140 (group A). Fluctuating low levels (<103 SIV RNA copies ml–1) remained detectable in A141/A142 from 28 days p.c. with SIVmac32H/L28.
|
SIV DNA levels in peripheral blood.
Levels of SIV gag DNA in peripheral blood were quantified by RT-PCR (Fig. 5). In challenge controls, levels of SIV DNA rose sharply after infection with SIVmac32H/L28, ranging between 104 and 105.5 SIV DNA copies per 105 PBMC at days 14 and 28 p.c. Peripheral SIV DNA levels decreased to 10–300 copies per 105 PBMC by day 84 p.c. Analysis of blood, spleen, MLN and PLN post-mortem (day 198 p.c.) detected 150–800 SIV DNA copies per 105 MNC in lymphoid tissues and slightly lower in blood (50–150 DNA copies per 105 PBMC). SIV DNA levels were consistently higher in MLN. In 20 week vaccinates (group A), SIV DNA was undetectable in the blood of A139/A140 throughout, but it was detected over the same period in A141/A142, though it never exceeded 103 SIV DNA copies per 105 PBMC. SIV sequences were detected in selected lymphoid, predominantly spleen, tissues post-mortem, though at very low levels in A139/A140 (<10 DNA copies per 105 MNC) and marginally higher levels in A141/A142 (10–20 DNA copies per 105 MNC).
|
).
Evidence of SIVmac32H/L28 superinfection.
SIV nef-specific PCR was used to detect and differentiate vaccine- and challenge-virus-derived DNA in blood 14 and 28 days p.c. with SIVmac32H/L28, and in blood and selected lymphoid tissue post-mortem (see Table 1). Wild-type SIV nef sequences were identified in blood and all lymphoid tissue samples from naïve controls that were challenged with SIVmac32H/L28. By contrast, in group A which was vaccinated with SIVmacC8 20 weeks prior to challenge, SIVmac32H/L28 was not identified in blood samples; only the vaccine virus SIVmacC8 was identified. SIVmac32H/L28 sequences were detected in the spleen and MLN from A141 and MLN and PLN from A142.
|
). In A144, only SIVmacC8 was detected. Analysis of lymphoid tissue detected SIVmac32H/L28 in spleen, MLN and PLN from A143, in PLN from A145 and in spleen and MLN from A146. No evidence of SIVmac32H/L28 was detected in any tissue sample from A144.
Restimulation of SIVmacC8 after SIVmac32H/L28 challenge.
Discriminatory SIV nef DNA-specific PCR revealed that reappearance of SIV DNA in the blood of A141/A142 after rechallenge with SIVmac32H/L28 was due to restimulation of SIVmacC8 alone (Table 1). This was investigated in greater detail in 3 week vaccinates (group B). The relative frequency of vaccine (SIVmacC8) to challenge virus (SIVmac32H/L28) in A143 and A146 was 1 : 4 and 1 : 12 at 14 days p.c. and at termination, respectively (Table 1). Sequence analysis of cloned PCR products from selected lymphoid tissues did not identify evidence of sequence repair. Indeed, for A143, the only changes observed were additional deletions in nef that would truncate any expression of Nef protein (data not shown).
Relative proportions of vaccine and wild-type virus RNA in the plasma of A143–A146 at 14, 28 and 170 days p.c. with SIVmac32H/L28 were determined by limiting dilution of template RNA and restriction digestion of product amplified by RT-PCR. Whenever plasma yielded a specific product, the vaccine virus SIVmacC8 was detectable, contributing between 20–100 % of the detectable vRNA signal (Table 2). Calculated vRNA loads of SIVmacC8 and SIVmac32H/L28, at peak and set-point viraemias, were based on the proportion of nef sequence and the total SIV RNA signal. SIVmac32H/L28 sequences were never detected by RT-PCR nef sequence analysis in A144.
|
). However, the distinctive pathology and distribution of SIVmac32H/L28 observed in Z3–Z6 (Supplementary Fig. S1) was also observed in challenge controls (group C), focused as large clusters of SIV-infected positive cells detected in the sinuses of lymphoid tissues, especially MLN and thymus (Supplementary Fig. S1). This was not observed in any vaccinated macaque, irrespective of evidence of infection with SIVmac32H/L28 (Supplementary Fig. S1). Changes in the proportion of CD3+/CD4+ cells following vaccination with SIVmacC8 and challenge with SIVmac32H/L28 revealed only minor fluctuations that remained within the normal range in CD3+/CD4+ cells in 20 week vaccinates, with more variation in 3 week vaccinates (data not shown). In challenge controls, CD3+/CD4+ cells for A148/A149 were in the normal range, though those in A147/A150 were in decline by the end of the study, accompanied by platelet loss to below the reference (150 µl–1). Occasional reduction in platelets were recorded for A139, A141, A142, A143, A148 and A149.
Anti-envelope responses
In challenge controls (group C), anti-SIV rgp130 responses detected 4–8 weeks after SIVmac32H/L28 challenge rose rapidly and plateaued with end-point titres between log10 3.5 and 4.5 (Fig. 2). Following challenge of group A with SIVmac32H/L28, anti-gp130 titres rose marginally in A141/A142 (end point titres >3.5 log10 at termination), remaining at lower levels in A139/A140 (log10 end point titres <3.0 at termination). Following SIVmac32H/L28 challenge of individuals in group B, end point titres for A143, A145 and A146 were >3.5 log10 at termination, whereas for A144 titres were <3.0 log10.
SIV envelope sequence
Partial SIV gp130 envelope sequences of SIVmac32H/L28 were derived from acute infection plasma samples (14 days) by RT-PCR from naïve macaques. Bulk sequence analysis of fragments spanning 1472 nucleotides of SIV gp130 envelope [nt 7739–9210 SIVmacJ5 clone SIVMM32H (GenBank accession number D01065.1
[GenBank]
)], spanning V1, V2 and V3 regions, indicated few nucleotide changes from the published sequence. Alignment of the first 370 amino acids of SIV gp130 generated by RT-PCR of plasma from A143/A146 (group B) 14 and 43 days p.c. with SIVmac32H/L28 indicated a high level (>99 %) of sequence conservation, with only a single substitution (A
T) identified in the V1 region (data not shown).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). The ability of SIVmacC8 to resist a highly potent, vigorously replicating virus was determined using a novel challenge virus stock (SIVmac32H/L28) 3 or 20 weeks after vaccination with SIVmacC8. SIVmac32H/L28, derived by ex vivo culture of splenocytes from a cynomolgus macaque infected for 8 weeks with uncloned SIVmac251 (32H reisolate) (Cranage et al., 1992; Almond et al., 1992), caused a reproducibly high peak and persisting viraemia in most vaccine-naïve Mauritian-derived cynomolgus macaques. The magnitude and duration of the viraemia induced by SIVmac32H/L28 is similar to that in rhesus macaques infected with pathogenic stocks of SIVmac (Abel et al., 2003; Lohman et al., 1994; Mascola et al., 2003; Schmitz et al., 2005), inducing CD4+ depletion, thrombocytopaenia and early signs of disease after 6 months.
Potent resistance to superinfection with SIVmac32H/L28 was identified in macaques challenged 20 weeks after vaccination with SIVmacC8. Two of four macaques showed no evidence of detectable infection with SIVmac32H/L28, the other two exhibited low, sporadic infection which was only detected in deep lymphoid tissues post-mortem. Compared with challenge controls, reductions in vRNA levels post-SIVmac32H/L28 challenge were statistically highly significantly different; if transferred through to a human setting, this would profoundly affect projected disease outcomes. The impact on the outcome of a challenge 3 weeks after vaccination was more complex. After challenge with SIVmac32H/L28, one macaque (A144) was protected from superinfection. In the three remaining macaques (A143/A145/A146), plasma RNA levels increased from readily detectable levels of the vaccine virus. Analysis of virus sequence indicated that up to 50 % of the increased p.c. vRNA signal in plasma was derived from the vaccine virus which replicated alongside the challenge virus. Moreover, in A144, the delayed rebound in viraemia, which exceeded 4.5 log10 SIV RNA copies ml–1, was composed entirely of the vaccine virus SIVmacC8. This phenomenon of vaccine virus restimulation has been noted previously (Mackay et al., 2004; Sharpe et al., 2004), though not in the detail nor at the levels observed in the 3 week vaccinates reported here. Analysis of total SIV RNA levels alone, therefore, was insufficient to fully explain the observations. Reports by different groups of the modulation of SIV RNA levels after challenge, assuming it to represent transient breakthrough of the challenge virus, may require reinterpretation in light of this observation. The factors that determine whether restimulation of the vaccine virus occurs and the magnitude of the phenomenon require further clarification.
Despite these confounding issues of vaccine-virus restimulation, more marked protection was observed 20 weeks after vaccination, compared with 3 weeks, which concurs with the reports of Connor et al. (1998) and Wyand et al. (1996) which demonstrated that longer periods of vaccination with other attenuated SIV vaccines conferred superior protection against uncloned SIVmac challenge. At the same time, applying the most rigorous definition of protection (no evidence of the challenge virus), the advantage of extended vaccination is minimal: 1/4 were protected 3 weeks after vaccination and 2 of 4 were protected 20 weeks after vaccination. These data bear similarities to the study by Norley et al. (1996) in which 1/4 SIVmacC8-vaccinated rhesus macaques were protected against uncloned SIVmac251 10 weeks after vaccination and also 20 weeks after vaccination. SIVmac32H/L28 is also an uncloned stock of SIVmac251, though analysis of env and nef sequences recovered p.c. did not indicate high levels of sequence divergence from the vaccine virus. However, SIVmac32H/L28 possesses distinct biological properties which may have contributed to its ability to bypass protection conferred by, or the restimulation of, SIVmacC8. The replication kinetics of SIVmac32H/L28 in the first 3–10 days and the period after this appeared to be reflected in differences in the distribution of virus-infected cells that were analysed post-mortem, compared with the distribution of cells infected with SIVmacC8 which are similar to the wild-type homologue SIVmacJ5 (Canto-Nogues et al., 2001). The differences were also more marked than those described in rhesus macaques infected with attenuated and pathogenic stocks of SIVmac (Lackner et al., 1994). It is unlikely that differences in envelope sequence relating to the pathogenesis of SIV stocks (Marthas et al., 1993) can account for these differences in distribution , as only occasional non-coding changes were identified in biologically functional regions.
While this vaccine study characterized the potency of protection conferred by SIVmacC8, it did not establish directly how vaccination with live attenuated SIV protects individuals. Our previous reports have not identified a central role for adaptive immunity in the protection conferred by SIVmacC8 (Stebbings et al., 2005; Almond et al., 1997). In this study, neutralizing antibody responses were poor and did not correlate with protection. Similarly, neither the breadth nor frequency of CD8+ T cells that targeted peptide epitopes across Nef correlated with the outcome of challenge. Moreover, the observation that challenge with wild-type virus was associated with restimulation of the vaccine virus whilst eliciting highly effective control of the challenge virus provides an intellectual challenge for the identification of an adaptive immune response as a correlate of this protection.
The apparent paradox of protection conferred by live attenuated SIV was first noted by Paul (1995). It is difficult to envisage how an adaptive immune response could protect against a wild-type challenge whilst failing to eliminate a genetically closely related and persisting attenuated vaccine virus. This would be possible if the persisting vaccine virus exists in an immunologically privileged site, yet the simultaneous detection of vaccine and challenge viruses argues against this. Our data questioning a role for adaptive immune responses in this study are supported by the observations of Whatmore et al. (1995), where repair of the attenuating mutation in nef was identified in rhesus macaques vaccinated with SIVmacC8. These revertants had a wild-type phenotype capable of outgrowing the vaccine virus, yet the same macaques were protected against challenge with exogenous wild-type virus. Furthermore, it was shown subsequently that revertant viruses were not vaccine escape mutants when they were used subsequently to challenge new groups of macaques vaccinated with SIV macC8 (Sharpe et al., 1997).
If adaptive immunity is not central to this vaccine protection, what other mechanisms may be involved? Persistence of the vaccine virus appears to be a key feature of live attenuated SIV vaccination (Mackay et al., 2004), supporting the hypothesis that replicative capacity is directly related to vaccine efficacy with highly attenuated vaccine strains that require longer to achieve protection (Johnson et al., 1999). SIVmacC8 is only minimally attenuated and persists at central sites, reflected by cells expressing vRNA in lymphoid tissues in protected vaccinates. Persistence of the vaccine virus may provide constant stimulation for what might otherwise be a short-lived anti-retroviral response, a major component of which may be non-immune. The target cell populations infected by SIVmacC8 at key sites early in the infection process, particularly gut-associated lymphoid tissue (Veazey et al., 1998), are currently under investigation. Memory T cell populations (CD4+ and CCR5+) in the gastrointestinal tract (Mattapallil et al., 2005; Li et al., 2005) are a major site for early wild-type virus replication. Depletion of these cells by the vaccine virus could limit the number of target cells available for subsequent wild-type virus infection. Alternatively, the presence of the vaccine could elicit retroviral interference mechanisms. Interference between retroviruses that share the same cellular receptor, mediated by the envelope protein, is recognized as a method by which superinfection with a second retrovirus may be prevented at the cellular level (Lama, 2003). It remains unclear as to how this could apply to attenuated SIV vaccines, when the frequency of SIV-infected cells would appear to be a very small proportion of the total number of susceptible cells.
Understanding the relative contribution of innate immunity or intrinsic anti-retroviral responses mediated through TRIM5
and APOBEC3G at local sites of infection such as the gut mucosae, driven by a persisting retrovirus infection, may elucidate further the vaccination mechanism of SIVmacC8. The observation that TRIM5 and APOBEC3G are both upregulated by alpha interferon (Sakuma et al., 2007; Wang et al., 2008) merits further investigation to clarify the role of antiviral innate and intrinsic immunity factors in the protective mechanisms in this model system. Detailed studies of the early pathogenesis, host responses to vaccination and target cell distribution of SIVmacC8 may provide clearer answers to these important questions.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Almond, N., Page, M., Mills, K., Jenkins, A., Ling, C., Thorpe, R., Kitchin, P. & Williams, M. (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.[CrossRef][Medline]
Almond, N., Jenkins, A., Slade, A., Heath, A., Cranage, M. & Kitchin, P. (1992). Population sequence analysis of a simian immunodeficiency virus (32H reisolate of SIVmac251): a virus stock used for international vaccine studies. AIDS Res Hum Retroviruses 8, 77–88.[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]
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.
Baba, T. W., Liska, V., Khimani, A. H., Ray, N. B., Dailey, P. J., Pennick, D., Bronson, R., Greene, M. F., McClure, H. M. & 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., 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]
Canto-Nogues, C., Jones, S., Sangster, R., Silvera, P., Hull, R., Cook, R., Hall, G., Walker, B., Stott, E. J. & other authors (2001). In situ hybridization and immunolabelling study of the early replication of simian immunodeficiency virus (SIVmacJ5) in vivo. J Gen Virol 82, 2225–2234.
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., Moore, J. P., Bonhoeffer, S., Gettie, A., Fenamore, E. A., Sheridan, K. E., Ho, D. D. & 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.
Cranage, M., Stott, J., Mills, K., Ashworth, T., Taffs, F., Farrar, G., Chan, L., Dennis, M., Putkonen, P. & other authors (1992). Vaccine studies with the 32H reisolate of SIVmac251: an overview. AIDS Res Hum Retroviruses 8, 1479–1481.[Medline]
Cranage, M. P., Whatmore, A. M., Sharpe, S. A., Cook, N., Polyanskaya, N., Leech, S., Smith, J. D., Rud, E. W., Dennis, M. J. & Hall, G. A. (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.
Dunn, C. S., Hurtrel, B., Beyer, C., Gloeckler, L., Ledger, T. N., Moog, C., Kieny, M. P., Mehtali, M., Schmitt, D. & other authors (1997). Protection of SIVmac-infected macaque monkeys against superinfection by a simian immunodeficiency virus expressing envelope glycoproteins of HIV type 1. AIDS Res Hum Retroviruses 13, 913–922.[Medline]
Gauduin, M. C., Glickman, R. L., Ahmad, S., Yilma, T. & Johnson, R. P. (1999). Immunization with live attenuated simian immunodeficiency virus induces strong type 1 T helper responses and β-chemokine production. Proc Natl Acad Sci U S A 96, 14031–14036.
Jin, X., Bauer, D. E., Tuttleton, S. E., Lewin, S., Gettie, A., Blanchard, J., Irwin, C. E., Safrit, J. T., Mittler, J. & other authors (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., 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]
Johnson, R. P., Lifson, J. D., Czajak, S. C., Cole, K. S., Manson, K. H., Glickman, R., Yang, J., Montefiori, D. C., Montelaro, R. & other authors (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., Kitchin, P., Mills, K. H., Page, M., Taffs, F., Corcoran, T., Silvera, P., Flanagan, B., Powell, C. & other authors (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 Retroviruses 10, 189–194.[Medline]
Lackner, A. A., Vogel, P., Ramos, R. A., Kluge, J. D. & Marthas, M. (1994). Early events during infection with pathogenic (SIVmac239) and nonpathogenic (SIVmac1A11) molecular clones of simian immunodeficiency virus. Am J Pathol 145, 428–439.[Abstract]
Lama, J. (2003). The physiological relevance of CD4 receptor down-modulation during HIV infection. Curr HIV Res 1, 167–184.[CrossRef][Medline]
Li, Q., Duan, L., Estes, J. D., Ma, Z. M., Rourke, T., Wang, Y., Reilly, C., Carlis, J., Miller, C. J. & Haase, A. T. (2005). Peak SIV replication in resting memory CD4+ T cells depletes gut lamina propria CD4+ T cells. Nature 434, 1148–1152.[Medline]
Lohman, B. L., McChesney, M. B., Miller, C. J., McGowan, E., Joye, S. M., Van Rompay, K. K., Reay, E., Antipa, L., Pedersen, N. C. & Marthas, M. L. (1994). A partially attenuated simian immunodeficiency virus induces host immunity that correlates with resistance to pathogenic virus challenge. J Virol 68, 7021–7029.
Mackay, G. A., Liu, Z., Singh, D. K., Smith, M. S., Mukherjee, S., Sheffer, D., Jia, F., Adany, I., Sun, K. H. & other authors (2004). Protection against late-onset AIDS in macaques prophylactically immunised with live simian HIV vaccine was dependent on persistence of the vaccine virus. J Immunol 173, 4100–4107.
Marthas, M. L., Ramos, R. A., Lohman, B. L., Van Rompay, K. K., Unger, R. E., Miller, C. J., Banapour, B., Pedersen, N. C. & Luciw, P. A. (1993). Viral determinants of simian immunodeficiency virus (SIV) virulence in rhesus macaques assessed by using attenuated and pathogenic molecular clones of SIVmac. J Virol 67, 6047–6055.
Mascola, J. R., Lewis, M. G., VanCott, T. C., Stiegler, G., Katinger, H., Seaman, M., Beaudry, K., Barouch, D. H., Korioth-Schmitz, B. & other authors (2003). Cellular immunity elicited by human immunodeficiency virus type 1/simian immunodeficiency virus DNA vaccination does not augment the sterile protection afforded by passive infusion of neutralizing antibodies. J Virol 77, 10348–10356.
Matano, T., Shibata, R., Siemon, C., Connors, M., Lane, H. C. & Martin, M. A. (1998). Administration of an anti-CD8 monoclonal antibody interferes with the clearance of chimeric simian/human immunodeficiency virus during primary infections of rhesus macaques. J Virol 72, 164–169.
Mattapallil, J. J., Douek, D. C., Hill, B., Nishimura, Y., Martin, M. & Roederer, M. (2005). Massive infection and loss of memory CD4+ T cells in multiple tissues during acute infection. Nature 434, 1093–1097.[CrossRef][Medline]
McMichael, A. J. (2006). HIV vaccines. Annu Rev Immunol 24, 227–255.[CrossRef][Medline]
Nilsson, C., Makitalo, 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-specific 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]
Paul, W. E. (1995). Can the immune response control HIV infection? Cell 82, 177–182.[CrossRef][Medline]
Rose, J., Silvera, P., Flanagan, B., Kitchin, P. & Almond, N. (1995). The development of PCR based assays for the detection and differentiation of simian immunodeficiency virus in vivo. J Virol Methods 51, 229–239.[CrossRef][Medline]
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.
Sakuma, R., Mael, A. & Ikeda, Y. (2007). Alpha interferon enhances TRIM5-mediated antiviral activities in human and rhesus monkey cells. J Virol 81, 10201–10206.
Sarkar, S., Kalia, V., Murphey-Corb, M. & Montelaro, R. C. (2002). Detailed analysis of CD4(+) Th responses to envelope and Gag proteins of simian immunodeficiency virus reveals an exclusion of broadly reactive Th epitopes from the glycosylated regions of envelope. J Immunol 168, 4001–4011.
Schmitz, J. E., Kuroda, M. J., Santra, S., Sasseville, V. G., Simon, M. A., Lifton, M. A., Racz, P., Tenner-Racz, K., Dalesandro, M. & other authors (1999). Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes. Science 283, 857–860.
Schmitz, J. E., Johnson, P. R., McClure, H., Manson, K. M., Wyand, M., Kuroda, M., Lifton, M. A., Khunkhun, R. S., McEvers, K. J. & other authors (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.
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. A., Cope, A., Dowall, S., Berry, N., Ham, C., Heeney, J. L., Hopkins, D., Easterbrook, L., Dennis, M. & other authors (2004). Macaques infected long-term with attenuated simian immunodeficiency virus (SIVmac) remain resistant to superinfection despite declining cytotoxic T lymphocyte responses to an immunodominant epitope. J Gen Virol 85, 2591–2602.
Silvera, P., Wade-Evans, A., Rud, E., Hull, R., Silvera, K., Sangster, R., Almond, N. & Stott, J. (2001). Mechanisms of protection induced by live attenuated simian immunodeficiency virus: III. Viral interference and the role of CD8+ T-cells and β-chemokines in the inhibition of virus infection of PBMCs in vitro. J Med Primatol 30, 1–13.[CrossRef][Medline]
Stebbings, R., Stott, J., Almond, N., Hull, R., Lines, J., Silvera, P., Sangster, R., Corcoran, T., Rose, J. & other authors (1998). Mechanisms of protection induced by attenuated simian immunodeficiency virus. II. Lymphocyte depletion does not abrogate protection. AIDS Res Hum Retroviruses 14, 1187–1198.[Medline]
Stebbings, R. J., Almond, N. M., Stott, E. J., Berry, N., Wade-Evans, A. M., Hull, R., Lines, J., Silvera, P., Sangster, R. & 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]
Stebbings, R., Berry, N., Stott, J., Hull, R., Walker, B., Lines, J., Elsley, W., Brown, S., Wade-Evans, A. & other authors (2004). Vaccination with live attenuated simian immunodeficiency virus for 21 days protects against superinfection. Virology 330, 249–260.[CrossRef][Medline]
Stebbings, R., Berry, N., Waldmann, H., Bird, P., Hale, G., Stott, J., North, D., Hull, R., Hall, J. & other authors (2005). CD8+ lymphocytes do not mediate early protection against superinfection by inoculation with a live attenuated simian immunodeficiency virus. J Virol 79, 12264–12272.
Stott, E. J., Chan, W. L., Mills, K. H., Page, M., Taffs, F., Cranage, M., Greenaway, P. & Kitchin, P. (1990). Preliminary report: protection of cynomolgus macaques against simian immunodeficiency virus by fixed infected-cell vaccine. Lancet 336, 1538–1541.[CrossRef][Medline]
Veazey, R. S., DeMaria, M., Chalifoux, L. V., Shvetz, D. E., Pauley, D. R., Knight, H. L., Rosenzweig, M., Johnson, R. P., Desrosiers, R. C. & Lackner, A. A. (1998). Gastrointestinal tract as a major site of CD4+ T cell depletion and viral replication in SIV infection. Science 280, 427–431.
Wang, F. X., Huang, J., Zhang, H., Ma, X. & Zhang, H. (2008). APOBEC3G upregulation by alpha interferon restricts human immunodeficiency virus type 1 infection in human peripheral plasmacytoid dendritic cells. J Gen Virol 89, 722–730.
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]
Wyand, M. S., Manson, K., Montefiori, D. C., Lifson, J. D., Johnson, R. P. & Desrosiers, R. C. (1999). Protection by live, attenuated simian immunodeficiency virus against heterologous challenge. J Virol 73, 8356–8363.
Received 3 March 2008;
accepted 28 April 2008.
This article has been cited by other articles:
|
|
 |
|
  A.-S. Beignon, K. Mollier, C. Liard, F. Coutant, S. Munier, J. Riviere, P. Souque, and P. Charneau Lentiviral Vector-Based Prime/Boost Vaccination against AIDS: Pilot Study Shows Protection against Simian Immunodeficiency Virus SIVmac251 Challenge in Macaques J. Virol., November 1, 2009; 83(21): 10963 - 10974. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. J. Burwitz, C. J. Pendley, J. M. Greene, A. M. Detmer, J. J. Lhost, J. A. Karl, S. M. Piaskowski, R. A. Rudersdorf, L. T. Wallace, B. N. Bimber, et al. Mauritian Cynomolgus Macaques Share Two Exceptionally Common Major Histocompatibility Complex Class I Alleles That Restrict Simian Immunodeficiency Virus-Specific CD8+ T Cells J. Virol., June 15, 2009; 83(12): 6011 - 6019. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. W. Yeh, P. Jaru-ampornpan, D. Nevidomskyte, M. Asmal, S. S. Rao, A. P. Buzby, D. C. Montefiori, B. T. Korber, and N. L. Letvin Partial Protection of Simian Immunodeficiency Virus (SIV)-Infected Rhesus Monkeys against Superinfection with a Heterologous SIV Isolate J. Virol., March 15, 2009; 83(6): 2686 - 2696. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
