In vitro replication kinetics of human immunodeficiency virus type 1 (HIV-1)
variants in relation to virus load in long-term survivors of HIV-1 infection
Blaak, H.; Brouwer, M.; Ran, L.J.; de Wolf, F.; Schuitemaker, J.
DOI
10.1086/514219
Publication date
1998
Published in
The Journal of Infectious Diseases
Link to publication
Citation for published version (APA):
Blaak, H., Brouwer, M., Ran, L. J., de Wolf, F., & Schuitemaker, J. (1998). In vitro replication
kinetics of human immunodeficiency virus type 1 (HIV-1) variants in relation to virus load in
long-term survivors of HIV-1 infection. The Journal of Infectious Diseases, 177, 600-610.
https://doi.org/10.1086/514219
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In Vitro Replication Kinetics of Human Immunodeficiency Virus Type 1 (HIV-1)
Variants in Relation to Virus Load in Long-Term Survivors of HIV-1 Infection
Hetty Blaak, Margreet Brouwer, Leonie J. Ran, Department of Clinical Viro-Immunology, Central Laboratory of theNetherlands Red Cross Blood Transfusion Service, Laboratory of
Frank de Wolf, and Hanneke Schuitemaker
Experimental and Clinical Immunology of the University of Amsterdam, and Department of Human Retrovirology, University of Amsterdam, Academic Medical Centre, Amsterdam, The Netherlands In 7 long-term survivors (LTS) and 8 progressors, all carrying solely non – syncytium-inducing
variants, a possible correlation between in vitro virus replicative capacity, virus load, and clinical course of human immunodeficiency virus type 1 (HIV-1) infection was analyzed. Late in infection, 3 LTS and 7 progressors had a high virus load, which coincided with the presence of rapid-replicating viruses. In contrast to progressors, LTS maintained relatively high and stable CD4 T cell counts. Four LTS persistently had relatively slow-replicating viruses and a low virus load, even after 6.6 – 9 years of seropositive follow-up. All virus isolates from 1 of these LTS had a 4-aa deletion in nef. These results suggest a correlation between the in vitro replicative capacity of non – syncytium-inducing HIV-1 variants and virus load. The presence of HIV-1 variants with relatively low replica-tive capacity throughout infection may have contributed to the beneficial clinical course in half of the LTS in this study.
Some human immunodeficiency virus type 1 (HIV-1) – in- Next to the immune response, host genetic factors may in-fluence the length of the asymptomatic period. Several combi-fected persons maintain high and stable CD4 T cell counts and
nations of histocompatibility complex genes [9] are associated normal T cell function and remain free of clinical symptoms for
with the length of the asymptomatic period. Also, heterozygos-prolonged periods of time [1]. Concomitantly, these persons,
ity for a 32-bp deletion in the gene of one of the cofactors classified as long-term survivors of HIV-1 infection (LTS),
for HIV-1 entry, the C-C chemokine receptor 5 [10 – 13], was have lower virus load than do persons with progressive disease
associated with longer AIDS-free survival [14, 15]. [2 – 4].
Viral characteristics, such as cytotropism, cytopathicity, and The absence of progression in LTS may be determined by
replicative capacity, have been associated with different stages an interplay between host factors and viral factors. Both
hu-of infection, suggesting an important role for HIV-1 phenotype moral and cellular immune responses have been implicated
in the clinical course of HIV infection [16 – 23]. On sexual, in long-term survival. In LTS, high titers of antibodies with
vertical, and parenteral transmission, infection seems to be initi-neutralizing activity have been detected [2, 3], although other
ated by macrophage-tropic variants [18, 24], while a shift from studies reported no differences in neutralizing capacity between
preferentially macrophage-tropic to preferentially T cell – tropic antibodies derived from LTS and progressors [4, 5]. Strong
HIV-1 populations is correlated with progression to disease and persistent cytotoxic T lymphocyte (CTL) responses against
[19]. In 50% of persons, this shift is associated with the emer-several HIV-1 epitopes have been detected in LTS; however,
gence of syncytium-inducing (SI) variants [25]. Persons devel-vigorous CTL responses are also readily detected during the
oping SI variants show an increased virus load, accelerated asymptomatic phase in progressors [6 – 8].
CD4 T cell loss, and rapid progression to AIDS compared with persons harboring solely non – syncytium-inducing (NSI) variants [21, 25 – 27].
Received 30 January 1997; revised 23 September 1997.
LTS in general and 50% of all persons who do progress to
This study was performed as part of the Amsterdam Cohort Studies on
AIDS carry solely NSI variants, suggesting that other viral
AIDS, a collaboration between the Municipal Health Service, the Academic
Medical Centre, and the Central Laboratory of the Netherlands Red Cross characteristics might contribute to the differences in the clinical
Blood Transfusion Service, Amsterdam, Netherlands. Written informed con- course of infection. Indeed, replication capacity of HIV-1 has sent was obtained from all participants. In the conduct of clinical research,
been shown to be associated with virus load, CD4 T cell
de-human experimentation guidelines of the authors’ institutions were followed.
Grant support: Netherlands Foundation for Preventive Medicine (grant no. cline, and disease progression [16, 17, 21, 23]. In these studies,
28-2547), within the Stimulation Program AIDS Research of the Dutch Pro- however, the rapid replication was associated with the SI phe-gramme Committee for AIDS Research (grant no. 94013).
notype or the phenotype was not determined. Whether
differ-Reprints or correspondence: Dr. H. Schuitemaker, Dept. of Clinical
Viro-ences in replicative capacity among NSI variants might account
Immunology, Central Laboratory of the Netherlands Red Cross Blood
Transfu-sion Service, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands for the differences in the course of infection between persons (clbkvi@xs4all.nl).
harboring solely NSI variants had not been studied before.
The Journal of Infectious Diseases 1998; 177:600 – 10
We hypothesized that NSI variants in LTS, in contrast to
q 1998 by The University of Chicago. All rights reserved.
136 months of follow-up, after a 10-year period of high and stable
reduced replicative capacity throughout the infection, thereby
CD4 T cell counts: ACH617 [P3]).
enabling LTS to maintain low virus load and high CD4 T cell
From L1 – L7 and P1 – P3, at least 2 peripheral blood
mononu-counts. Therefore, we analyzed virus load in relation to the in
clear cell (PBMC) samples were used for analysis. The first sample
vitro replicative capacity of biologic virus clones obtained early
was obtained as early as possible after seroconversion or entry in
and late in infection from 7 LTS and 8 persons who progressed
the cohort studies. The other sample was obtained as late as
possi-to AIDS in the presence of solely NSI variants. In addition,
ble, which wasÇ9 years after seroconversion or entry for most
since defective nef genes have been implicated as being instru- LTS, 6.6 years for L4, and about the time of AIDS diagnosis for mental for low replicative capacity and long-term survival [28 – the progressors. From P4 – P8, a sample derived from a time point 31], the nef sequences of the biologic variants isolated from close to AIDS diagnosis was analyzed (table 1).
LTS and progressors in this study were analyzed. Analysis of CD4 T cell counts. T lymphocyte immunophenotyp-ing for the CD4 T cells was carried out at 3-month intervals by flow cytofluorometry. PBMC were stained with CD4 monoclonal antibody according to standard procedures for cell cytometric analysis.
Materials and Methods
Virus isolation and determination of infectious cellular virus load. Virus was isolated under limiting diluting conditions as
Subjects. By October 1994, 12 participants of the Amsterdam
previously described [19, 27]. Briefly, participant PBMC (0.5 – Cohort studies on AIDS had an asymptomatic follow-up of at least
4 1 104
cells/well in 48 or 96 wells) were cocultivated with 9 years with a relatively normal mean CD4 T cell count (ú400/
phytohemagglutinin (PHA)-stimulated healthy donor PBMC (105/ mm3). Additionally, these persons harbored solely NSI variants
well) in 96-well microtiter plates. Every week, culture supernatants and did not receive any anti-HIV drug treatment during the course
were tested for p24 antigen by an in-house p24 antigen-capture of infection. These persons were termed LTS [1]. Seven of these
ELISA [32]. At the same time, one-third of the culture volume 12 LTS were studied here (ACH78 [L1], ACH441 [L2], ACH583
was transferred to new 96-well plates, and 105
fresh PHA-stimu-[L3], ACH709 [L4], ACH68 [L5], ACH337 [L6], ACH750 [L7]).
lated healthy donor PBMC were added to propagate the culture. L5 underwent splenectomy 72 months after entry in the cohort
The proportion of productively infected CD4 T cells was calculated studies.
with the formula for Poisson distribution, FÅ 0ln(F0), in which For comparison, 8 progressors were selected on the basis of the
F0is the fraction of negative cultures. presence of solely NSI variants during their clinical course. Three
PBMC from wells tested positive were transferred to 25-mL of them were rapid progressors (AIDS diagnosis after 43 – 76
culture flasks containing 51 106fresh PHA-stimulated PBMC in months of follow-up: ACH424 [P1], ACH537 [P4], ACH53 [P5]),
5 mL of medium to grow virus stocks. From these cultures, the 4 were typical progressors (AIDS diagnosis after 99 – 109 months
cell-free supernatant was stored at0707C until use. One million of follow-up: ACH19 [P2], ACH38 [P6], ACH1081 [P7], ACH142
[P8]), and 1 was termed a slow progressor (AIDS diagnosis after infected PBMC were used for DNA analysis. To the remaining
Table 1. Characteristics of subjects in HIV-1 study.
Early sample Late sample
Interval Disease stage by end of Months after No. of biologic Months after No. of biologic between Entry Date of entry or follow-up†
(months after seroconversion virus clones seroconversion virus clones samples Participant* serostatus seroconversion seroconversion or entry) or entry analyzed or entry analyzed (months)
L1 / 8 Nov 84 Asypmtomatic (124) 17 2 115 5 98 L2 / 21 Jan 85 Asymptomatic (149) 16 3 111 6 95 L3 / 18 Feb 85 Asymptomatic (138) 24 3 109 5 85 L4 0 14 Aug 85 Asymptomatic (122) 43 2 80 3 37 L5 / 6 Nov 84 Asymptomatic (143) 18 1 100 5 82 L6 / 2 Jan 85 Asymptomatic (138) 24 4 113 5 89 L7 / 23 Mar 85 Asymptomatic (148) 16 5 112 5 96 P1 0 14 Mar 88 AIDS (43) 6 2 43 5 37 P2 / 25 Oct 84 AIDS (99) 24 5 98 5 74 P3 / 27 Feb 85 AIDS (136) 15 2 111 5 96 P4 0 14 Jul 86 AIDS (43) NA NA 37 4 NA P5 / 5 Nov 84 AIDS (76) NA NA 77 6 NA P6 / 24 Nov 84 AIDS (100) NA NA 102 5 NA P7 0 22 Sep 85 AIDS (101) NA NA 94 6 NA P8 / 22 Nov 84 AIDS (109) NA NA 93 6 NA
NOTE. NA, not applicable.
* Long-term survivors (L) or progressor to AIDS (P).
†
PBMC, MT2 cells (11 106
) were added to analyze syncytium- buffer containing guanidinethiocyanate, Triton X-100, and Tris-HCl. Three synthetic RNAs (QA, QB, QC) of known high, me-inducing capacity of the virus clones [33].
Quantification of RNA in serum. HIV-1 RNA was quantified dium, and low concentration, respectively, were added to the lysis buffer containing the released nucleic acid. These Q-RNAs served in serum by using a nucleic acid sequence – based amplification
assay (HIV-1 RNA QT; Organon Teknika, Boxtel, Netherlands). as internal calibrators, each differing from the HIV-1 wild type RNA by only a small sequence [34].
their replicative capacity. From most persons,õ5 clones from the early time point were analyzed (table 1), becauseõ5 virus clones could be recovered as a result of low virus load. The selected viruses had different time points of first detection during clonal isolation. The titer of the virus stocks was quantified by determina-tion of the TCID50in PHA-stimulated healthy donor PBMC.
From each virus clone, 1250 TCID50 was added to 5 1 106 PHA-stimulated PBMC derived from the same donor on which the TCID50was determined. To keep the volume of the inocula from exceeding 1.25 mL, virus stocks withõ103TCID
50/mL were excluded from replication analysis. Virus and PBMC were incu-bated in a 1.5-mL volume for 2 h at 377C. PBMC were then washed, resuspended in 5 mL of fresh recombinant interleukin-2 (interleukin-20 U/mL; PROLEUKIN; Chiron, Amsterdam) – supplemented medium, and cultured for 16 days.
To determine the kinetics of virus production, 75 mL of the culture supernatant was harvested every second day and stored at 47C until analysis. At day 8 of culture, 4 1 106 freshly PHA-stimulated PBMC in 2 mL of medium were added to the cultures. Supernatants harvested at all different time points were analyzed for the presence of virus in an in-house p24 antigen – capture ELISA [32]. p24 production per milliliter of supernatant was
deter-Figure 1. Replication kinetics of biologic virus clones. Healthy do- mined and corrected for the differences in volume of culture
super-nor peripheral blood mononuclear cells that had been stimulated with natants between the moments of sampling.
phytohemagglutinin were infected with 1250 TCID50of biologic virus nef sequence analysis. From 106 infected PBMC, obtained
clones obtained from LTS (A) and disease progressors (B) early (I)
after propagation of the clonal virus stocks, proviral DNA was
and late (II) in infection. Cultures were maintained for 16 days, and
isolated. PBMC were lysed in L6 lysis buffer (0.08 M GuSCN
new target cells were added on day 8. p24 production in supernatant
[Life Technologies Gibco BRL, Gaithersburg, MD], 0.08 M
Tris-harvested every second day was measured. Replication phenotype
Cl, pH 6.4, 0.035 M EDTA, 2% (wt/vol) Triton X-100) [35] and
(C) was based on mean values for increase in p24 production during
stored at0707C until use. DNA was precipitated by addition of
days 4 and 8 of culture and maximal p24 production reached within
14 days of culture. Open symbols, slow-replicating; solid symbols, isopropanol in a 1:1 ratio (vol/vol) and centrifugation for 15 min
rapid-replicating; shaded symbols, intermediate. at 13,000 g. DNA pellets were washed twice with 70% ethanol and resuspended in water.
nef DNA was amplified by a nested polymerase chain reaction
Subsequently, RNA was isolated as previously described [4].
(PCR) with primers Nef A (nt 2408 – 2428 in relation to the SF2 Amplification of wild type HIV-1 RNA and Q-RNAs is based on
env sequence: 5*-GTCTAGAACTAAAGAATAGTG-3*, sense primer extension of primer 1 (nt 682 – 711 in relation to the HX2B
orientation) and LTR BCAT (nt 519 – 545 of the LTR sequence: 5 *-gag sequence,
5*-ACTCTCTTGGTTCCCCTTCACTGTATCGT-GCACTCAAGGCAAGCTTTATTGAGGC-3*, antisense) in the
GCCATATCACTCAGCATAATCTTAA-3*; antisense; T7-RNA
first reaction and primers Nef B (nt 2526 – 2546 of the env se-polymerase recognition site underlined), by avian myeloblastosis
quence: 5*-ATCTAGAAGAATAAGACAGGG-3*, sense) and Nef virus reverse transcriptase (AMV-RT). Extension is followed by
C (nt 350 – 370 of the LTR sequence: 5 *-AAGTCTAGAGCG-degradation of the template RNAs by RNase H, synthesis of the
GAAAGTCCC-3*, antisense) for the second reaction. second DNA strand through extension of primer 2 (nt 569 – 590: 5
*-For both reactions, DNA was denatured for 5 min at 957C,
AGTGGGGGGGACATCAAGCAGCCATGCAAA-3*) by
AMV-followed by 30 cycles of 1.5 min of denaturation at 957C, 1.5 min RT, and cyclic and isothermal (417C for 90 min) RNA synthesis
of annealing at 487C, and 1.5 min of extension at 727C, and a by T7-RNA polymerase.
subsequent extra 5-min extension at 727C and soaking at 47C. Five Amplificates were hybridized with an HIV-1 – specific
bead-microliters of DNA was amplified in 50-mL reactions containing oligo (i.e., a biotin-oligo bound to streptavidin-coated magnetic
11 Taq buffer (Promega, Madison, WI), 0.2 mM each dNTP, 100 beads acting as solid phase) and ruthenium-labeled probes, each
ng of each primer, 1.5 mM MgCl2, and 1 U of Taq DNA polymer-specific for the wild type and control amplificates. Magnetic beads
ase (Promega). Five microliters of the first reaction was used as carrying the hybridized amplificate-probe complex are captured
input for the nested PCR reaction. PCR products were purified by on the surface of an electrode by means of a magnet. Voltage
use of a spin PCR purification kit (Qiagen, Chatsworth, CA). applied to this electrode triggers the electrochemoluminescence
The positive strands were sequenced by use of Sequenase DNA reaction, and the light emitted by the hybridized ruthenium-labeled
polymerase (United States Biochemicals, Cleveland) with primers probes is proportional to the amount of amplificate.
Nef B, LTR 1A (nt 412 – 438 of the nef sequence: 5*-AGATAT-On the basis of the relative amounts of the four amplificates,
CCACTGACCTTTGGATGGTGC-3*), and Nef 1C (nt 96–126 of the original amount of wild type RNA in the sample was calculated.
the nef sequence: 5
*-AGCATCTCGAGACCTGGAAAAACA-Testing of replicative capacity of virus clones. From each
Both DNA purification and sequencing procedures were per- Virus load in relation to replicative capacity of HIV-1. To
formed according to the instructions of the manufacturer. study whether the replicative capacity of HIV-1 variants
corre-Statistical analysis. Differences between the 3 groups of phe- lated with virus load, the infectious cellular load at the time notypically distinct virus variants in the increase in p24 production points of virus isolation was calculated. The contribution of between days 4 and 8 of culture and the maximal p24 production
HIV-1 variants with different replicative capacity to the virus
were analyzed by one-way analysis of variance. The
Mann-Whit-load was estimated for each participant at each time point.
ney U test was used to analyze the relationship between the
pres-This was achieved by extrapolating the relative contribution of
ence of rapid-replicating virus variants and the infectious cellular
rapid-, intermediate-, and slow-replicating viruses to the total
load. The correlation between the infectious load and the RNA
infectious cellular load (figure 2).
load in serum was analyzed by use of Spearman’s correlation
Early in infection, all participants had a low cellular virus
coefficient.
load (1 – 16 TCID/106CD4 T cells), which consisted of
slow-and/or intermediate-replicating viruses in most persons. The 2 LTS (L6 and L7) in whom viruses with high replicative
capac-Results
Correlation between HIV-1 replication characteristics and clinical course. Biologic virus clones of 7 LTS and 3 prog-ressors, obtained from PBMC that originated from an early and a late time point during follow-up (table 1), were analyzed with respect to their replicative capacity (figure 1A, B). The replicative capacity of each virus was reflected by the rate of p24 accumulation early in culture (between days 4 and 8) and the maximum levels of p24 production reached within 14 days of culture.
The mean increase in p24 production between days 4 and 8 of culture (0.25 mg/mL) and the mean maximal p24 production (1.26 mg/mL) of all viruses analyzed were used as reference for determining the replication phenotype of each single virus clone (figure 1C). Variants with a p24 increase between days 4 and 8 and a maximal p24 production above average were typed rapid (upper right quadrant in figure 1C; mean values, 0.5 mg/mL and 1.6 mg/mL, respectively); variants with both measures below average were typed slow (lower left quadrant in figure 1C; mean values, 0.1 mg/mL and 0.9 mg/mL). Viruses with only one measure above average were typed as variants with intermediate replicative capacity (upper left and lower right quadrants; mean values, 0.2 mg/mL and 1.2 mg/mL). The mean values of both measures were significantly different be-tween the 3 phenotypically distinct groups (Põ .001).
At the early time point, all participants carried viruses with relatively low replication kinetics. Only LTS L6 and L7 addi-tionally had viruses with high replication kinetics at this time point. In 4 of the 8 LTS (L1 – L4), all late-stage viruses that were analyzed also had relatively low replication kinetics; L4 even seemed to have developed less-replication-competent vi-ruses over time.
Three LTS (L5 – L7) and the progressors (P1 – P3) had vi-ruses with high replicative capacity late in infection. In these subjects (with the exception of L7), the proportion of rapid-replicating viruses had increased compared with that at the
Figure 2. Contribution of biologic virus clones with different
repli-early time point, suggesting a shift to
more-replication-compe-cative capacity to infectious cellular load expressed as TCID/106
CD4
tent variants during the course of infection. In L6 and P2, all
T cells. Proportion of each phenotype (open bars, slow-replicating;
late-stage virus variants analyzed had high replication kinetics, solid bars, rapid-replicating; shaded bars, intermediate) within ana-while in the other subjects, viruses with high replication kinet- lyzed group of viruses was extrapolated to total cellular load early
(A) and late (B) in infection.
ity were detected had the highest infectious cellular load (fig- course at a time point close to AIDS diagnosis (P4 – P8; table 1). For each subject, the infectious cellular load at this time ure 2A).
Because of the low virus load, õ5 biologic clones were point, consisting solely of NSI variants, is given in table 2. For comparison of replicative capacity, slow- (nÅ 7) and rapid-obtained at the early time point for most persons (table 1).
From 3 of these (L4, L5, and P3), the replication kinetics of replicating viruses (n Å 7) from the first experiment were included.
virus clones from an additional time point were analyzed,Ç1
year after the early time point. Also at this time point, only In contrast to the rapid-replicating control viruses, which first showed detectable p24 production at days 2 –6 of culture, the viruses with relatively low replicative capacity were present
(data not shown), and the cellular virus load was still low (see slow-replicating control viruses did not produce detectable p24 levels until days 10–14 of culture. Therefore, the viruses from also figure 3).
The 4 LTS (L1 – L4) who only had slow- and intermediate- P4–P8 were not classified by the combination of their increase in p24 production between days 4 and 8 of culture and the maxi-replicating viruses late in infection also maintained a low
infec-tious cellular load (1 – 29 TCID/106CD4 T cells). Interestingly, mum p24 production, as in the first experiment, but by the
combi-nation of the first day of detection and maximal p24 production all 3 progressors and the 3 LTS (L5 – L7) with rapid-replicating
variants late in infection had a high cellular load at this time relative to the means of the control viruses (day 8 and 0.2 mg/ mL respectively). In progressors P5–P8, but not in P4, who had point (83 – 288 TCID/106CD4 T cells) (figure 2B).
We additionally analyzed the replicative capacity of biologic a low infectious cellular load at the time point of analysis, viruses with high replicative capacity were detected (table 2).
virus clones derived from 5 persons with a progressive disease
Figure 3. Longitudinal analysis of CD4 T cell counts and virus load. Nos. of CD4 T cells (r) were routinely measured every 3 months during follow-up. Infectious cellular load (l) was calculated from outcome of clonal isolation procedure and is expressed as TCID/106
CD4 T cells. HIV-1 RNA copy numbers (s) were determined by nucleic acid sequence–based amplification assay. Follow-up is indicated in months after HIV-1 seroconversion or seropositive entry in cohort studies. Arrowheads on x-axis indicate time points from which biologic virus clones analyzed for replicative capacity were obtained.
Table 2. Replicative capacity and infectious cellular load around AIDS diagnosis.
Replicative capacity, no. (%)
No. of biologic virus Infectious cellular load Participant clones analyzed Slow Intermediate Rapid (TCID/106CD4 T cells)
P4 4 0 4 (100) 0 32
P5 6 1 (16.67) 4 (66.67) 1 (16.67) 169
P6 5 1 (20) 3 (60) 1 (20) 182
P7 6 0 3 (50) 3 (50) 1543
P8 6 0 1 (16.67) 5 (83.33) 315
Both early and late in infection, the infectious cellular load Together, these data show that some LTS maintain high and relatively stable CD4 T cell counts for an extended period of was significantly higher in subjects with rapid-replicating
vi-ruses than in subjects from whom no rapid-replicating vivi-ruses time in the presence of high numbers of infected cells and relatively high levels of viral RNA in serum.
were detected (early median load, 12.5 [n Å 2] vs. 2.5 [n
Å 8]; P Å .04; late median load, 171 [n Å 10] vs. 27 [n Å 5]; nef sequence analysis. It has previously been demonstrated that some LTS harbor HIV-1 variants with a defective nef gene
Põ .001).
Infectious cellular load and RNA load in serum. It could be [28 – 31]. We analyzed whether the slow replication kinetics of viruses studied here also resulted from the presence of defective argued that a high infectious cellular load, in particular the high
load observed in 3 of the LTS, might be an in vitro artifact nef genes. nef sequences of the slow-replicating biologic virus
clones of L1 – L4 were analyzed. In addition, nef sequences of caused by activation of latently infected cells during culture
procedures. Therefore, we analyzed the RNA levels in serum both slow- and rapid-replicating virus clones of L6 and of progressors P1 – P3 were analyzed (figure 4).
samples obtained at the late time points for all study participants
(with the exception of P6). In agreement with our previous Intersubject variation in nef sequences was observed, yet previously described functional domains [37] were generally observations [36], the log-transformed values of both measures
of virus load were significantly correlated (Spearman correlation intact. No specific mutations could be related to the viruses from either progressors or LTS nor to slow- or rapid-replicating coefficientÅ .76, P Å .002). The low infectious cellular load
in L1 – L4 was accompanied by a low RNA serum load (õ104 viruses. In 4 subjects, the majority of viruses had an insertion
of 4 aa at position 25. However, this insertion was observed RNA copies/mL of serum), while L5 – L7 and the progressors
had both a high infectious cellular and a high serum RNA load in viruses with different phenotypes. Mutations in the (PxxP)3
domain (proline repeat sequence; position 69 – 78) were ob-(ú104RNA copies/mL of serum) (see also figure 3).
Virus load in relation to clinical course. The finding of served in all viruses of L1 and in all late viruses of L2. The R-to-K substitution at position 71 in this domain has been LTS with high virus load raised the question of whether this
high load reflected the onset of disease progression in these described previously for variants from LTS [37], although the
nef sequences of these particular variants were shown to be
subjects. However, during the 1- to 2-year follow-up period
beyond the late time point, the CD4 T cell counts in L5 – L7 functional in vitro [38]. All viruses of L3 had a cysteine at position 138, recently described to affect replication capacity remained relatively stable (figure 3). Analysis of the infectious
cellular virus load and viral RNA in serum at several additional in H9 cells [30]. However, this same mutation was also present in the nef of the rapid-replicating viruses isolated from the time points during follow-up of these LTS revealed a relatively
high cellular load (78 and 74 TCID/106CD4 T cells, respec- typical progressor P2 at the moment of AIDS diagnosis.
Possible dysfunctional nef genes were observed in the viruses tively), even at earlier time points in L6 and L7.
The time span between detection of a high load and the most with slow kinetics of L4, which had a 4-aa deletion at positions 156 – 159. This deletion was present in at least one of the viruses recent measurement of high CD4 T cell counts was 43, 70,
and 59 months for L5, L6, and L7, respectively. During this isolated from PBMC in 1989 and was still present in all 3 viruses isolated from PBMC in 1992.
period, the number of CD4 T cells remained relatively stable (decline 41, 13, and 1 CD4 T cell/mL/year for L5, L6, and L7 respectively). In contrast, the CD4 T cell decline observed in
Discussion
the progressors (P1 – P3 and P8) appeared to coincide with or
even precede the moment of first detection of high infectious A small number of HIV-infected persons harboring solely NSI variants show no signs of progression to disease. However, cellular load (CD4 T cell decline between moment of first
detection of high infectious cellular load and AIDS diagnosis: 50% of all AIDS patients also harbor solely NSI variants. The absence of SI variants therefore cannot be the sole explanation 117, 82, 97, and 66 CD4 T cells/mL/year for P1, P2, L3, and
Figure 4. nef consensus sequences of biologic virus clones derived early and late in infection from 3 progressors (P) and 5 LTS (L), aligned with consensus sequence (cons) derived from all clones. Locations of putative functional domains [37] are shown above consensus sequence. PxxP Å proline repeat sequence; PKC Å protein kinase C phosphorylation site; PPT Å polypurine tract. * 2 sequences of late time point are depicted, since both genotypes were representative for 50% of biologic virus clones analyzed (n Å 4).
factors [2, 6 – 8, 14, 15], viral characteristics other than syncy- sioned that increased viral replicative capacity, which causes the increase in virus load, is not necessarily associated with tium-inducing capacity might contribute to the differential
clin-ical course observed between HIV-1 – infected persons. Here increased cytopathicity of these viruses. Indeed, viruses iso-lated from persons with differing progression rates yet similar we tested the hypothesis that NSI variants present in LTS may
have relatively attenuated replication capacity and are unable virus load were found to be differentially cytopathic in a SCID-hu mouse model [51].
to achieve a high virus load in vivo, thereby explaining at least
in part the beneficial clinical course observed in LTS. After 6.6 (L4) or evenú9 years of infection, 4 LTS still harbored slow-replicating variants. To explain the absence of Biologic virus clones were isolated from 7 LTS and 8
sub-jects with progressive HIV-1 infection and analyzed for their evolution to variants with a more rapid phenotype, several hypotheses might be envisioned. First, viruses are under selec-replicative potential. Early in infection, all participants
ana-lyzed had relatively slow-replicating viruses. Two LTS who tive pressure by host cellular immune responses. The persisting slow-replicating viruses in our study subjects may be best had the highest infectious cellular load at the early point of
analysis additionally had rapid-replicating variants at this time adapted to withstand cellular immunity, whereas changes asso-ciated with more rapid replicative capacity may generate epi-point.
At the time point late in infection, still only relatively slow- topes that are well-recognized by CTL, causing immediate elimination of that variant. It has been postulated previously replicating viruses could be detected in 4 LTS. Accordingly,
these LTS maintained low levels of RNA in serum as well as that the presence of CTL directed against conserved epitopes prevents the evolution of virus populations [52]. In this view, a low infectious cellular load. This is in agreement with the
recently described low levels of viral mRNA transcripts late the slow-replicating variants may have mutations in relatively conserved sequences that allow them to escape immune surveil-in surveil-infection surveil-in LTS [39]. In contrast, from the other 3 LTS and
the progressors late in infection, either solely rapid-replicating lance yet at the same time result in severe attenuation. Next to specific immune pressure, viral characteristics may viruses or coexisting rapid- and relatively slow-replicating
vi-ruses were detected. These persons all had an increased virus play a role in virus evolution. It is conceivable that the virus variants that establish infection in a particular person, compared load, suggestive of a correlation between in vitro HIV-1
replica-tive capacity and virus load in vivo. with the initial variants in another person, are genetically closer to a genotype that is associated with more rapid replication. In Increasing cellular load as well as RNA load has been
associ-ated with CD4 T cell decline and disease progression [2, 3, this case, it would be just a matter of time to allow accumulation of relevant mutations [53]. Finally, low replication kinetics might 21, 27, 40 – 43]. It is therefore conceivable that the high load
in 3 of the LTS is a sign of progression. However, these 3 be due to aberrations in HIV-1 regulatory genes [54, 55]. The best-studied in this respect is the nef gene. The absence LTS maintained high and relatively stable CD4 T cell counts
for a subsequent 43 – 70 months, suggesting that some LTS of functional nef was associated with a lack of viral pathogenic-ity in the SCID-hu mouse model [56] and in simian immunode-remain healthy for prolonged periods of time despite the
pres-ence of rapid-replicating viruses and a high virus load. In sup- ficiency virus strain mac – infected rhesus monkeys [57]. This is in agreement with the observation that nef is required for port of this, Rump et al. [44] recently reported the existence
of LTS with continuously high virus load as measured by serum efficient replication in primary cells [58 – 60]. More recently,
nef was associated with HIV-1 pathogenesis in humans by the
p24 antigen.
The maintenance of relatively stable CD4 T cell numbers in finding that some LTS have HIV-1 variants with defective nef genes [28 – 31].
the face of increasing infectious load can have several
explana-tions. In agreement with the low frequencies of infected cells Some of the slow-replicating viruses in our study carried changes in nef. Some of these changes have been observed in lymph nodes [45], the magnitude of CD4 T cell turnover
seems to be much smaller [46] than previously suggested [47, before in LTS [37] but were not associated with attenuated function in vitro [38]. It has been suggested that a cysteine 48]. The loss of CD4 T cells might then be explained from a
combination of HIV-1 – mediated killing and a failing homeo- residue at position 138 may attenuate nef function [30]. How-ever, the presence of a cysteine at this position in nef of slow-stasis. The capacity by which new T cells can be generated
might be genetically determined, thereby contributing to differ- as well as rapid-replicating viruses in our study supports the finding that this mutation is irrelevant for nef function [38]. ences in CD4 T cell decline between individuals.
Additionally, the low degree of CD4 T cell depletion might With the exception of L4, who had viruses with a relatively large deletion in the nef gene, it seems unlikely that deviations be due to the absence of an effective anti-HIV CTL response
[49]. In support of this, only low frequencies of CTL precursors in nef contribute to the absence of disease progression in the group of LTS studied here.
directed against epitopes of multiple HIV-1 proteins have been
observed in participant L6 (Pontesilli O, personal communica- In conclusion, we have shown that some persons classified as LTS persistently harbored slow-replicating viruses associ-tion).
Finally, since viral cytopathicity may be a mechanism ated with a low virus load, which might explain the absence of progression in these persons. The existence of LTS with through which CD4 T cells are depleted [50], it could be
envi-14. Dean M, Carrington M, Winkler C, et al. Genetic restriction of HIV-1
rapid-replicating viruses and a high virus load, however,
sug-infection and progression to AIDS by a deletion allele of the CKR5
gests that the basis for long-term survival of HIV-1 infection
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