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Immunology Letters 57 (1997) 63–68
Dynamics of HIV variants and specific cytotoxic T-cell recognition in
nonprogressors and progressors
Gaby Haas *, Anne Hosmalin, Fabienne Hadida, Jo¨rg Duntze, Patrice Debre´, Brigitte Autran
Laboratoire d
’
Immunologie Cellulaire et Tissulaire,URA CNRS
625
,Baˆ timent CERVI,Hoˆpital Pitie´ -Salpe´trie`re,
75651
Paris Cedex
13
,France
Abstract
Infection with the human immunodeficiency virus (HIV) results in a disease characterized by a rapid viral replication,
immunodeficiency and chronic immune activation. The vigorous polyspecific cytotoxic T-cell (CTL) response directed against
multiple HIV epitopes reduces HIV-infected cell numbers, although unable to eradicate the virus. The plasticity of the specific
CTL repertoire ensures adaptation to the high rate of viral variation that can be found in CTL epitopes of several HIV-1 proteins.
However, viral persistence occurs despite continous CTL recognition and although functional importance of conserved sites in the
different HIV proteins may impose constraints to viral variation. In the reverse transcriptase (RT) which is a major target for
antiretroviral therapy, the impact of the continous pressure of drug therapy is more obvious than that of the CTLs. Shifts in
immunodominant RT regions seem to allow the maintenance of the HIV-1 RT CTL recognition with disease progression and
antiretroviral therapy. In respect to new highly active drug combinations, understanding the capacity of virus-specific CTLs to
control residual viral variants seems very important and may allow development of efficient immunotherapies to prevent
drug-induced viral resistance. © 1997 Elsevier Science B.V.
Keywords
:
AIDS; HIV; Cytotoxic T-cells; HIV variants; HIV-1-Nef; HIV-1-reverse transcriptase
1. Introduction
Most individuals who are infected with HIV first
undergo a phase of primary infection that is character-
ized by a strong viral replication rapidly followed by a
strong immune response. During the first month after
entry of the virus, a strong cytotoxic T-cell (CTL)
response develops even before the appearance of HIV-
specific antibodies. A second phase without clinical
symptoms, but with persistent viral replication, eventu-
ally results in the development of acquired immunodefi-
ciency syndrome (AIDS). Temporal association
between HIV-specific CTL activity and decrease in viral
load in several patients with primary infections indi-
cates a role for CTL in controlling the initial viremia
[1,2]. Polyclonal CTL responses directed concurrently
against multiple proteins and a variety of epitopes in
the context of several HLA molecules persist for several
years in patients that do not show signs of disease
progression [3–6]. There are few individuals with low
viral loads and high CD4 counts even after a long
history of HIV infection. CTLs can contribute to slow
down disease progression by multiple mechanisms: by
elimination of virus-producing cells and by production
of cytokines and suppressive factors.
The impact that HIV-specific CTLs may have on
viral variation is still controversial [7–10]. A delicate
balance between viral elimination and replication seems
to exist, but the factors that determine disease progres-
sion are not clear. However the high turn-over of HIV
and infected cells at all stages of the infection indicates
that CTLs are efficient at eliminating actively replicat-
ing virus in infected cells though unable to eradicate
latently infected cells [11,12]. These CTL responses,
even if protective, eventually fail in most cases at
preventing the reascension of viral load before the onset
* Corresponding author. Tel.: +33 1 42177403; fax: +33 1
42177490; e-mail: ghaas@ccr.jussieu.fr
0165-2478/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved.
PII
S01 5-2478(97)00076-X
G.Haas et al.
/
Immunology Letters
57 (1997) 63–68
64
of AIDS. In this article we consider the aspect of
specific CTL activity and viral variation.
2. Disease progression and HIV-specific CTL
High levels of HIV-specific effector CTLs are de-
tectable in the periphery and in the tissues of HIV
infected individuals and appear effective in maintaining
the chronic asymptomatic stage [13]. Indeed, we could
show that CTLs infiltrate infected tissues [3,14] with
exceptionally high frequencies of effector CTLs (10
−4
–
10
−6
) specific for HIV-nef epitopes in pauci-symp-
tomatic individuals [15]. Assuming a frequency of
HIV-infected cells of about 1×10
−3
, an in vivo ratio
of at least 1:1 should be established between effector
CTLs and target cells to ensure efficient killing of
infected cells. These in vivo activated CD8
+
CTLs also
produce IFN-g[16] but control of HIV seems to require
in addition their secretion of suppressive factors or
chemokines [17–19]. However these effector CTLs do
not eradicate HIV since a major viral burden persists in
CD4
+
T-cells from lymphoid organs, as shown by our
group [20]. HIV-specific CTL precursor (CTLp) and
memory cell frequencies also range from 10
−2
–10
−6
as
measured by different methods [6,21,22]. An inverse
correlation between CTL frequency and cell-associated
viremia, as well as broad CTL responses have been
associated with long-term nonprogression (LTNP) [23–
26], while a decline of gag-specific CTLp was shown to
coincide with a CD4 count drop, increasing viral load
and disease progression [21]. Moreover, recent associa-
tions between some HLA-class I or class II genes and
slow or lack of progression have recently been shown
[27]. On the other hand, presence of activated anti-HIV
CTLs may not be required for LTNP since no anti-HIV
CTLs were found in non-progressors with extremely
low viral load [24]. The persistent replication of HIV in
immunologically active tissues probably sustains per-
manent CTL activation, although the need for antigen
to maintain CTL memory is still a matter of debate
[28–30]. While neither a high viral load nor antigenic
diversity seemed to be required for generating multispe-
cific CTL responses, a threshold for the induction of
specific CTL may exist that is different in the various
individuals. Thus it appears difficult to evaluate protec-
tion conferred by HIV-specific CTLs on the single basis
of their correlation to viral load (Fig. 1). Finally the
question of which factors drive disease progression and
how the virus finally avoids immune control remains
debated. The continuous exponential viral replication
and generation of variants may simply overload the
capacities of specific CTLs or induce CTL anergy while
virus persistance in sanctuary cells may provide a reser-
voir.
3. CTL adaptation to natural viral variants
Rapid turnover of virus particles is accompanied by a
high degree of viral mutations, thus generating a broad
range of quasispecies with heterogeneous patterns of
immunogenicity. A dynamic interaction between viral
variants and the immune system seems to take place
after initial viral entry and spread, during which the
virus itself appears to influence the properties of im-
mune activation, processing and recognition of viral
antigens in an attempt to avoid the host immune re-
sponses [31]. In addition stochastic processes like anti-
gen-driven or cytokine-induced activation of viral
replication can also influence the evolution of viral
quasispecies and the impact on disease progression
remains unclear [8]. Changes in the viral population
seem already to occur early after primary infection [9]
and differences in viral diversity have been found in
correlation to rapid and slow disease progression [32].
Recent findings of Wolinsky et al. show that slow and
non-progressors display highly variable viral sequences
in the en6gene accompanied by strong CTL responses
specific for HIV-1 proteins, while rapid progressors
show rather limited variation and no CTL responses to
viral proteins [33]. In a longitudinal study in four
HIV-infected slow and nonprogressors, we could show
a high degree of viral variation in three CTL epitopes in
the regulatory protein HIV-1 Nef: variant-specific
CTLs seemed to adapt over time to the high rate of
viral variation in context of HLA-A2, A3 and B7 and
correlated with the elimination of such variants [34].
Such adaptation could also take advantage of switches
in HLA restriction from HLA-A2 to HLA-A3. Fre-
quencies of CTLs specific for new viral variants were
augmented over time from 30 per 10
6
to 100 per 10
6
concomitantly with the in vivo expansion of those
Fig. 1. Dynamics of spontaneous HIV variants and HIV-specific
CTL. A model for the evolution of spontaneous HIV variation and
the induction of specific CTL. After an initial expansion of HIV
during primary infection, epitope-specific CTLs arise and may be
detectable over years. Other viral variants arise in successive waves
and are followed by waves of specific CTLs. The whole set of
variant-specific CTLs forms polyspecific responses that decline
only at late stages of disease.
G.Haas et al.
/
Immunology Letters
57 (1997) 63–68
65
Fig. 2. Evolution of natural and drug-induced mutations and induction of HIV-specific CTLs. Left panel: (A) Examples for variation and
persistence of major HIV-1 Nef PCR clones (,A and B). (B) Specific CTL adapted to viral variants from 1992 to 1994. Right panel: (C)
RT variants changed from wildtype RT 215 T to mutant RT215 Y in the course of AZT therapy. (D) Polyclonal CTL recognition switched from
epitope RT 191–215 to RT 514–524. CTL lines were generated after polyclonal expansion with autologous variants. Specific lysis is shown at E/T
ratios of 50/1 using 1 mM peptide concentration and background lysis (0–18%) is subtracted.
variants. This suggests that the polyclonal CTL re-
sponse simultaneously directed at diverse epitopes may
contribute to the absence of progression by eliminating
a large number of infected cells. On the other hand, in
epitopes that are known to be conserved, viral persis-
tence could be observed despite CTL recognition. This
may be either related to functional constraints or to
inefficient antigen processing (B. Maier, personal com-
munication). The strong CTL responses in nonprogres-
sors with high viral diversity and low viral load may
suggest also an influence on viral variation. However,
the only examples showing a selective pressure of CTL
on virus variants, were found in an SIV-infected mon-
key and in a HIV-infected patient who received in vivo
transfers of single CTL clones directed against a nef or
agag epitope [35,36].
Viral persistence may therefore result from inefficient
specific responses or from temporal lag of efficient CTL
responses in vivo that are always late in hunting after
the virus (Fig. 1). Considering only single epitopes,
some immunodominant or conserved epitopes that may
impose functional constraints for the virus may persist
despite continuous CTL recognition [34,37,38]. Viral
variation can result in non recognition of potential
epitopes by alteration of MHC binding or epitope
processing ([39,40], B. Maier, personal communication)
or by generation of altered peptide ligands with
modified T-cell receptor (TCR) contact sites [41–43].
The HLA context that has been shown to be important
for disease progression, may also the possibil-
ities of viral variants to avoid immune recognition.
Strongly restricted CTL responses directed against sin-
G.Haas et al.
/
Immunology Letters
57 (1997) 63–68
66
gle immunodominant epitopes have been described in
context of HLA-B8 and B27 [10,44], whereas polyspe-
cific responses were often observed in context of HLA-
A2 or A3 or B7 [15,34,45,46]. Evasion from CTL
recognition was found on the level of single epitopes
either in primary infection for a HLA B44 restricted
epitope or in late disease progression for HLA-B27
restricted epitopes at stages of high viral replication and
turnover [9,10].
4. CTL responses directed against target epitopes of
antiretroviral drugs
In the natural course of infection, HIV reverse tran-
scriptase (RT) is fairly conserved and a major target for
HIV-specific CTLs. RT is also a major target of an-
tiretroviral therapy with AZT, ddc, ddI or 3TC. After
several weeks to months of antiretroviral mono-ther-
apy, viral variants occur frequently that reduce drug
susceptibility. Such drug resistance mutations occur in
the NH
2
segment of the molecule at codons RT 41, 65,
70, 74, 181, 184 or 215, depending on the drug [47]. In
respect to HIV-1 RT variation, the selective pressure of
anti-retroviral drugs seems far more potent than that of
CTL. Compared to epitope variations observed in the
HIV-1 Nef protein that is not a target of antiretroviral
drugs, RT epitope variations in the NH
2
-segment of the
molecule seem to occur mostly with antiretroviral ther-
apy. One explanation could be the proximity of some of
the epitopes to functional important sites of the RT
that may impose sequence constraints which do not
tolerate natural variation. For example, in models to
relate functional sites and sequence features, taking into
account recent X-ray spectroscopy data of the RNase
H domain, RT 181 lies directly in the substrate binding
pocket in close relation with RT 215 [48]. Other sites of
CTL epitopes in the RT may also be of functional
importance, as described for persisting epitopes in nef
and gag [37,45].
When comparing CTL recognition of four individu-
alized regions of the RT protein in a cross-sectional
study of 47 patients and a longitudinal study of ten
patients, differences in immunogenicity could be ob-
served with significantly higher frequencies of recogni-
tion of the NH
2
-segment in nonprogressors or in
untreated patients (G. Haas et al., submitted). Thus,
when analyzing the different regions of the RT
molecule, a shift in immunodominance seemed to occur
from the NH
2
to the COOH-segment of the RT
molecule with disease progression and antiretroviral
therapy as shown in Fig. 2 (Haas et al., in preparation).
In such a cross-sectional study, differences in distribu-
tion of HLA types that have been associated with
progression and nonprogression [27] might influence the
spectrum of epitopes recognized by CTL. However, no
obvious bias in HLA representation between the differ-
ent groups of the study has been found, although the
number of patients is too small to allow a significant
analysis. Switches in epitope recognition might result
from alterations in dominant CTL epitopes as proposed
in a mathematical model [44]. Following this hypothe-
sis, CTL recognition of COOH epitopes of the RT may
not be detectable, because of the dominant recognition
of NH
2
ones. Such subdominant epitopes may then be
unmasked after the occurrence of viral mutations in the
NH
2
epitopes, thus contributing to maintain CTL
recognition of the whole protein (Fig. 3)
Long term mono-therapy with inhibitors of RT such
as AZT, ddI or 3TC favors the emergence of drug
resistance mutations in the NH
2
segment of the
molecule at codons RT41, RT 184 and RT215. Non-
recognition of NH
2
epitopes that include such muta-
tions have been found in the course of disease
progression in correlation to the occurrence of viral
variation, especially when viral load was still high de-
spite treatment. (Haas et al., in preparation). The
strong antiretroviral activity of RT inhibitors when
combined with protease inhibitors limits significantly
viral replication and can already lower the emergence
of viral mutations. At a time when such new highly
active antiretroviral drug combinations open perspec-
tives for the elimination of the virus, it appears of
major importance to evaluate CTL that can control
residual viral variants. Further studies on this topic
may provide a basis for immunotherapy to induce CTL
against drug-related viral mutants, thus preventing drug
resistance.
Fig. 3. Antiretroviral treatment (ART)-induced RT variants and
specific CTL. Drug sensitive HIV strains decrease after drug
monotherapy. Over time drug-resistant strains with mutations in the
NH
2
segment of the RT arise. When such strains are no longer
recognized by NH
2
-specific CTLs, COOH-specific CTLs are ex-
panded and can thus ensure permanent CTL recognition of the
molecule.
G.Haas et al.
/
Immunology Letters
57 (1997) 63–68
67
Acknowledgements
The authors acknowledge the help of O. Bonduelle,
J.M. Bouley, K. Dott, Y. Dudoit, E. Gomard, H.G.
Ihlenfeldt, G. Jung, K. Katlama, B. Maier, A. Meyer-
hans, U. Plikat, A. Samri, S. Scheveiler and K.H.
Wiesmu¨ller.
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