understanding cytotoxic t-lymphocyte escape during simian immunodeficiency virus infection

David O’Connor Understanding cytotoxicThomas Friedrich

T-lymphocyte escape during simianAustin HughesTodd M. Allen immunodeficiency virus infectionDavid Watkins

Authors’ address

David O’Connor1, Thomas Friedrich1, Austin Hughes2,Todd M. Allen1, David Watkins1,1Madison, WI, USA.2Columbia, SC, USA.

Correspondence to:

David H. O’Connor1220 Capitol CourtMadison, WIUSA

Immunological Reviews 2001Vol. 183: 115–126Printed in Denmark. All rights reserved

Copyright C Munksgaard 2001

Immunological ReviewsISSN 0105-2896

115

Summary: Infection of rhesus macaques with simian immunodeficiencyvirus (SIV) is an excellent model system for studying viral adaptation toimmune responses. In this review, we discuss how the SIV-infected ma-caque has provided unequivocal evidence for cytotoxic T-lymphocyte(CTL) selection of viral escape variants. This improved understanding ofCTL escape may influence human immunodeficiency virus (HIV) vaccinedesign as well as our understanding of HIV pathogenesis.

Introduction

The utility of non-human primate models in AIDS research

has long been understood and will be described in detail

throughout this issue. These models hold particular promise

for understanding the role that viral variation plays in the

subversion of the host cytotoxic T-lymphocyte (CTL) re-

sponse. There are several key advantages to using the non-

human primate models for these types of studies. First, the

asymptomatic period which normally lasts between 6 and 10

years in an untreated human immunodeficiency virus (HIV)-

infected individual is typically less than 3 years in a simian

immunodeficiency virus (SIV)-infected macaque (1). This ac-

celerated timecourse of disease is ideal for longitudinal

studies of immune responses and evaluation of candidate vac-

cine.

Second, the genetic characterization of SIV enables detailed

studies of viral pathogenesis, particularly those involving viral

evolution. For experimental use, several SIV isolates have been

cloned and sequenced in their entirety (2–6). Infecting ma-

caques with these molecularly cloned, well-defined patho-

genic stocks facilitates straightforward evaluation of in vivo

viral evolution (7–12). Moreover, cohorts of animals can be

infected with the same stock of the same virus, revealing

subtle, individual differences in the immune responses to the

virus. Infection with a homogenous, pathogenic molecular

clone also approximates heterosexual transmission of HIV,

O’Connor et al ¡ Cytotoxic T-lymphocyte escape during SIV infection

where the transmitted viral variant is generally homogenous

(13).

Third, the number of difficulties associated with studying

naturally occurring HIV infection is reduced. The route of

challenge, the dose of challenge, and the composition of the

challenge virus are regulated in SIV-challenge studies. Ad-

ditionally, the absence of highly active antiretroviral treatment

(HAART) and other therapeutic interventions allows for ex-

tensive characterization of natural infection.

It is only recently, however, that SIV-infected macaques

have been widely used in CTL escape studies. One limitation

of studying escape in the macaque is the lack of well-defined

SIV CTL epitopes. For identification of CTL epitopes to be

useful, the MHC class I alleles that bind and present the epi-

topes also need to be identified. Unfortunately, less than 20

classical MHC class I alleles have been described in the rhesus

macaque (14). The most recent HIV Molecular Immunology

Database contains over 600 CTL epitopes for HIV-1 and

HIV-2, but only 49 SIV CTL epitopes (15). Of those 49, 14 do

not have defined MHC class I-restricting molecules. However,

improving technologies such as ELISPOT assays and intra-

cellular staining for interferon-g production are revolutioniz-

ing epitope mapping in both HIV and SIV (16). These tools,

combined with the increased usage of the macaque model,

are contributing to a rapid increase in the number of iden-

tified CTL epitopes in SIV.

How does SIV escape cytotoxic T-lymphocyte responses?

SIV and HIV employ multiple mechanisms to avoid recog-

nition by CTL. These include downregulation of MHC class I

molecules on the cell surface by the viral Nef protein (17–

19), upregulation of Fas-ligand on the surface of infected

CD4 T lymphocytes (17–19), infection of target cells that are

resistant to CTL lysis (20), destruction of CD4π lymphocytes

that undermines the function of CTL (21, 22) and mutational

escape. While this review focuses on mutational escape from

SIV, several reviews on other mechanisms of CTL escape have

recently been published (23–25).

HIV and SIV avoid CTL by stochastically generating resis-

tant viruses that reduce CD8π lymphocyte recognition of

MHC class I:peptide complexes. This can be achieved by ac-

cumulating amino acid replacements that either reduce pep-

tide binding to MHC class I molecules or reduce peptide rec-

ognition by the T-cell receptor.

To study mutational escape, one typically starts by sequenc-

ing regions of HIV or SIV containing known CTL epitopes

and monitoring viral evolution in these regions throughout

116 Immunological Reviews 183/2001

infection. HIV and SIV reverse transcription is error-prone

and it is known that these viruses can tolerate at least some

of the errors introduced during this process (26–29). Indeed,

mathematical modeling of HIV replication dynamics suggests

that every possible viral variant is generated daily during in-

fection (30). If many variants are well tolerated, rapid and

dramatic antigenic drift should occur throughout the viral

genome (31–33), leading to changes within CTL epitopes.

Alternately, if most variant viruses are crippled by the vari-

ation and cannot function properly, minimal sequence vari-

ation should be observed (34, 35). A third possibility is that

mutational variation may accumulate due to positive selec-

tion, whereby CTL select for resistant viruses that are more

fit than susceptible viruses (36, 37).

In order to differentiate between these possibilities, statisti-

cal tests are applied to viral sequence data. The most widely

utilized of these methods compares the rate of synonymous

nucleotide substitutions (dS) with the rate of non-synony-

mous nucleotide substitutions (dN) (38, 39). If a nucleotide

change encodes a variant amino acid (dN) with a deleterious

consequence on the structure and function of the protein,

the nucleotide change should be selected against (purifying

selection). In this case, the dN to dS ratio is expected to be

less than 1, since synonymous changes will predominate. If,

alternately, nucleotide changes encoding variant amino acids

are localized within a protein domain that has little effect on

protein structure or function, the substitution rates are neu-

tral and dS will be roughly equal to dN. In the rare case when

an external selective pressure encourages the maintenance of

variant amino acid sequences, dN will exceed dS.

It should be emphasized that positive selection by CTL on

a region of the virus does not necessarily preclude a fitness

cost to the viruses harboring the escape variants. It does,

however, imply that the benefit of CTL resistance is greater

than the loss of replicative fitness relative to CTL-susceptible

viruses.

An abridged history of CTL escape studies

Direct antigenic variation capable of inhibiting CTL responses

was first documented in 1990 (40). Researchers infected

mice transgenic for a single T-cell receptor with either high

or low doses of lymphochoriomeningitis virus (LCMV).

While the group that received the low-dose challenge effec-

tively resolved their primary viremia, the high-dose group

developed a persistent infection and failed to control acute

viremia. When the investigators looked at the epitope recog-

nized by the transgenic CTL, they realized that all of the mice


in the high-dose group harbored virus with amino acid re-

placements within this epitope. They then showed that these

epitope variants were poorly recognized by the CTL that effec-

tively controlled the initial viremia in the low-dose challenge

group. These results were extended a year later when it was

shown that LCMV accumulated epitope substitutions when

co-cultured with CTL clones in vitro (41). Taken together,

these studies showed that CTL could select for antigenic vari-

ants both in vivo and in vitro.

In the first study documenting CTL escape during LCMV

infection, the authors theorized that escape might also be a

mechanism for viral persistence in HIV infection (40). Many

studies on CTL escape were conducted on samples from HIV-

infected patients during the ensuing decade. While several of

these studies document sequence variation within CTL anti-

gens (42–50), others questioned the relevance of these find-

ings (35, 51–60).

CTL escape during chronic SIV infection

The first effort to resolve this controversy using experimen-

tally infected rhesus macaques was published in 1992. That

study examined a CTL epitope TPYDINQML (Gag TL9) re-

stricted by the MHC class I allele Mamu-A*01. Three Mamu-

A*01-positive animals were infected with SIV and viral se-

quence evolution within the epitope and the flanking region

were studied. Though epitope variation was seen in two of

the animals, the frequency of variation within the epitope

was not statistically higher than the frequency of variation in

the sequence flanking the epitope (61). Moreover, CTL re-

acted equally well against both the predominant epitope vari-

ant peptides and the wild-type TL9 epitope. Since 1992, tech-

nological improvements have enabled a more precise defi-

nition of this minimal, optimal Mamu-A*01-restricted

epitope as Gag CTPYDINQM (Gag CM9) and not Gag TL9

(62). In light of this finding, the authors re-evaluated their

data and discovered that the observed epitope variants dis-

sociate more rapidly from Mamu-A*01 than from wild-type

epitope peptides (63).

In 1998, Mortara and colleagues vaccinated several ma-

caques with a mixture of Nef- and Gag-derived lipopeptide

constructs (64). Within several months of challenge with

pathogenic SIV, the Nef-specific CTL responses elicited by im-

munization were lost. Sequencing of virus derived from these

animals revealed a substantial number of viral variants within

the recognized CTL epitopes. These variants were poorly rec-

ognized by the CTL from these animals, again suggesting es-

cape from the vaccine-induced responses. A recent follow-up

117Immunological Reviews 183/2001

report from the same group suggests that additional Nef-spe-

cific responses eventually selected for epitope variants during

chronic SIV infection (65).

Building on this earlier work, recent studies in the SIV-

infected macaque have unequivocally demonstrated CTL es-

cape during chronic infection. In each of five recognized Env

and Nef CTL epitopes (66), amino acid replacements that

reduced the recognition of CTL accumulated by late-stage in-

fection (67). Statistical analysis of dN:dS showed that nucle-

otide variants encoding amino acid replacements preferen-

tially occurred within each of the CTL epitopes, providing

strong evidence for positive selection driven by CTL.

Interestingly, the three animals in this study had differing

rates of disease progression. The basis for most differences in

survivorship following HIV or SIV infection remains un-

known, though compelling associations between certain HLA

class I alleles and HIV disease progression have been identified

(68–72). It is possible that the two animals with better out-

comes retained the capacity to generate de novo CTL responses

for a longer period than the rapid progressor, balancing the

loss of existing CTL due to escape with the gain of new re-

sponses (54). Alternately, the CTL epitopes initially targeted

by the slow progressors may have been significantly more

effective at controlling the virus than the epitopes targeted

by the rapid progressor. Recent work has shown that new

specificities of CTL can be generated throughout HIV infec-

tion. In approximately 70% of HLA-A201-positive individ-

uals, the HLA-A0201 response SLYNTVATL (SL9) is detectable

by 3 years post-infection (73–75). During acute infection,

however, this response is rarely detected (75).

CTL escape during acute SIV infections

Since the above results clearly showed that escape occurred

during late-stage chronic infection, we reasoned that the

earliest emergence of escape variants should coincide with

the time at which CTL responses began exerting an antiviral

effect. Previous studies had shown the emergence of escape

mutants during early infection (48, 50). These studies, how-

ever, were restricted to small cohorts of HIV-positive patients.

To rigorously examine CTL escape during acute infection,

we infected a large cohort of macaques with identical stocks

of the molecularly cloned SIVmac239. Discriminating im-

mune selection from genetic drift can be extremely difficult

in animals infected with biological isolates, particularly when

considering infections across mucosal surfaces (76). Six CTL

responses restricted by the MHC class I molecule Mamu-A*01

were followed during acute infection. Two of these responses,


Fig. 1. Rapid emergence of epitope variants in Tat SL8. Irrespectiveof the other class I alleles expressed in these 10 Mamu-A*01-positiveanimals, strong Tat SL8-specific responses selected for escape variantsduring acute infection with SIVmac239. The frequency of a particularsequence within the overall set derived from each animal is shown tothe right of the sequence.

Gag CM9 and Tat STPESANL (Tat SL8), dominated during

early infection of Mamu-A*01-positive animals. When we

examined the epitopes recognized by these CD8π T cells, we

discovered remarkable sequence variation within Tat SL8 by

8 weeks post-infection (Fig. 1). This sequence variation did

not occur in animals that did not express the Mamu-A*01

molecule and did not occur consistently elsewhere in the vi-


rus. dN:dS analysis of this data demonstrated that this epitope

was evolving under strong positive selection by CTL.

Since this escape occurred coincident with the decline in

primary viremia, we reasoned that this response might be

effectively containing the virus and exerting selective pressure

analogous to treatment with antiviral monotherapy. In con-

trast to more durable CTL responses that do not select for

escape variants until later in infection, the wild-type

SIVmac239 apparently could not tolerate this primary CTL

response. This was evidenced by the near-complete absence

of wild-type virus in these animals. Therefore, we reasoned

that this CTL response, and others that rapidly select for es-

cape variants during primary infection, might be the most

‘‘effective’’ responses mounted by the immune system.

Understanding these responses may be important for vaccine

design.

Why does the Tat SL8-specific CTL response select for

variants so quickly?

There are at least three non-exclusive reasons why the Tat SL8

response selects for variants more rapidly than other, well-

studied CTL responses.

Multiple open reading frames: the non-intuitive constraint

on Tat SL8 variation

First, the SL8 epitope may be located within a functionally

disposable domain of Tat, thereby enabling the maintenance

of epitope variants that avoid CTL while having minimal ef-

fect on replicative fitness. However, the Tat SL8 epitope over-

laps the open reading frame of the viral protein Vpr, so any

nucleotide changes that encode amino acid replacements in

Tat SL8 may also affect amino acids in Vpr.

This phenomenon of viral proteins encoded by overlapping

reading frames has attracted the attention of evolutionary bi-

ologists since its discovery (77–81). One question of evol-

utionary interest raised by this phenomenon is how natural

selection can act simultaneously on two different protein

products encoded in different reading frames by the same

DNA sequence. Fig. 2 illustrates the mean number of synony-

mous nucleotide substitutions per synonymous site (dS) and

mean number of non-synonymous substitutions per non-syn-

onymous site (dN) (38) for comparisons between samples

from Mamu-A*01-positive monkeys and the inoculum in a

sliding window analysis of tat and vpr reading frames. In the

tat reading frame, a strong peak in dN was observed in the

region of the SL8 epitope, while in the vpr reading frame there

was a corresponding peak in dS in the same region (Fig. 2).


Fig. 2. Nucleotide substitutions. Meannumbers of synonymous (dS) (dotted line)and non-synonymous (dN) (solid line)nucleotide substitutions per site incomparisons of tat and vpr reading frames ina sliding nine-codon window. Comparisonbetween samples from Mamu-A*01-positivemonkeys with the inoculum. Horizontal bar –location of the SL8 epitope in tat. (From A.Hughes et al. J Virol 2001;75:7966-7972).

Table 1 summarizes the means of dS and dN in epitope and

non-epitope regions in both tat and vpr reading frames. In

comparisons of the tat reading frame of samples from infected

monkeys with that of the viral inoculum, mean dN for

Mamu-A*01-positive monkeys significantly exceeded mean

dS in the SL8 epitope but not elsewhere in the gene (Table 1).

No such pattern was seen in the SL8 epitope in the case of

Mamu-A*01-negative monkeys (Table 1). Likewise, in com-

parisons within samples from Mamu-A*01-positive monkeys,

mean dN significantly exceeded mean dS (Table 1). Again, no

difference was seen in the case of Mamu-A*01-negative mon-

keys (Table 1).

In the vpr reading frame, in Mamu-A*01-negative mon-

keys, no significant difference between mean dS and mean

dN was seen in either the nine codons overlapping the SL8

epitope or in the remainder of the gene (Table 1). However,

in Mamu-A*01-positive monkeys, mean dS was significantly

greater than mean dN in the region corresponding to the SL8


epitope (Table 1). Thus, positive selection favoring amino acid

changes in the SL8 epitope of the Tat protein in virus in-

fecting Mamu-A*01-positive monkeys evidently resulted in a

burst of synonymous changes in the vpr reading frame (Fig. 2,

Table 1).

This finding was further analyzed by considering all poss-

ible non-synonymous changes that might occur in the SL8

epitope. There were 78 such possible changes, of which 34

would also cause a non-synonymous change in the vpr reading

frame, while the remaining 44 would cause a synonymous

change in the vpr reading frame. Of the 34 possible non-

synonymous changes in tat that are also non-synonymous in

vpr, only six were actually observed in the viral sequences

from Mamu-A*01-positive monkeys. On the other hand, 22

of 44 possible non-synonymous changes in tat that are syn-

onymous in vpr were observed. The difference between ob-

served and expected is highly significant (pΩ0.0018; Fisher’s

exact test). This result shows that positively selected non-syn-


Table 1. Mean numbers (∫SEM) of synonymous (dS) and non-synonymous (dN) numbersof nucleotide substitutions per 100 sites in comparisons of tat/vpr protein regions

Epitope RemainderdS dN dS dN

tat reading frame

A*01ª vs. Inoc 0.2∫0.2 1.1∫0.5 0.5∫0.2 0.3∫0.1

within 0.5∫0.5 0.9∫0.3 0.6∫0.2 0.4∫0.1

A*01π vs. Inoc 0.4∫0.4 5.6∫0.4***††† 0.4∫0.1 0.1∫0.0

within 0.8∫0.8 7.2∫1.1***††† 0.5∫0.2 0.2∫0.1

vpr reading frame

A*01ª vs. Inoc 0.4∫0.3 1.5∫0.7 0.7∫0.4 0.2∫0.1

within 0.9∫0.6 1.2∫0.6 0.9∫0.4 0.4∫0.2

A*01π vs. Inoc 9.3∫1.6††† 2.0∫0.5** 0.3∫0.1 0.1∫0.0

within 13.9∫2.7†† 2.2∫0.6** 0.6∫0.2 0.3∫0.1

In the tat reading frame, the epitope region encompasses the eight codons aligned with the Tat SL8epitope. In the vpr reading frame, it encompasses nine codons overlapping those eight codons in tat.InocΩinoculum.

Paired sample t-tests of the hypothesis that mean dSΩmean dN: **p,0.01; ***p,0.001.

t-tests of the hypothesis that mean dS or mean dN in Mamu-A01-positive animals equals thecorresponding value in Mamu-A01-negative animals: ††p,0.01; †††p,0.001.

onymous changes in the tat gene occurred disproportionately

in such a way as not to change the amino acid sequence of

Vpr (82).

Tat may be a superior antigenic target

Alternately, the expression kinetics of the viral Tat protein

may make epitopes derived from this protein more anti-

genic than epitopes derived from other proteins. The in-

creased antigenicity could be the result of greater protein

abundance in infected cells or the early expression of Tat

in the viral life cycle. Since Tat is one of the first viral pro-

teins synthesized in an infected cell, CTL epitopes derived

from this protein will be expressed on the cell surface well

before the assembly and release of mature virions. CTL di-

rected against these epitopes, then, may be more able to

effectively contain viral replication.

Recent experiments by Gruters and colleagues have shown

that expression kinetics may play an important role in CTL

effectiveness (83). They examined the ability of a CTL clone

specific for a reverse transcriptase to contain in vitro viral repli-

cation. The wild-type virus replicated well in vitro in both the

presence and absence of the CTL clone. In contrast, a recom-

binant virus engineered to express this same epitope as part

of the Nef protein replicated well by itself but did not grow

to high titer in the presence of CTL. This directly demon-

strates that the antigenic context of CTL epitopes can influ-

ence their efficacy.


Tat SL8-specific CTL are fundamentally different than

most HIV-specific CTL

Another explanation for the rapid and dramatic escape ob-

served from Tat SL8-specific responses is that this response

is of high avidity. Preliminary evidence from our laboratory

suggests that much lower concentrations of peptide are

needed to elicit Tat SL8 responses than Gag CM9 responses ex

vivo (84). In other viral systems, similar high avidity CTL have

been shown to be particularly effective at containing viral

replication (85–87). This leads to the tempting hypothesis

that selection for Tat SL8 variants by the immune system is

induced by qualitatively different CTL than those that have

been well studied during chronic HIV and SIV infections.

Understanding the transmission of ‘‘escaped’’ viruses

The existence of CTL escape raises important questions for the

design of future vaccines. Can CTL escape variants be passed

between hosts? If so, do viruses that have escaped immuno-

dominant immune responses exhibit increased pathogenicity

when passaged to hosts of the same genetic background? Do

such viruses retain variant sequences in MHC-disparate hosts,

when, in the absence of a specific CTL response, there is no

selection on these sequences? If this is so, will viruses circu-

lating in populations accumulate escape mutations in epitopes

restricted by MHC class I alleles common to those popula-

tions?


The well-studied evolution of resistance to antiviral drugs

may illustrate fundamental principles that are broadly appli-

cable to the study of CTL-induced variation. Currently avail-

able antivirals target one of two enzymatic activities required

for virus replication, those of reverse transcriptase (RT) and

the viral protease. Evolution of resistance to either of these

classes of drugs can be expected to have detrimental effects

on viral fitness, as it requires alterations in the active sites of

these two enzymes. Such a loss of fitness with respect to par-

ental, drug-susceptible strains has been reported as dimin-

ished in vitro replicative capacity for viruses resistant to pro-

tease inhibitors (88–90) and RT inhibitors (91, 92).

During the first weeks of infection, HIV and SIV replicate

to extremely high titer, generally in the absence of antiretrovi-

ral treatment or specific cellular immune responses. This high

turnover, coupled with the viruses’ intrinsically high error

rate, might be expected to generate de novo variants with

higher replicative fitness than the infecting strain. However,

in a cohort of patients with acute HIV-1 infection, mutations

conferring resistance to RT inhibitors were detected (91).

These viruses showed high in vitro replicative capacity, but also

acquired drug-resistant phenotypes when the patients were

placed on antiviral therapy. Interestingly, transmission of vi-

ruses resistant to both RT- and protease-inhibitors has been

documented (93), including a single case in which virus

from the probable source patient exhibited a high frequency

of wild-type sequences (94).

No study to date has investigated the transmission of CTL

escape viruses, or the consequences of escape on disease

course when such viruses are transmitted. Neither is it clear

whether escape variations are retained in viral sequences

when variant viruses are passaged among hosts. The answers

to these questions will be important in understanding the role

of viral evolution in AIDS pathogenesis and in HIV-vaccine

development.

Perspectives on CTL escape and the notion of

‘‘effective’’ CTL responses

The correlates of protective immunity against HIV and SIV

remain poorly defined, despite 20 years of intense investiga-

tion. Cellular and humoral immune responses have now been

extensively documented in both HIV-infected individuals and

SIV-infected macaques (95, 96). However, the efficacy of

these responses in ameliorating the disease course remains

controversial.

Much of this controversy is caused by a failure to identify

a single response, or category of responses, that can unam-


biguously improve HIV and SIV disease prognosis (94, 95),

largely because these viruses have a remarkable capacity to

adapt to their surroundings and establish persistent infection.

Traditionally, CTL responses that are immunodominant in

multiple individuals have been considered ‘‘effective’’ im-

mune responses. In the setting of HIV/SIV infection, however,

an ‘‘effective’’ CTL response is likely to be short-lived, as viral

adaptation and escape will favor the emergence of CTL-resis-

tant viruses. While many transiently ‘‘effective’’ CTL re-

sponses are likely elicited throughout infection, no singularly

‘‘effective’’ immune responses to the virus have been iden-

tified and infection universally ends in progressive immuno-

deficiency and mortality.

The analysis of ‘‘effective’’ CTL responses is difficult since

the composition of the virus is usually unknown. Most

studies of HIV-specific CTL involve ex vivo manipulations of

lymphocytes derived from infected individuals. The responses

measured by these ex vivo assays are stimulated with synthetic

peptides that represent a ‘‘best guess’’ or ‘‘consensus’’ se-

quence from circulating strains that are present in a geo-

graphic region. This approach may not detect CTL responses

to portions of the virus that do not share sequence similarity

with the synthetic peptide set, such as those responses that

rapidly select for viral variants during acute infection. There-

fore, a very important subset of CTL responses is not being

properly accounted for using conventional techniques.

The only way to ultimately assess the ‘‘effectiveness’’ of a

particularly immune response is to measure its impact in vivo.

Because the overall immune response to HIV is multifactorial,

gross analyses of disease progression (i.e. CD4π T-lymphocyte

counts, development of opportunistic infections, changes in

viral load, etc.) will not usually illuminate the contribution

of any single response. Therefore, it may be more instructive

to monitor the virus for evidence of adaptation to the im-

mune system as a measurement of an effective CTL response.

When a CTL response effectively responds to a particular viral

strain and limits its replication, variant viruses resistant to

this same CTL response will increasingly dominate the viral

population in the host. Therefore, sequencing can often

identify the regions of the virus ‘‘marked’’ by the immune

system of a particular host, allowing one to determine the

specificity of immune responses that were, at least at one

time, ‘‘effective’’.

Do all potent CTL epitopes accumulate escape variants?

Prior to accepting the premise that viral escape is an inevi-

table consequence of an ‘‘effective’’ CTL response, it is


reasonable to question whether CTL escape is a universal

feature of SIV and HIV infections. There are examples in

the literature, as well is in experiments from our lab, that

suggest CTL escape occurs in rapid, intermediate and slow

progressors. Additionally, these studies show that CTL es-

cape can occur during both chronic and acute infections.

An outstanding question is, under what, if any, circum-

stances does CTL escape not occur?

It is possible that certain CTL epitopes, located within do-

mains that are structurally or functionally conserved, may be

refractory to amino acid substitution. If the number of toler-

able variants within a given epitope is low, the generation of

the unique variants that maintain adequate viral fitness while

also evading the CTL response should require more rounds

of viral replication. Similarly, escape from regions under tight

structural constraints may require the concomitant presence

of one or more compensatory substitutions, outside the actual

CTL epitope, to maintain fitness. If compensatory substi-

tutions are required, a double mutant virus would need to be

generated. The likelihood of a double mutant arising simul-

taneously at both sites is several orders of magnitude lower

than the odds of either single mutant arising individually. For

the compensatory substitutions to arise sequentially, the com-

pensatory substitution would need to be selectively neutral to

compete with the wild-type virus. Once the compensatory

substitutions is present at high frequency, the second site

mutation within the actual epitope can occur. It is important

to remember that the situation can not occur in reverse, as

the epitope substitution alone will probably likely generate a

low fitness virus. Since the generation of genomic errors by

the viral reverse transcriptase is essentially random, the num-

ber of replication cycles needed to spawn an escape virus that

requires compensatory substitutions should be greater than

the number of cycles needed to spawn a single site mutant.

However, evidence suggests that compensatory substitutions

do eventually occur and result in the escape of highly con-

strained epitopes from CTL recognition (97).

Anecdotal supporting evidence for this concept can also be

found in the SIV-infected macaque. The Mamu-A*01-re-

stricted Gag CM9 response is directed against an epitope in

the viral capsid. This epitope is well conserved in HIV-1,

HIV-2 and SIV isolates, implying an important functional role

for this region for the virus. However, escape from Gag CM9

has recently been documented (98). Notably, only a single

site within this epitope was changed in multiple animals. Ad-

ditionally, escape within this epitope was tightly linked to

the presence of two additional amino acid replacements, one

upstream and one downstream of the epitope ((61) and D.


H. O’Connor, D. I. Watkins, unpublished data). These putative

compensatory substitutions may be needed to maintain

viability of viruses containing the Gag CM9 escape variant.

In this case, the response may be very effective and durable

throughout infection, but the ability of the virus to escape is

limited by constraints on the virus.

This category of responses that does not escape rapidly

stands in marked contrast to the category of CTL responses

that escape extremely rapidly during acute infection. We have

now identified five CTL responses that appear to eliminate

virus containing wild-type epitope sequences by 4 weeks

post-infection ((99) and D. H. O’Connor, T. Allen, T. Vodel,

P. Sing, I. DeSouza, E. Podds, E. Dunphy, C. Melsaether, B.

Mothe, K. Westover, H. Horton, A. Hughes, D. Watkins,

manuscript in preparation). The only precedent for viral evo-

lution this rapid is found in HIV-infected individuals treated

with antiviral monotherapy (100). In therapy, drug failure is

so common that a cottage industry for tracking and monitor-

ing drug resistance has emerged. This leads us to speculate

that CTL responses that select for new variants during the

acute phase exert an effect analogous to treatment with anti-

retrovirals such as AZT. Unlike drug treatment, however,

where the failure of one medication usually results in the

prescription of an alternate medication, the failure of acute

phase CTL may lead to the emergence of previously subdom-

inant responses or the generation of novel responses. While

these later responses clearly contribute to viral containment

as evidenced by their eventual selection for variants, their po-

tency may not be as great as the responses that engender acute

escape.

Implications of CTL escape for HIV vaccine development

The identification of CTL responses that select for escape vari-

ants at different rates has implications for HIV vaccine devel-

opment. At the most basic level, these escape studies have

prompted some to revisit the question of what constitutes an

‘‘effective’’ CTL response against HIV. Does rapid CTL escape

imply an extraordinarily important role for a particular re-

sponse in viral containment or simply an extraordinary ability

of the virus to tolerate variation in particular regions of its

genome? While the answer to this question remains contro-

versial, several lines of evidence suggest that acute CTL re-

sponses are particularly important in the early containment

of SIV by the immune response. First, acute CTL responses

that select for escape variants have now been detected in three

viral proteins, suggesting that the occurrence of rapid Tat SL8

escape is not solely attributable to its location in a variable


region of Tat (D. H. O’Connor, T. Allen, T. Vodel, P. Sing, I.

DeSouza, E. Podds, E. Dunphy, C. Melsaether, B. Mothe, K.

Westover, H. Horton, A. Hughes, D. Watkins, manuscript in

preparation). Secondly, three of the five acute CTL responses

identified so far are located in regions of the genome that

overlap multiple reading frames. In all three cases, non-syn-

onymous variation was largely limited to the reading frame

that encoded the CTL epitope. Therefore, the virus selects for

and maintains only those variants that confer a selective ad-

vantage in the open reading frame that is being recognized

by CTL. The most striking example of this phenomenon is

observed in a Vpr epitope. The CTL epitope in Vpr overlaps

the Mamu-A*01-restricted Tat SL8 epitope. In this animal,

changes that were non-synonymous in Vpr were primarily

synonymous in Tat, though it has been established that this

region of the Tat open reading frame can also accommodate

non-synonymous substitutions.

Finally, we have directly sequenced entire viral genomes

from SIV-infected macaques during the first weeks of infec-

tion. The predominant sequences are relatively invariant dur-

ing early infection despite enormous levels of replication.

Alexander et al. have recently described a series of suboptimal

References1. 6. 11.Gardner MB. SIV infection of macaques: a Kestler H, et al. Induction of AIDS in rhesus Kimata JT, Kuller L, Anderson DB, Dailey P,

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2. Science 1990;248:1109–1112.Kestler HWd, et al. Comparison of simian variants influence AIDS progression.immunodeficiency virus isolates. 7. Burns DP, Desrosiers RC. Selection of Nat Med 1999;5:535–541.Nature 1988;331:619–622. genetic variants of simian 12. Heidecker G, et al. Macaques infected with

3. immunodeficiency virus in persistentlyRegier DA, Desrosiers RC. The complete cloned simian immunodeficiency virusnucleotide sequence of a pathogenic infected rhesus monkeys. show recurring nef gene alterations.molecular clone of simian J Virol 1991;65:1843–1854. Virology 1998;249:260–274.immunodeficiency virus. 8. Hirsch VM, et al. Viral genetic evolution in 13. Greenier JL, et al. Route of simianAIDS Res Hum Retroviruses macaques infected with molecularly cloned immunodeficiency virus inoculation1990;6:1221–1231. simian immunodeficiency virus correlates determines the complexity but not the

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5. monkeys.Naidu YM, et al. Characterization of Immunology Database 1999. Los Alamos:infectious molecular clones of simian J Virol 1993;67:4104–4113. Los Alamos National Laboratory, Theoreticalimmunodeficiency virus (SIVmac) and 10. Mortara L, Letourneur F, Gras-Masse H, Biology and Biophysics, 1999.human immunodeficiency virus type 2: Venet A, Guillet JG, Bourgault-Villada I. 16. Goulder PJ, et al. Rapid definition of fivepersistent infection of rhesus monkeys with Selection of virus variants and emergence novel HLA-A*3002-restricted humanmolecularly cloned SIVmac. of virus escape mutants after immunization immunodeficiency virus-specific cytotoxicJ Virol 1988;62:4691–4696. with an epitope vaccine. T-lymphocyte epitopes by Elispot and

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nucleotide substitutions in the molecular clone of SIVmac239

that routinely revert during natural infection with this virus

(100). On average, less than one nucleotide site in the 9000

nucleotide SIV genome accumulates variation that cannot be

attributed to CTL escape or to these suboptimal nucleotides

(D. H. O’Connor, T. Allen, T. Vodel, P. Sing, I. DeSouza, E.

Podds, E. Dunphy, C. Melsaether, B. Mothe, K. Westover, H.

Horton, A. Hughes, D. Watkins, manuscript in preparation)

by 4 weeks post-infection. Genetic drift does not appear to

drive neutral variants to high frequency at any particular site

within the genome, suggesting that it is unlikely that any

region is inherently tolerant to acute phase variation.

In summary, the preliminary evidence may suggest that the

acute phase CTL responses constitute the ‘‘most effective’’

group of responses marshaled by the host immune system. If

this is true, identification of other ‘‘effective’’ CTL responses

in acute HIV and SIV infections will be extremely useful. Vac-

cine trials using these types of epitopes, which are capable of

exerting significant and rapid selective pressure, are still in

their infancy, but additional work, utilizing more epitopes

and improved delivery systems, may provide hope for an ef-

fective vaccine against HIV.


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understanding cytotoxic t-lymphocyte escape during simian immunodeficiency virus infection

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