Cross-clade T cell recognition of HIV.1

388

Cross-clade T cell recognition of HIV.1 Frances Gotch Despite

considerable

infection,

successful

effective

vaccine.

lymphocyte

(subtypes)

Current

responses

such vaccination. lymphocytes

advances global

in antiviral

intervention evidence

suggests

will be an important

Recent

evidence

raised against

may be a correlation

an

that cytotoxic

T

of

that cytotoxic

from different

extensively

for HIV vaccine

for HIV

component

suggests

viral antigens

of HIV.1 can cross-react

have major implications

therapy

will require

T

clades

and such data

design.

Address Department of immunology, Chelsea and Westminster Hospital, 369 Fulham Road, London SW1 0 9NH, UK Current Opinion in Immunology

1998, 10:388-392

http://biomednet.com/elecref/0952791501000388 0 Current Biology Publications ISSN 0952-7915 Abbreviations CTL cytotoxic T lymphocyte recombinant glycoprotein r9p

Introduction In December 1997 it was reported that there had been an estimated 5.8 million new infections with HIV during that year, bringing the total number of people living with HIV world-wide to some 30 million [l]. The greatest growth in AIDS cases is in the densely populated developing countries of south-east Asia, India and sub-Saharan Africa [Z]. Whereas recent advances in antiretroviral therapy provide great optimism, issues related to cost and availability mean that such interventions are unlikely to have a major impact on the global epidemic. The need for an effective vaccine is paramount. The extraordinary genetic diversity of HIV.1 is a major obstacle to overcome if an effective prophylactic vaccine which can be used world-wide is to be developed. Based sequence analysis, eight sequence on envelope subtypes - clades - of HIV.1 (A to J) and one outlier group (0) of HIV.1 have been identified as distinct phylogenetic lineages in evolutionary trees [3]. In no country of the world is a single clade of virus circulating. While correlates of immune protection against HIV.1 are not yet completely defined, it is widely held that a broadly cross-reactive immune response, capable of recognising multiple HIV. 1 clades, must be a desirable feature of a vaccine-induced response. Most vaccines currently in development are based on B-clade immunogens and for these to be of global use it will be necessary for them to induce cross-reactive responses. Neutralising antibodies are thought to be important in blocking infection of cells by free virus particles and there

between

antibodies and protection models [4,5]. To date, most only been able to generate ing homologous laboratory

the presence

of neutralising

from infection in some animal candidate HIV.1 vaccines have antibodies capable of neutralisstrains of HIV.1 [6,7], although

there has been a recent report of neutralisation B primary isolate by sera from HIV.l-uninfected of a recombinant glycoprotein containing canarypox vaccine; with rgp1’20sF_z [8].

(rgp)lZO,t, both groups

Cellular

that

immune

responses

recognise

of a clade recipients or rgp160,tNwere boosted

conserved

epitopes within the virus’ structural and regulatory elements might provide much broader immunologic specificities than humoral responses recognising variable envelope proteins. T-helper function is necessary for both memory antibody responses and clonal expansion of specific cytotoxic T lymphocytes (CTLs). Cross-reactive T-cell proliferative responses to V3 peptides (from a variable region of HIV.1 envelope protein) corresponding to different geographical HIV.1 isolates have been observed in HIV.l-infected individuals [9]. CTLs have been shown to play a major role in effective virus-specific host immune responses. Data from studies of cytomegalovirus, Epstein-Barr virus, HIV and simian immunodeficiency virus suggest a protective role for anti-viral CTLs [lO,ll]. HIV-specific CTL recognition of short viral peptide fragments from HIV - bound to MHC class I molecules on an infected cell - can result in lysis of the infected cell, activation of the T cell and the release of cytokines. Although some CTL epitopes have been described within the envelope of HIV.1, such epitopes are mainly located in the core (e.g. gag and pol) and regulatory (e.g. nef) proteins, which are more conserved than envelope proteins among clades [12]. Correlations have been made between the rapid decrease in plasma HIV viraemia following primary infection with HIV and the emergence of high levels of virus-specific HLA-restricted CTLs [ 131. A negative correlation has been shown between HIV&specific CTL activity and HIV.2 viral load [14], while in HIV.1 infection high levels of CTLs have been associated with nonprogression to disease [15]. HIV-specific CTL activity has been observed in HIV- babies born to seropositive mothers [16] and in highly exposed seronegative adults [17]. In animal models it has been shown that the precursor frequency of vaccine-induced CTLs negatively correlates with viral load following challenge [18]. Clinical trials of candidate HIV.1 vaccines in the past five years have been for the most part based on envelope-subunit vaccines. These trials have confirmed that such vaccines, although capable of inducing high titre but noncross-reactive humoral activities, were inefficient at

Cross-clade T cell recognition of HIV.1 Gotch

eliciting

CD8+ CTLs

[19,20]. Vaccines

using live, recombi-

nant poxvirus constructs, DNA immunisation or attenuated live virus are more potent CTL imunogens, particularly in conjunction with a ‘prime-boost’ strategy involving an initial immunisation using the vector followed by a subunit boost [Zl]. Such vaccines are mostly based on clade-B immunogens which predominate in Europe and the ITSA. If such potentially effective vaccines have to be reformulated for different clades, delivery of promising interventions to the neediest

populations

could

cytotoxic T lymphocytes in HIV

Four recent studies have assessed the ability of HIV.l-specific cytotoxic T cells in HIV.l-infected individuals to recognise viral sequences from clades of virus other than those with which they are infected. The extent of crossreactivity within such individuals, each infected with a characterised virus, might reflect the kind of cross-reactive immune responses inducible by a monovalent vaccine.

be delayed.

It had been widely assumed’that there would be limited cross-reactivity for CTL responses amongst different clades of HIV.1, such that different vaccines might be needed for different geographical areas; however several recent publications have examined in more detail the spectra of crossreactivity of CTLs between HIV.l-infected individuals

Cross-reactive infection

389

the different clades of HI\<1 in or in vaccinated volunteers and

it now seems that CTLs raised against viral antigens from different clades can cross-react extensively. Such data has major implications for HIV vaccine design.

Vaccine studies Early vaccine studies in the murine and nonhuman primate systems demonstrated that single immunisations with DNA constructs encoding HIV.1 gp16O membranebound glycoproteins may induce cross-clade cytotoxic T cell responses specific for the envelope [ZZ]. Recent vaccine studies in humans have investigated immune responses to canarypox-clade-B based ALVAC/HI\‘. 1 vaccines containing full length gp160,,,; or gp120 plus the transmembrane portion of gp41, full length gag and protease [U”]. The specificity of vaccine-induced CTLs against autologous CD4+ lymphoblast target cells that were infected with representative primary HI\‘.1 isolates from clade A-F was tested in a small subset of volunteers using AL\‘AC vaccines. One out of two volunteers - immunised Lvith ALVACIHIV.1 gp16O showed a broad pattern of CTL activity, lysing target cells infected with cladc A-F isolates. The CTL reactivvolunteers - immunised ities from two with ALVAC/HI\:.l gp120 plus gp41, gag and protease recognised select populations of primary HIV.l-infected target cells; one volunteer had CTLs recognising A-, B-, C- and E-isolates whereas the other had CTLs recognising A-, B- and F-clade isolates. It was observed that vaccine-induced cross-reactive CTL acti\,ity could be measured up to 20 weeks post vaccination. Thus both the gplhO-based canarypox immunogen and the one containing additional gag/protease genes could elicit CTL reactivities capable of recognising CD4+ cells infected with genetically diverse HIV.1 primary isolates. These findings suggest that vaccine-induced CTLs are not constrained by existing barriers that have prevented the exploitation of neutralising antibodies in vaccine development - namely their inability to neutralise field isolates on their natural target cells and to effectively cross-neutralise diverse strains of HIV.1 [7].

Betts CTL

et N/. [24”] examined activity in the peripheral

cross-clade HIV.l-specific blood of Zambian individ-

uals infected restimulated

with clade-C HIL’.l. CTLs using autologous B-cell

transformed ITV/psoralen

by and

nants

containing

Epstein-Barr infected with clade-B

virus, vaccinia

HIV.1 (strain

were lines

selectively that were

inactivated by virus recombiIIIB)

gag/pol/env

(vvCPE) or nef (vvnef). Such a restimulation protocol may not reflect the normal it/ Gzo response, as only crossreactive CTLs are likely to be restimulated and CTLs specific for the C clade will not be apparent. Target cells were autologous B cell lines infected with recombinant vaccinia viruses conaining the same clade B as was used for restimulation. Six of the eight persons infected by clade C demonstrated highly significant levels of HIV.lspecific CTI, activit) ro HI\‘.1 clade B vvGPE but only one out of the three that were tested demonstrated specific CTL activity specific for HI\‘.1 nef, which was low anyway. Further analysis of the eight persons revealed that two individuals had CTLs which recognised B-clade gag, pol and env; one individual had CTLs only recognising gag; and one had CTLs recognising only pal. Results thus indicate that cross-clade CTL activity is not limited to a single HIV protein. Examination of the Los Alamos National Laboratory database reveals many conserved CTL epitopes between Bclade and C-clade isolates of HIV.l. Sixteen gag epitopes, nine pol epitopes and seven envelope epitopes are seen to be fully conserved between B- and C-clade isolates. This is likely to be an underestimate of functional conservation since some CTL epitopes are tolerant of substitutions [2.5,26]. The completely conserved epitopes bind to a variety of HLA class I alleles [27]. A second study [Z-3”] examined the ability of nine wellcharacterised CTL clones - isolated from individuals infected with B-clade virus, that were specific for HIV1 gag, reverse transcriptase or env - to recognise analogous A-, C-, D- and E-clade viral sequences. Clones selected had specific HLA-restricting alleles representative of HLA distributions in populations where vaccines may be of most use. All CTL clones were cross-reactive with at least one non-B-clade strain, in some cases due to sequence conservation and in others to CTL recognition of a variant peptide. CJse of target cells, infected with a vaccinia virus recombinant expressing A-clade envelope, established that cross-clade recognition persists with

390

HIV

intracellular processing and presentation of antigens. This study also examined polyclonal HIV.l-specific CTL responses in 14 individuals infected with A-, C- or Gclade virus, all of whom demonstrated cross-reactivity

In the fourth study [30”], recognition of non-clade-B peptides (based on an 8-mer peptide in p17 of gag) by a CTL clone restricted to HLA-BB, which was derived from a patient infected with clade B and was restimulated with a

with clade-B nef proteins. accomplished

peptide based on the evaluated. All peptides

with

viral constructs Restimulation using either

recombinant

vaccinia

expressing of enriched autologous viruses

gag, env, pol and CDX+ cells was B cells, infected

expressing

antigens

from HIV.1 B clade and inactivated with UV/psoralen; or using autologous irradiated populations with expanded CD4+ cells, that were superinfected with HIV.1 IIIB. Both restimulation protocols would be expected the elicitation of cross-reactive CTL responses.

to favour

Although epitope conservation may be one explanation of the cross-clade recognition seen in this study it was shown that single, or even double, amino-acid substitutions frequently did not abrogate recognition. Analyses performed indicated that the proteins most likely to stimulate crossclade immune responses were the capsid protein gag ~24 and reverse transcriptase - the most conserved proteins. A third study [29] examined the cross-reactivity of CTLs induced in eight patients infected with clade-A virus and seven patients infected with the B clade. CTLs were restimulated using autologous virus and target cells were infected with recombinant vaccinia viruses expressing env, gag, pol and nef from A- or B-clades. CTLs from all individuals cross-reacted extensively with proteins from the heterologous clade. Epitopes conserved between the viruses of the A- and B-clades were especially frequent in gag ~24, gag ~18, integrase and the central region of nef.

B-clade consensus sequence, was tested for recognition had one or

two amino-acid changes compared with the index peptide. None of the variant peptides was recognised. &‘hile this degree of specificity has been described for many other CTL clones and lines, it was not reported by Cao et ~1. [28”] although they did not evaluate CTL clones specific for HLA-B8 in their study. Patients with a particular HLA class I type (such as HLA-B8) may make CTL responses which are specific for variable regions of the virus [31] and such individuals might be expected to show less cross-reactivity. Such a lack of cross-reactivity or specificity may give a misleading impression and the polyclonal responses that a patient actually makes itzaiw to a whole antigen or series of antigens may be far more heterogeneous.

This study [30”] also evaluated cross-clade recognition of p5.S antigen by CTLs in persons infected with diverse clades of HIV.l. Experiments were designed to evaluate whether individuals - infected with A-, B-, C-. or D-clade virus-had CTLs (in this case inducible by autologous virus) that were capable of recognising and killing autologous target cells that were infected with recombinant vaccinia viruses expressing the gag p% protein from A-, B-. Cand D-clade HI\‘.l. Again extensive cross-reactivity in CTL responses was demonstrated in the majority of individuals between four of the major clades of HI\‘.l; these data are summarised in Table 1.

Table 1 The majority of HIV.1 infected patients show cross-reactivity of the clade with which they are infected. *Recognition

by CTLs of autologous

to HIV.1 core protein ~55 gag from A-, B-, C- and D-clades, regardless

target

cells infected

with recombinant

vaccinia

viruses expressing:

Clade of HIV.1 with which

Influenza

HIV.2

HIV.1

HIV.1

HIV.1

CTL donor is infected

PB2 or NP

~55 gag

clade A

clade B

clade C

HIV.1 clade D

~55 gag

~55 gag

~55 gag

p55 gag

A

_t

++* ++

++ ++

++

++

4-f

++

++ ++ ++

++ +

+§ +

++ +

++ ++ ++ ++ ++ ++ ++ ++ ++ ++

++

++

++

A A A/C A/C D AlClD B B B B B B B

_

+

ND ND ND

NK *Each set of results represents lytic CTL responses vaccinia construct. Effector : target ratio was 5O:l. known; NP, nucleoprotein; PB2, polymerase-binding

Mock infection

_

+ ++ ++ N”D ND ND +

++ ++ ++ ++ ++ ++ ++ ++ +

++

++ ++ ++ ++ +

of one individual. Lysis is compared to that of the same target cell infected with an irrelevant no specific lysis or < 10% lysis; I+, > 10% lysis; *++, >15% lysis. ND, not done; NK, not protein 2. Adapted with permission from [30**1 and F Gotch, unpublished data.

t-,

Cross-clade

At least

two of the patients

documented

single

described

exposure

in this paper

to HIV.1 so it seems

had a

recent tive

studies

demonstrated

between

HI\<1

CTLs

and

which

were cross-reac-

Such

data

HIV.2

make

described, although not confirmed, putative offered by prior infection by HIV.2 to infection [32] unlikely

to be mediated

Conclusions The

relative

strategies

Further

studies

clade-B

viruses

are truly

es

need

viral-peptide

to be performed

of CTL

if cross-reactive

class I molecules

must

continue,

in

in genetically order

may be easily

CTL

Safrit J, Fung M, Andrews C: Hu-PBL-SCID mice can be protected from HIV.1 infection by passive transfer of monoclonal antibody to the principle neutralising determinant of envelope gp120. AlDS 1993, 7:15-21.

6.

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7.

Mascola J, Snyder S, Weislow 0, Belay S, Belshe R, Schwartz A, Clements M-L, Dolin R, Graham B, Gorse G et al.: lmmunisation with envelope subunit vaccine products elicits neutralising antibodies versus lab-adapted but not primary isolates of human immunodeficiency virus type I. J Infect Dis 1996, 173:340-348.

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that

diverse

9.

Fernandez M, Fidler S, Pitman R, Weber J, Rees A: CD4+ T cell recognition of diverse clade B HIV.1 isolates. A//IS 1997, 11:281-288.

vaccine-

quantitated

10.

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12.

Myers G, Korber B, Berzofsky J, Smith R, Pavlakis G, Wain-Hobson S (Eds): Human refroviruses and AIDS 1993. A compilation and analysis of nucleic acid and amino acid sequences. Los Alamos, New Mexico: Los Alamos National Laboratory; 1993.

13.

Borrow P, Lewicki H, Wei X, Horwitz M, Peffer N, Meyers H, Nelson J, Gairin J, Hahn B, Oldstone M, Shaw G: Antiviral pressure exerted by HIV.1 specific cytotoxic T lymphocytes (CTLs) during primary infection demonstrated by rapid selection of CTL escape virus. Nat Med 1997, 3:205-21 1.

14.

Arioyoshi K, Berry N, Jafar S, Sabally S, Corrah T, Whittle H: HIV.2 specific CTL activity is inversely related to proviral load. AlDS 1995, 9:555-559.

15.

Moss P, Rowland-Jones S, Frodsham P, McAdam S, Giangrande P, McMichael A, Bell J: Persistent high frequency of HIV specific cytotoxic T cells in peripheral blood of infected donor;. Proc Nat/ Acad Sci USA 1995, 92:5773-5777.

16.

Rowland-Jones S, Nixon D, Aldous M, Gotch F, Ariyoshi K, Kroll S, Froebel K, McMichael A: HIV specific CTL activity in an HIV exposed but uninfected infant. Lancet 1993, 341:860-861.

17.

Rowland-Jones S, Sutton J, Ariyoshi K, Dong T, Gotch F, McAdam S, Whitby D, Sabally S, Gallimore A, Corrah T et a/.: HIV-specific cytotoxic T cells in HIV-exposed but uninfected Gambian women. Nat Med 1995, 1:59-64.

18.

Gallimore A, Cranage M, Cook N, Almond N, Bootman J, Rud E, Silvera P, Dennis M, Concoran C, Stott J, Gotch F: Early suppression of SIV replication by CD8+ nef-specific cytotoxic T cells in vaccinated macaques. /Vat Med 1995, 1 :l 167-l 173.

19.

Graham B, Keefer M, McElrath M, Gorse G, Schwartz D, Weinhold K, Matthews T, Esterlitz J, Sinangil F, Fast P: Safety and immunogenicity of a candidate HIV.1 vaccine in healthy adults: recombinant glycoprotein (rgp) 120. A randomised, double blind trial. Ann lntem Med 1996, 125:270-279.

20.

Keefer M, Graham B, McElrath M, Matthews T, Stablein D, Corey L, Wright P, Lawrence D, Fast P, Weinhold K et al.: Safety and immunogenicity of env2-3. A human immunodeficiency virus type 1 candidate vaccine, in combination with a novel adjuvent, MTPPE/MF59. AlDS Res Hum Retronrus 1996, 12:683-693.

21.

Fleury B, Janvier G, Pialoux G, Buseyne F, Robertson M, Tartaglia J, Paoletti E, Kieny M-P, Excler J, Riviere Y: Memory cytotoxic T

that non-

respons-

through

CTL common

populations

induced

CTL

[33]. It will also be

necessary to examine CTL activity in individuals infected with recombinant viruses; and to elucidate whether CTL responses

are cross-reactive

clonal

responses

potent

CTL-inducing

better

at the clonal

reflect

the i/l a&

immunogens

lope subunit

vaccines

of live-vector

unit boosting

may result in the generation

HIV.1

in eliciting

may lead

to good

responses, a vaccine less cross-reactivity. virus might [34]. The

challenge

protective

responses.

followed

The

by env-sub-

of multiple,

good

possible that, whilst natural of dynamic virus such as

polyclonal

to induce

remains

against

responses

Some

cross-reactive

cellular

based on a single sequence may induce A vaccine based on live, attenuated

be expected

ing reactivities

situation.

humoral

priming

responses. It remains with a quasispecies

or if poly-

may fall short of enve-

combination immune infection

level

cross-reacti\,e

of broadening

primary

isolates

responses

virus-neutralisand ensuring

that

are long lasting.

Thus significant cross-reactivity for CTL responses with evidence of either epitope conservation or cross-clade recognition has been demonstrated and this provides support for the strategy of initiating trials with vaccines based on Bclade immunogens in areas of the world where the B clade is not common

but where

the need

for a vaccine

References

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is greatest.

reading

Papers of particular interest, published within the annual period of review, have been highlighted as: l l

1.

2.

I abolishes

5.

responses.

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that are restricted

HLA

Emini E, Nara P, Schlief W: Antibody mediated in vitro neutralisation of Human lmmunodeficiency virus type infectivity for chimpanzees. J Viral 1990, 64:3674-3678.

immunity

with additional

Identification

epitopes

responses

the induction

to determine

global.

to protective

but it is of clear importance

include

4.

by CTLs.

of CTLs

is still not fully understood, vaccine

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the

protection with HI\:1

and future directions

contribution

391

3.

unlikely

that rhe cross-reactivity described in this and the other studies is a function of multiple exposure. None of these

T cell recognition of HIV.1 Gotch

of special interest * of outstanding interest Decock K: Monitoring the epidemic of HIV/AIDS [Abstract L21. In Proceedings of the Fiffh Conference on Retroviruses and opportunistic infections: 7998 Feb 1-5; Chicago. Edited by Richman DD et al. Alexandria, Virginia: The Foundation for Retrovirology and Human Health; 1998:l. Over M, Piot P: Human immunodeficiency virus infection and other sexuallv transmitted diseases in develooina countries: public health-importance and priorities for r&o&e allocation. J infect Dis 1996,174:Sl62-S175.

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Wang B, Boyer J, Srikantan V, Ugen K, Agadjanian M, Merva M, Gilbert L, Dang K, McCallus D, Moelling K: DNA inoculation induces cross clade anti-HIV.1 responses. Ann N YAcad Sci 1995, 772:186-l 97.

Ferrari G, Humphrey W, McElrath M, Excler J-L, Duliege A-M, Clements M-L, Corey L, Bolognesi D, Weinhold K: Clade B-based HIV.1 vaccines elicit cross clade cytotoxic T lymphocyte reactivities in uninfected volunteers. Proc NaN Acad Sci USA 1997, 94:1396-l 401. This is the first demonstration that canarypox vaccines based on clade (subtype) B can elicit broad CTL reactivities capable of recognising viruses belonging to genetically diverse HIV.1 clades. The results reinforce the impact of viral core elements in a vaccine as well as the pattern of MHC class I allelic expression by the vaccine recipient, in determining the relative breadth of the cellular response. The results suggest that CTL cross-recognition among HIV.1 clades is more widespread than previously anticipated and that a vaccine based on a single clade may be broadly applicable. 23 ..

24. *

Betts M, Krowka J, Santamaria C, Balsam0 K, Gao F, Mulundu G, Luo C, N’Gandu N, Sheppard H, Hahn B et al.: Cross-clade human immunodeficiency virus (HIV)-specific cytotoxic T lymphocyte responses in HIV.1 infected Zambians. J Viral 1997, 71:8908-891 1. This is a demonstration that individuals infected with HIV.1 clade C can mount vigorous HIV.l-CTL responses reactive to HIV.1 clade B and that such responses are not limited to a single HIV protein. The results suggest that CTL cross-recognition among HIV.1 clades is more widespread than previously anticipated and that a vaccine based on a single clade may be broadly applicable. l

25.

26.

27.

Pogue R, Eron J, Frelinger J, Matsui M: Amino-terminal alteration of the HLA-A*OZOi-restricted human immunodeficiency virus pol peptide increases complex stability and in vitro immunogenicity. froc Nat/ Acad Sci USA 1995,92:8166-8170. Sette A, Vitiello A, Reherman B, Fowler P, Nayersina R, Kast W, Melief C, Oseroff C, Yuan L, Ruppert J: The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes. J lmmunoll994, 153:5586-5592. Tsuji K, Aizawa M, Sasazuki T (Eds): Proceedings of the Eleventh Histocompatibility Workshop and Conference. Yokohama, Japan. Oxford: Oxford Science Publications; 1991.

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Cao H, Kanki P, Sankale J-L, Dieng-Sarr A, Mazzara G, Kalams S, Korber B, Mboup S, Walker B: Cytotoxic T-lymphocyte crossreactivity among different human immunodeficiency virus Type I clades: Implications for vaccine development. J Viral 1997, 71:8615-8623. This provides data from a cohort of patients infected with viruses of the B clade (subtypes) and non-B clade. The patients demonstrate significant cross-reactivity of CTL responses, with evidence of epitope conservation or cross-clade recognition. The results suggest that CTL cross-recognition among HIV.1 clades is more widespread than previously anticipated and that a vaccine based on a single clade may be broadly applicable. 29.

Durali D, Morvan J, Letourneur F, Gomard E, Venet A: Large crossreactivities of CTL responses from HIV-infected African and European patients. In Proceedings of the eleventh Colloquium Des Cent Gardes: 1997 Ott 27-29; blames-la-coquette, France. Edited by Girard. M, Dodet B. Amsterdam: Elsevier; 1998: 88.

McAdam S, Kaleebu P, Krausa P, Goulder P, French N, Collin B, Blanchard T, Whitworth J, McMichael A, Gotch F: Cross clade recognition of ~55 by CTL in HIV.1 infection. A//X 1998, 12:57 l-579. Cross-clade recognition of p55 protein antigen was evaluated in persons infected with diverse clades (subtypes) of HIV.1 and most patients were shown to mount cross-clade cytotoxic T lymphocyte (CTL) responses recognising gag proteins from clades of HIV.1 other than those with which they were infected. The results suggest that CTL cross-recognition among HIV.1 clades is more widespread than previously anticipated and that a vaccine based on a single clade may be broadly applicable. 30. ..

31.

McAdam S, Klenerman P, Tussey L, Rowland-Jones S, Lalloo D. Phillips R, Edwards A, Giangrande P, Brown A, Gotch F: Immunogenic variants that bind to HLA-BI but fail to stimulate CTL responses. J lmmunoll995,155:2729-2’736.

32.

Marlink R, Kanki P, Thior I, Travers K, Eisen G, Silby T, Traore I, Hsieh C. Dia M. Gueve E: Reduced rate of disease development after HIV.2 infection’as compared to HIV.1. Science 1994: 265:1587-l 590.

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Gotch F, Koup R, Safrit J: New observations on cellular immune responses to HIV and T-cell epitopes. A/LX 1997, 11 (suppl A):S99 -SlO7.

34.

Wyand M, Manson K, Garcia M, Montefiori D, Desrosiers R: Vaccine protection by a triple deletion mutant of SIV. J Viol 1996, 7013724.3733.