AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 8, Number 8, 1992 Mary Ann Liebert, Inc., Publishers
Human
Virus Variants That T-Cell Recognition
Immunodeficiency
Escape Cytotoxic
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SARAH L. ROWLAND-JONES, R.E. PHILLIPS, D.F. NIXON, F.M. GOTCH, J.P. EDWARDS, A.O. OGUNLESI, J.G. ELVIN, J.A. ROTHBARD, CR.M. BANGHAM, C.R. RIZZA, and A.J. McMICHAEL
Cytotoxic
T lymphocytes (CTL) appear early in the immune response to viral infection, and have been shown to have a role in clearing virus in acute infections and containing virus replication in persistent infections. In the early stages of HIV infection, there is a vigorous HIV-specific CTL response which, it has been suggested, may contribute to the long asymptomatic period. However, this response usually has declined to undetectable levels by the time disease develops. It is not clear what causes the HIV-specific CTL response to decline: possible explanations include loss of cytolytic cells, either as part of a general effect or specific loss of HIV-CTL precursors, or the emergence of virus variants that escape T-cell
recognition.
We have studied the CTL response to the gag-protein in 6 hemophiliac patients who were first identified as HIV infected in
1985.' Three of these have HLA-B8, which as part of the extended haplotype Al B8 DR3 has been associated with more rapid progression of disease in some studies. Three peptide epitopes were identified in gag (see Fig. 1 ) which were restricted by HLA-B8 and the response to these peptides was studied over time. It was observed that the response to these peptides fluctuated with time: for example, donor 020 initially made a strong response to peptide p24-13 which subsided to undetectable levels over a two-year period, and was replaced by a strong response to peptide pi7-3. This donor is now symptomatic and an HIV-gag-specific CTL response has not been demonstrable at all on recent bleeds. Similar patterns of fluctuating response have been seen in the other patients with HLA-B8. By contrast, only one peptide epitope in gag restricted by HLA-B27 has been identified. When the response to this peptide was studied over time in donor 007, a consistent response was observed over several years. In order to investigate the possibility that antigenic variation in the virus might account for the shift in dominant B8-restricted responses, the sequence of proviral DNA was analyzed after polymerization chain reaction (PCR) amplification in the vicinity of the B8- and B27-restricted epitopes in the three patients
Molecular
Immunology Group.
with HLA-B8, and compared with similar regions of the virus in three patients with a dominant HLA-B27-restricted response. From each patient, proviral DNA was extracted at several time points, and the sequence of 20 clones was analyzed at each time point. The results are summarized in the figure. Considerable variation was observed in the B8-restricted epitopes in patients with HLA-B8. particularly in the regions of peptides pi 7-3 and p24-13. In contrast, virtually no variability in these regions was seen in the patients with HLA-B27, even in the B27 donor who is also symptomatic. Similarly, variation in the B27 epitope was seen in several sequences from the B27 patients but was not observed in the patients with HLA-B8. Wc then studied the potential impact of such sequence varition on CTL recognition. Peptides were synthesized based on two variants of p24-13, and when first tested for CTL recognition, were recognized as efficiently as the index peptide sequence by CTL from donor 020. A year later, peripheral blood lymphocytes (PBL) from this donor still showed recognition of the variant sequences, but the index peptide was no longer recognized, suggesting that the dominant CTL response was now directed toward the prevailing sequence variants. Synthetic peptides based on variant sequences of peptide pi 7-3 also were tested for recognition by CTL from two donors, and in several cases the variant sequences did not sensitize target cells for lysis. A relatively conservative amino acid change from lysine to arginine, seen in several sequences from all three HLA-B8 donors, appeared to be sufficient to abolish CTL recognition. A similar phenomenon was observed using CTL from donor 008, who made a good CTL response to peptide p24-20 but failed to recognize a variant peptide with a single arginine for lysine substitution. By contrast, a peptide based on the only mutation seen in the B27-restricted epitope, peptide p24-14, with a change from leucine to methionine, considerably enhanced CTL recognition. Previous studies of patients with HLA-B27 also have failed to demonstrate sequence variants which could not be recognized by CTL from the patients, despite advancing disease. This differ-
Institute of Molecular Medicine, John Radcliffe
1353
Hospital. Headington. Oxford, OX3 9DU, England.
1354
ROWLAND-JONES ET AL.
P24-14 KRWIILGLNKIVRMY KRWIILGMNKIVRMY
Downloaded by UPPSALA UNIVERSITETSBIBLIOTEK from www.liebertpub.com at 01/09/19. For personal use only.
LRP66KKYKLKHIV
LRP6GKKKYKLKHIV
NNPPIPVGEIYKRWII
LRP6GRKKYKLKHIV LRPGGRKKYKFKHII LRPGGRKKYKFKHIV LRPGGRKKYKLKHII LRPGGKKRYKLKHII LRPGGKKKYKLKHIL
NNPPIPVGDIYKRWII
VQNANPDCRTILKAL
HLA-B8
patients
P24-13 +
P24-20
indicates whether or not
or
patients
VQNANPDCKTILKAL
HNPPIPVGDIYKRwïl NNLPIPVGDIYKRWII NNPPIPVGDIYKRWIT HNSPIPVGDIYKRWII NNSPIPVGEIYKRWII
PI7-3
HLA-B27
recognised by patients' CTL
-
FIG. 1.
Variation in the sequences of CTL epitopes in
between HLA types may reflect differing requirements for peptide-binding between HLA-B8and B27, or differences in the functional or structural significance of the peptide epitopes restricted by these molecules to the virus. Thus changes in the B27 epitope which would allow escape from CTL recognition may not be tolerated by the virus because they would impair gag protein function. These data are consistent with a protective role for CTL in HIV infection, but imply that protective immunization will require stimulation with antigen derived from many diverse strains of virus. For rational vaccine design, it becomes important to locate CTL epitopes in regions of the virus which remain conserved for functional or structural regions. ence
HIV-gag in patients with HLA-B8 and
HLA-B27.
REFERENCE I.
Phillips RE. Rowland-Jones SL, Nixon DF, Gotch FM. Edwards JP, Ogunlesi AO, Elvin JE, Rothbard JA, Bangham CRM, Rizza CR, and McMichael AJ: Human immunodeficiency virus genetic variation that can escape cytotoxic T cell recognition. Nature 1991 ;354. Address reprint requests to: Sarah L. Rowland-Jones Molecular Immunology Group Institute of Molecular Medicine John Radcliffe Hospital Heading ton, Oxford, OX3 9DU England
Human
Virus Variants That T-Cell Recognition
Immunodeficiency
Escape Cytotoxic
Downloaded by UPPSALA UNIVERSITETSBIBLIOTEK from www.liebertpub.com at 01/09/19. For personal use only.
SARAH L. ROWLAND-JONES, R.E. PHILLIPS, D.F. NIXON, F.M. GOTCH, J.P. EDWARDS, A.O. OGUNLESI, J.G. ELVIN, J.A. ROTHBARD, CR.M. BANGHAM, C.R. RIZZA, and A.J. McMICHAEL
Cytotoxic
T lymphocytes (CTL) appear early in the immune response to viral infection, and have been shown to have a role in clearing virus in acute infections and containing virus replication in persistent infections. In the early stages of HIV infection, there is a vigorous HIV-specific CTL response which, it has been suggested, may contribute to the long asymptomatic period. However, this response usually has declined to undetectable levels by the time disease develops. It is not clear what causes the HIV-specific CTL response to decline: possible explanations include loss of cytolytic cells, either as part of a general effect or specific loss of HIV-CTL precursors, or the emergence of virus variants that escape T-cell
recognition.
We have studied the CTL response to the gag-protein in 6 hemophiliac patients who were first identified as HIV infected in
1985.' Three of these have HLA-B8, which as part of the extended haplotype Al B8 DR3 has been associated with more rapid progression of disease in some studies. Three peptide epitopes were identified in gag (see Fig. 1 ) which were restricted by HLA-B8 and the response to these peptides was studied over time. It was observed that the response to these peptides fluctuated with time: for example, donor 020 initially made a strong response to peptide p24-13 which subsided to undetectable levels over a two-year period, and was replaced by a strong response to peptide pi7-3. This donor is now symptomatic and an HIV-gag-specific CTL response has not been demonstrable at all on recent bleeds. Similar patterns of fluctuating response have been seen in the other patients with HLA-B8. By contrast, only one peptide epitope in gag restricted by HLA-B27 has been identified. When the response to this peptide was studied over time in donor 007, a consistent response was observed over several years. In order to investigate the possibility that antigenic variation in the virus might account for the shift in dominant B8-restricted responses, the sequence of proviral DNA was analyzed after polymerization chain reaction (PCR) amplification in the vicinity of the B8- and B27-restricted epitopes in the three patients
Molecular
Immunology Group.
with HLA-B8, and compared with similar regions of the virus in three patients with a dominant HLA-B27-restricted response. From each patient, proviral DNA was extracted at several time points, and the sequence of 20 clones was analyzed at each time point. The results are summarized in the figure. Considerable variation was observed in the B8-restricted epitopes in patients with HLA-B8. particularly in the regions of peptides pi 7-3 and p24-13. In contrast, virtually no variability in these regions was seen in the patients with HLA-B27, even in the B27 donor who is also symptomatic. Similarly, variation in the B27 epitope was seen in several sequences from the B27 patients but was not observed in the patients with HLA-B8. Wc then studied the potential impact of such sequence varition on CTL recognition. Peptides were synthesized based on two variants of p24-13, and when first tested for CTL recognition, were recognized as efficiently as the index peptide sequence by CTL from donor 020. A year later, peripheral blood lymphocytes (PBL) from this donor still showed recognition of the variant sequences, but the index peptide was no longer recognized, suggesting that the dominant CTL response was now directed toward the prevailing sequence variants. Synthetic peptides based on variant sequences of peptide pi 7-3 also were tested for recognition by CTL from two donors, and in several cases the variant sequences did not sensitize target cells for lysis. A relatively conservative amino acid change from lysine to arginine, seen in several sequences from all three HLA-B8 donors, appeared to be sufficient to abolish CTL recognition. A similar phenomenon was observed using CTL from donor 008, who made a good CTL response to peptide p24-20 but failed to recognize a variant peptide with a single arginine for lysine substitution. By contrast, a peptide based on the only mutation seen in the B27-restricted epitope, peptide p24-14, with a change from leucine to methionine, considerably enhanced CTL recognition. Previous studies of patients with HLA-B27 also have failed to demonstrate sequence variants which could not be recognized by CTL from the patients, despite advancing disease. This differ-
Institute of Molecular Medicine, John Radcliffe
1353
Hospital. Headington. Oxford, OX3 9DU, England.
1354
ROWLAND-JONES ET AL.
P24-14 KRWIILGLNKIVRMY KRWIILGMNKIVRMY
Downloaded by UPPSALA UNIVERSITETSBIBLIOTEK from www.liebertpub.com at 01/09/19. For personal use only.
LRP66KKYKLKHIV
LRP6GKKKYKLKHIV
NNPPIPVGEIYKRWII
LRP6GRKKYKLKHIV LRPGGRKKYKFKHII LRPGGRKKYKFKHIV LRPGGRKKYKLKHII LRPGGKKRYKLKHII LRPGGKKKYKLKHIL
NNPPIPVGDIYKRWII
VQNANPDCRTILKAL
HLA-B8
patients
P24-13 +
P24-20
indicates whether or not
or
patients
VQNANPDCKTILKAL
HNPPIPVGDIYKRwïl NNLPIPVGDIYKRWII NNPPIPVGDIYKRWIT HNSPIPVGDIYKRWII NNSPIPVGEIYKRWII
PI7-3
HLA-B27
recognised by patients' CTL
-
FIG. 1.
Variation in the sequences of CTL epitopes in
between HLA types may reflect differing requirements for peptide-binding between HLA-B8and B27, or differences in the functional or structural significance of the peptide epitopes restricted by these molecules to the virus. Thus changes in the B27 epitope which would allow escape from CTL recognition may not be tolerated by the virus because they would impair gag protein function. These data are consistent with a protective role for CTL in HIV infection, but imply that protective immunization will require stimulation with antigen derived from many diverse strains of virus. For rational vaccine design, it becomes important to locate CTL epitopes in regions of the virus which remain conserved for functional or structural regions. ence
HIV-gag in patients with HLA-B8 and
HLA-B27.
REFERENCE I.
Phillips RE. Rowland-Jones SL, Nixon DF, Gotch FM. Edwards JP, Ogunlesi AO, Elvin JE, Rothbard JA, Bangham CRM, Rizza CR, and McMichael AJ: Human immunodeficiency virus genetic variation that can escape cytotoxic T cell recognition. Nature 1991 ;354. Address reprint requests to: Sarah L. Rowland-Jones Molecular Immunology Group Institute of Molecular Medicine John Radcliffe Hospital Heading ton, Oxford, OX3 9DU England
