Immunology and Cell Biology (1992) 70, 215-221
Diversity and variation in human immunodeficiency virus: Implications for immune control ALISTAIRJ. RAMSAY Division of Cell Biology, fohn Curtin School ofMedical Research, Australian National University, Canberra, Australian Capital Territory, Australia
Introduction The central problems in understanding the progression of infection with HIV to AIDS stem from the long latent period in the host; the median time from infection to development of disease is currently about 8 years. This raises two important questions. First, what is the role of the immune response in progression to AIDS: is the decline in the immune response the cause or the consequence of the rise in HIV titre? Second, what is the effect of the extreme sequence diversity of HIV: do variants arise that escape the immune response? It is now clear that HIV-1 exists, in the individual, as a population of viruses exhibiting a significant degree of genomic variation.''^ This implies that a dynamic balance exists between host defence mechanisms against the virus and the virus itself, which is not surprising given that HIV infects the very cells that initiate antiviral immune responses. The genomic diversity and antigenic variability of HIV and the problems that this presents for immune control of the virus are the subjects of this paper.
HIV mutates rapidly during replication HIV is a member of the lentivirus subfamily of the retroviruses: small enveloped viruses
which contain a diploid single-stranded RNA genome. Entry to target cells occurs by fusion of virion and cell membrane, following which the viral RNA genome is converted into double-stranded DNA by the action of reverse transcriptase and viral ribonuclease H.^''* Viral DNA is then integrated into the host chromosome to form a provirus via the action of virus-encoded integrase and remains permanently associated with host genetic material. Virus expression begins with the transcription of pro viral DNA in the activated cell by host RNA polymerase. The resultant viral RNA is either translated to produce viral proteins or packaged as the viral genome. Retroviral passage in vivo is a complex process. There is potential for a high rate of homologous recombination due to the packaging oftwo retroviral RNA molecules in one virion. Thus, insertions, duplications and deletions may occur in single genes with major effects on virus phenotype, but, in addition, recombination of whole virus segments may also alter virus characteristics.'* Clearly then, HIV should be added to the list of viruses that undergo genetic 'shift'. Reverse transcription during lentivirus replication is the basic mechanism generating recombinants and point mutations. Owing to the infidelity of this process, it is likely that a pool of genotypes is generated, with viable ones surviving as progeny virus.^ Selection of viral genomes from replicating progeny
Correspondence: Dr Alistair Ramsay, Division of Cell Biology, John Curtin School of Medical Research, PO Box 334, Canberra, ACT 2601, Australia. Presented at the 4th Frank and Bobbie Fenner Conference in Medical Research on Genetic Variation and Selection in Microbiology and Immunology, held on 6-8 November 1991 at the John Curtin School of Medical Research, Canberra, Australia. Accepted for publication 6 November 1991.
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would then allow their evasion of and survival in the host immune system. Evolution of viral genes is characterized by enormously high speed compared to nuclear genes of eukaryotic ^organisms. It has been estimated that evolution of the HIV gag gene occurs at a similar rate to that of the haemagglutinin gene of human influenza virus, faster than in other retroviruses and many orders of magnitude faster than in eukaryotic genes.^ The gag gene appears to evolve according to the neutral theory of evolution,^ in that silent mutations predominate over non-silent (or amino acid-changing) mutations. This theory holds that nucleic acid substitutions result from the random selection and fixation of neutral or near-neutral variants. In marked contrast, the rate of non-silent changes occurring in the hypervariable regions of the HIV env gene (17.2 xlO~^ base substitutions/site per year) almost equals the rate of silent changes (14x10"^), with both exceeding the estimate for silent gag mutations (9.7 x 10"^).^ Clearly, evolution of the HIV envelope occurs at an extremely high rate and not according to the neutral theory. It is likely that this is due to intensive immunological selection pressure.
The third variahle envelope domain dominates humoral immunity As outlined above, mutation in hypervariable regions of the env glycoprotein occurs rapidly, although the overall rate of occurrence of silent mutations still exceeds that of non-silent changes. This may imply that different regions of the envelope (a structure built from alternating domains separated by cysteine bridges) either allow, or do not allow, amino acid substitutions.^ Regions that allow substitutions resulting in changes in amino acid sequence may do so where the substitutions confer benefit on the virus (for example, allowing escape from host immunity) or where substitutions are chemically equivalent, with no resultant functional change.^ There are, of course, functional constraints on the virus. In the case of the envelope, evolution around a conserved cellular receptor must dictate the limit of variation possible in the overall structure and
shape of the glycoprotein. Recent work, however, suggests that HIV has overcome this problem and evolved a means of escape from the host neutralizing antibody through a convergence of the structural and functional roles of its third variable (V3) domain.""'^ Analyses of sera from HIV-infected individuals have led to the definition of a number of immunogenic sequences of env against which neutralizing antibody responses are directed.'^"'^ One of these sequences, occurring in the V3 domain, has been shown to dominate the neutralizing antibody response to infection or immunization. Thus, although infected individuals mount responses to a number of different regions of the env glycoprotein, each responds to the V3 domain and it is this response which is predominant in their antibody repertoire. The sequence is defined by a cysteine-cysteine bridge formed between amino acids 296-309 and 331-343,^ depending on the strain of virus, and occurs in a region exhibiting the exceptionally high rate of over 10"^ non-silent substitutions/site per year. Despite this enormous variation, one consensus V3 sequence predominates in viruses circulating during the current AIDS epidemic in the United States and Europe.^°'"'^^ This amino acid conservation is most pronounced in the central tetrapeptide, GPGR, which is found on the tip of the V3 loop in a position exposed to antibody. The great majority of isolates from both continents display this motif. Evidence from several laboratories suggests that this site, while not influencing the ability of the envelope to bind to the virus receptor, CD4, is a substrate for hydrolysis by cell surface enzymes.'^"'^ The V3 loop may, therefore, represent a fusion domain, playing a direct role in virus infectivity and the potent neutralizing effect of V3-speciflc antibody may reflect blocking of this activity.^ Indeed, it has become clear that this region of the envelope represents a multifunctional molecule and a number of demonstrated structural and functional properties, together with some proposed roles, are outlined in Table 1.
y3 mediates escape from humoral immunity Initially, it was thought that the V3 region represented a simple, linear epitope and that
Antigenic variation and immunity to HIV
Table 1. Structural and functional properties ofthe V3 domain. Established properties Immunodominant B cell epitope'^''"* Cross-reactive epitope^''^" Flexible contiguous neutralization epitope^ CD8 CTL/CD4 T helper cell epitope^' Component of an ordered oligomeric Proposed roles Determinant of cellular tropism' Functional proteolytic substrate'^ Protease inhibitor"
knowledge of the variable amino acids in V3 loops of different HIV strains would lead to a successful peptide-based vaccine. It has now become clear that this is not the case. Viruses exhibiting mutations from their parental amino acid sequences both within and outside the V3 domain, which also have escaped from the neutralizing antibody, have now been derived following passage of the virus both in vitro and in vivo. First, McKeating et al.^^ passaged cloned HIV-1 in vitro in the presence of an isolate-specific neutralizing antibody against V3 determinants and found that variants resistant to neutralization were rapidly selected. More interesting, however, are recent findings of in vivo antigenic drift of HIV-1. In the first of these studies, the emergence of escape mutants was found in sequential viral isolates recovered from chimpanzees up to 32 weeks after infection.^'* Mutants were defined by their relative resistance to neutralization by V3 strain-specific neutralizing antibodies. Surprisingly, there was complete conservation of the parental V3 primary sequence in all cases, although sequence analysis revealed multiple amino acid substitutions in the 32 week isolate, especially in the fourth constant and variable regions. It appeared, therefore, that non-silent mutations, distant from the V3 neutralizing antibody-binding site, were conferring resistance to neutralization. Similar results have been reported from studies of acute seroconversions in humans.^^ Here, neutralizing antibody titres against the homotypic viral strain first isolated from an
217
infected individual were high in all sequential serum samples tested. In contrast, the appearance of a neutralizing antibody response to a particular viral variant occurred only around the time of its isolation. These findings suggest that viral variants in infected individuals may not be effectively neutralized by antibodies generated against previously dominant viral strains. This presents a scenario in which the antibody response follows emerging variants without ever affecting them at the point of emergence.^ Antigenic drift occurring in this manner, within infected individuals, contrasts with that exhibited by influenza virus, which occurs at the population level, though the capacity of these variants to infect individuals with circulating antibody to earlier strains requires further study. In the light of these data, Nara and Goudsmit have recently proposed a scheme whereby the envelope, and the V3 loop in particular, controls host-directed antibody responses to the benefit of the virus.^•'^ Briefiy, they suggest that rapid replication of the virus upon infection leads to the production of neutralizing antibody and subsequent control of primary viraemia (as in other lentiviral infections). The control of acute viral replication in this manner, however, may be viewed as a survival property of the virus, in that the neutralizing antibody selects for viruses which have optimized transmission and longevity. It is proposed that this paradoxical situation is mediated by the V3 loop. Thus, antibody to this region dominates the repertoire, rendering the host less able to respond to emerging viruses with closely related V3 structures and resulting in a relatively restricted dominance of the neutralization response. These authors suggest that the demonstrated flexibility of the V3 domain (involving distant-site amino acid mutations) provides for subtle conformational variability of this region which does not infiuence its critical functional role in infeetivity, but provides escape from host immunity. Thus, molecular evolution may have allowed for the selection of this multifunction region, capable of the kind of conformational flexibility necessary to allow escape from neutralization, while preserving functional integrity.
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HIV may evade cellular immune responses Clearly, humoral immunity is unable to control HIV infection, although it remains unclear exactly which immune responses are important in this regard; at this stage there are no individuals known to have recovered who are available for study. It is apparent, however, that HIV elicits a vigorous, sustained cytotoxic T cell (CTL) response soon after infection and that this response is greatly diminished in individuals who have developed AIDS.^^ In general, CTL responses are directed against only a few short sequences of different proteins and each is apparently restricted to recognition by effector cells from individuals of one or two baplotypes. Several HIV proteins have now been shown to be immunogenic for CTL and some details are presented in Table 2. Comparatively little is known of the role played by CTL reactivity in HIV infection or of the evolutionary pressure which it may exert on the virus. In general, viruses appear to have two potential mechanisms of escape from CTL immunity, that is by mutating to evade either tbe antiviral T cell receptor or host class
I major histocompatibility complex (MHC) molecules. T cell repertoires have been widely considered to be broad enough to allow recognition of mutated viral proteins, providing that antigen processing and MHC association and presentation can occur.^^ Mutations at sites critical for binding to MHC molecules, however, would be expected to render proteins invisible to existing antiviral CTL, as the repertoire of class I MHC molecules of each individual is very small and cannot be expanded. A number of recent studies have focused on the relationship between sequence variation and CTL immunogenicity of HIV. Data from the first of these suggest that a single amino acid residue can contribute most of the binding energy which defines the specificity of the T cell receptor and show that mutations at such a critical site can abrogate recognition of an immunogenic sequence by CTL. Briefiy, murine CTL clones were raised against an H-2D''-restricted immunogenic sequence of env gpl60 (Table 2) in two different strains of HIV-1 (i.e. III-B and MN) and were found to be totally non-cross-reactive. However, reciprocal changes in a single amino acid residue
Table 2. CTL target sites of HIV proteins Protein
aa sequence
env
315-329 112-124 315-329 428-443 834-848 5 sequences 265-279 193-203 219-233 418-433 446-460 172-196 325-349 461-485 495-519 203-219 203-219 73-97 113-128
pol
nef
Class I restriction molecule
Reference
Mouse Human
H-2D'' HLA-A2
(21) (27)
Human Human Human
not determined HLA-B27 HLA-A2
(28) (29) (30)
Human
HLA-B8 HLA-All HLA-B8 HLA-All H-2^ HLA-B17, HLA-B37 HLA-A3 HLA-B17, HLA-B37
(31)
Mouse Human Human Human
(32) (33) (34)
Antigenic variation and immunity to HIV
219
(at the hypervariable position 325) of the respective sequences was sufficient to completely reverse the specificity of CTL recognition, despite five other sequence differences. When considered together with earlier studies by the same group,'^^ these findings have established that as few as two to four amino acid residues in a minimal immunogenic sequence are crucial for the specificity of binding to MHC molecules and T cell recognition. In light of the established potential for change in HIV, even within relatively conserved regions, it seems that mutation at a few critical sites could indeed be an important mechanism whereby HIV might evade cellular immunity. Indeed, the persistence of HIV in infected individuals may rely, at least in part, upon such processes.^^ Evidence that HIV mutants which have escaped CTL immune control may arise during natural infection has now been found.^^ In these studies, fluctuations in the specificity of CTL specific for sequences of the structural protein gag in HIV-seropositive individuals were shown to be matched by variability in proviral gag DNA immunogenic sequences found in the patients' peripheral blood lymphocytes. In vitro analysis, using peptides synthesized to represent each of the proviral variants, confirmed that some of the naturally occurring amino acid substitutions had abolished sequence recognition by circulating CTL, although the degree of variability differed in individuals of differing haplotypes. Thus, variation in immunogenic viral protein sequences may be selected by cellular immune responses, while the accumulation of changes in CTL targets may eventually lead to escape from immune control. Should the number of possible viral target sequences be low, it is conceivable that such variation may eventually outstrip the capacity of the antiviral CTL response to control iniection.
ciation with different class I MHC molecules from the repertoire of that individual.'''"' In addition, uncloned CTL from a single individual may recognize multiple sequences ofgag,''''^ of the pol gene product, reverse transcriptase'*' and of the «e/^ regulatory protein.'''' Our own studies of conserved regions of the env glycoprotein, in this case in the murine system, have also revealed overlapping immunogenic sequences which are recognized by distinct CTL populations in association with different class I alleles of the same inbred strain (Ramsay, Blanden and Cowden, unpubl-data).
Notwithstanding these important findings, it now appears that there may be greater heterogeneity of CTL responses to HIV proteins than previously thought. Using histoeompatibility locus antigen-matched allogeneic target cells, Johnson et al. have recently shown that distinct CTL clones, derived from an infected individual, recognize short, overlapping immunogenic sequences ofgag in asso-
This work was supported by grants from the National Health and Medical Research Council of Australia and the Commonwealth AIDS Research Grants Committee.
Conclusions There is now clear evidence that both humoral and cell-mediated immune responses exert pressure for mutational change in HIV and that escape mutants, resistant to existing immunity, may arise within infected individuals. Data have been presented which suggest that the virus, through its hypervariable envelope glycoprotein, has evolved the capacity to direct host antibody responses and to accommodate mutational changes while retaining infectivity. Recognition by antiviral CTL may be abrogated by even single amino acid changes in relatively conserved immunogenic sequences. The accumulation of variants harbouring such altered sequences may be an important factor in the ultimate progression to AIDS. Despite increasing evidence of heterogeneity in the immune response of infected individuals to various HIV proteins, these findings also have profound implications for vaccination. Protective immunization will almost certainly require immunogens representing a wide diversity of antigenic variants of HIV. Acknowledgements
References 1. Hahn, B. H., Shaw, G. M., Taylor, M.E. eC al. 1986. Genetic variation in HTLV-III/LAV
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over time in patients with AIDS or at risk for proteins with long (19- to 36-residue) AIDS. Sciefice 232: 1548-1553. synthetic peptides./ Cen. Virol. 71: 85-95. 2. Saag, M. S., Hahn, B. H., Gibbons, J., Li, Y., 16. Katzenstein, D. A., Vuujcic, L. K., Latif, A. ct Parks, E. S., Parks, W. P. and Shaw, G. M. al. 1990. Human immunodeficiency virus neu1988. Extensive variation of human immunotralising antibodies in sera from North Amerideficiency virus type 1 in vim. Nature 334: cans and Africans./ AIDS 3: 810-816. 440-444. 17. Koito, A., Hattori, T., Murakami, T. et al. 3. Weiss, R., Teich, N., Varmus, H. and Coffm, 1989. A neutralizing epitope of human immuJ. 1982. RNA Tumour Viruses. Cold Spring nodeficiency virus type 1 has homologous Harbor Laboratory, Cold Spring Harbor, NY. amino acid sequences with the active site of 4. Hu, W. and Temin, H. M. 1990. Retroviral inter-a-trypsin inhibitor. Int. Immunol. 1: 613recombination and reverse transcription. Science 618. 250: 1227-1233. 18. Kido, H., Fukotomi, A. and Katanuma, N. 5. Goudsmit, J., Back, N. K. T. and Nara, P. 1990. A novel membrane-bound serine esterase 1991. Genomic diversity and antigenic variin human T4 * lymphocytes immunologically ation in HIV-1: links between pathogenesis, reactive with antibody inhibiting syncitia epidemiology and vaccine development. induced by HIV-1. / . Biol. Chem. 265: FASEBJ. 5: 2427-2436. 21 979-21 985. 6. Gojobori, T., Noriyama, E. N. and Kimura, N. 19. Clements, G. J., Price-Jones, M. J., Stephens, 1990. Molecular clock of viral evolution, and P. E. et al. 1991. The V3 loop of the HIV-1 the neutral theory. Proc. Natl Acad. Sci. USA surface glycoprotein contains proteolytic cleav87: 10 015-10 018. age sites: a possible function in viral fusion. 7. Kimura, M. 1968. Evolutionary rate at the AIDS Res. Hum. Retroviruses 7: 3-16. molecular level. Nature 217: 624-626. 20. Zwart, G., Langedijk, H., van der Hoek, L. et 8. Li, W-H., Tanimura, M. and Sharp, P. M. al. 1991. Immunodominance and antigenic 1988. Rates and dates of divergence between variation of the principal neutralization doAIDS virus nucleotide sequences. Mol. Biol. main of HIV-1. Virology 181: 481-489. Evol. 5: 313-330. 21. Takahashi, H., Germain, R. N., Moss, B. and 9. Nara, P. L., Garrity, P. R. and Goudsmit, J. Berzofsky, J. A. 1990. An immunodominant 1991. Neutralization of HIV-1: a paradox of class I-restricted cytotoxic T lymphocyte deterhumoral proportions. FASEBJ. 5: 2437-2455. minant of human immunodeficiency virus type 10. LaRosa, G. J., Davide,J. P., Weinhold, K. et al. I induces CD4 class Il-restricted help for itself 1990. Conserved sequence and structural el/ Exp. Med. 171: 571-576. ements in the HIV-1 principal neutralising 22. Leonard, C , Spellman, M., Riddle, L., Harris, domain. Science 249: 932-935. R., Thomas, J. and Gregory, T. 1990. Assign11. Wolf, T. R, Dejong, J., Van Den Berg, H., ment of intrachain disulfide bonds and characTijnagel, N. G. H., Krone, W. and Goudsmit, terization of potential glycosylation sites of the J. 1990. Evolution of sequences encoding the type I recombinant human immunodeficiency principal neutralisation epitope of human imvirus envelope glycoprotein (gpl20) expressed munodeficiency virus type 1 is host-dependent, in Chinese hamster ovary cells. / Biol. Chem. rapid and continuous. Proc. Natl Acad. Sci. USA 265: 10 373-10 382. 87: 9938-9942. 23. Willey, R. L, Ross, E. K., Buckler-White, 12. McKeating, J. A., Gow, J., Goudsmit, J., Pearl, A. J., Theodore, T. S. and Martin, M. A. 1989. L. H., Mulder, C. and Weiss, R.A. 1989. Functional interaction of constant and variable Characterisation of HIV-1 neutralisation esdomains of human immunodeficiency virus cape mutants. AIDS 3: 777-784. type 1 g p l 2 0 . / Virol. 63: 3595-3600. 13. Goudsmit, J. 1988. Immunodominant B-cell 24. Nara, P. L, Smit. L., Dunlop, N. et al. 1990. epitopes of the HIV-1 envelope recognised by Emergence of viruses resistant to neutralization infected and immunised hosts. AIDS 2: 5 4 1 by V3 specific antibodies in experimental 545. HIV-1 IIIB infection of chimpanzees./ Virol. 14. Niedrig, M., Hinkula, J., Weigelt, W. et al. 64: 3779-3791. 1989. Epitope mapping of monoclonal antihodies against human immunodeficiency virus 25. Tremblay, M. and Wainberg, M. A. 1990. type 1 structural proteins hy using peptides. Neutralization of multiple HIV-1 isolates from J. Virol. 63: 3525-3528. a single subject by autologous sequential sera./ 15. Neurath, A R , Strick, N. and Lee, E. S. Y. Infect. Dis. 162: 735-737. 1990. B-cell epitope mapping of human im26. Hoffenbach, A., Langlade-Demoyen, P., Dadmunodeficiency virus (HIV-1) envelope glycoaglio, G. et al 1989. Unusually high fre-
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quencies of HlV-specific cytotoxic T lymphocytes in humans. J. Immunol. 142: 452-462. Clerici, M., Lucey, D. L, Zajac, R. A., Takahashi, H., Berzofsky, J. A. and Shearer, G. M. 1991. Detection of cytotoxic T lymphocytes specific for synthetic peptides of gpl60 in HIV-seropositive individuals,/ Immunol. 146: 2214-2219. Earl, P. L, Koenig, S. and Moss, B. 1991. Biological and immunological properties of human immunodeficiency virus type I envelope glycoprotein: analysis of proteins with truncations and deletions expressed by recombinant vaccinia viruses./ Virol. 65: 31-41. Nixon, D. F., Townsend, A. R. M., Elvin, J. G., Rizza, C. R., Gallway, J. and McMichael, A. J. 1988. HIV-1 ^a^-specific cytotoxic T lymphocytes defined with recombinant vaccinia virus and synthetic peptides. Nature 336: 484-487. Claverie, J. M. Kourilsky, P., LangladeDemoyen, P. et al. 1988. T-immunogenic peptides are constituted of rare sequence patterns. Use in the identification of T epitopes in the human immunodeficiency virus gag protein. Eur.J. Immunol. 18: 1547-1553. Walker, B. D., Flexner, C, Birch-Limberger, K. et al. 1989. Long-term culture and fine specificity of human cytotoxic T-lymphocyte clones reactive with human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA 86: 9514-9518. Hosmalin, A., Clerici, M., Houghten, R. et al. 1990. An epitope in human immunodeficiency
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virus 1 reverse transcriptase recognised by both mouse and human cytotoxic T lymphocytes. Proc. Natl Acad. Sci. USA 87: 2344-2348. Koenig, S., Fuerst, T. R., Wood, L. V. et al. 1990. Mapping the fine specificity of a cytolytic T cell response to HIV-1 nef protein./ Immunol. 145: 127-135. Culmann, B., Gomard, E., Kieny, M. P. et al. 1991. Six epitopes reacting with human cytotoxic CD8 * T cells in the central region of the HIV-1 nef protein. / Immunol. 146: 15601565. Schwartz, R. H. 1985. T-lymphocyte recognition of antigen in association with gene products of the major histocompatibility complex. Annu. Rev. Immunol. 3: 237-261. Takahashi, H., Merli, S., Putney, S. D. et al. 1989. A single amino acid interchange yields reciprocal CTL specificities for HIV-1 gpl60. Science 246: 118-121. Takahashi, H., Houghten, R., Putney S. D. et al. 1989. Structural requirements for class I MHC molecule-mediated antigen presentation and cytotoxic T cell recognition of an immunodominant determinant of the HIV envelope protein./ Exp. Med. 170: 2023-2035. Phillips, R. E., Rowland-Jones, S., Nixon, D. F. et al. 1991. Human immunodeficiency virus genetic variation that can escape cytotoxic T cell recognition. Nature 354: 453-460. Johnson, R. P., Trocha, A., Yang, L. et al. 1991. HIV-1 ^fl^-specific cytotoxic T lymphocytes recognize multiple highly conserved epitopes./ Immunol. 147: 1512-1521.
Diversity and variation in human immunodeficiency virus: Implications for immune control ALISTAIRJ. RAMSAY Division of Cell Biology, fohn Curtin School ofMedical Research, Australian National University, Canberra, Australian Capital Territory, Australia
Introduction The central problems in understanding the progression of infection with HIV to AIDS stem from the long latent period in the host; the median time from infection to development of disease is currently about 8 years. This raises two important questions. First, what is the role of the immune response in progression to AIDS: is the decline in the immune response the cause or the consequence of the rise in HIV titre? Second, what is the effect of the extreme sequence diversity of HIV: do variants arise that escape the immune response? It is now clear that HIV-1 exists, in the individual, as a population of viruses exhibiting a significant degree of genomic variation.''^ This implies that a dynamic balance exists between host defence mechanisms against the virus and the virus itself, which is not surprising given that HIV infects the very cells that initiate antiviral immune responses. The genomic diversity and antigenic variability of HIV and the problems that this presents for immune control of the virus are the subjects of this paper.
HIV mutates rapidly during replication HIV is a member of the lentivirus subfamily of the retroviruses: small enveloped viruses
which contain a diploid single-stranded RNA genome. Entry to target cells occurs by fusion of virion and cell membrane, following which the viral RNA genome is converted into double-stranded DNA by the action of reverse transcriptase and viral ribonuclease H.^''* Viral DNA is then integrated into the host chromosome to form a provirus via the action of virus-encoded integrase and remains permanently associated with host genetic material. Virus expression begins with the transcription of pro viral DNA in the activated cell by host RNA polymerase. The resultant viral RNA is either translated to produce viral proteins or packaged as the viral genome. Retroviral passage in vivo is a complex process. There is potential for a high rate of homologous recombination due to the packaging oftwo retroviral RNA molecules in one virion. Thus, insertions, duplications and deletions may occur in single genes with major effects on virus phenotype, but, in addition, recombination of whole virus segments may also alter virus characteristics.'* Clearly then, HIV should be added to the list of viruses that undergo genetic 'shift'. Reverse transcription during lentivirus replication is the basic mechanism generating recombinants and point mutations. Owing to the infidelity of this process, it is likely that a pool of genotypes is generated, with viable ones surviving as progeny virus.^ Selection of viral genomes from replicating progeny
Correspondence: Dr Alistair Ramsay, Division of Cell Biology, John Curtin School of Medical Research, PO Box 334, Canberra, ACT 2601, Australia. Presented at the 4th Frank and Bobbie Fenner Conference in Medical Research on Genetic Variation and Selection in Microbiology and Immunology, held on 6-8 November 1991 at the John Curtin School of Medical Research, Canberra, Australia. Accepted for publication 6 November 1991.
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would then allow their evasion of and survival in the host immune system. Evolution of viral genes is characterized by enormously high speed compared to nuclear genes of eukaryotic ^organisms. It has been estimated that evolution of the HIV gag gene occurs at a similar rate to that of the haemagglutinin gene of human influenza virus, faster than in other retroviruses and many orders of magnitude faster than in eukaryotic genes.^ The gag gene appears to evolve according to the neutral theory of evolution,^ in that silent mutations predominate over non-silent (or amino acid-changing) mutations. This theory holds that nucleic acid substitutions result from the random selection and fixation of neutral or near-neutral variants. In marked contrast, the rate of non-silent changes occurring in the hypervariable regions of the HIV env gene (17.2 xlO~^ base substitutions/site per year) almost equals the rate of silent changes (14x10"^), with both exceeding the estimate for silent gag mutations (9.7 x 10"^).^ Clearly, evolution of the HIV envelope occurs at an extremely high rate and not according to the neutral theory. It is likely that this is due to intensive immunological selection pressure.
The third variahle envelope domain dominates humoral immunity As outlined above, mutation in hypervariable regions of the env glycoprotein occurs rapidly, although the overall rate of occurrence of silent mutations still exceeds that of non-silent changes. This may imply that different regions of the envelope (a structure built from alternating domains separated by cysteine bridges) either allow, or do not allow, amino acid substitutions.^ Regions that allow substitutions resulting in changes in amino acid sequence may do so where the substitutions confer benefit on the virus (for example, allowing escape from host immunity) or where substitutions are chemically equivalent, with no resultant functional change.^ There are, of course, functional constraints on the virus. In the case of the envelope, evolution around a conserved cellular receptor must dictate the limit of variation possible in the overall structure and
shape of the glycoprotein. Recent work, however, suggests that HIV has overcome this problem and evolved a means of escape from the host neutralizing antibody through a convergence of the structural and functional roles of its third variable (V3) domain.""'^ Analyses of sera from HIV-infected individuals have led to the definition of a number of immunogenic sequences of env against which neutralizing antibody responses are directed.'^"'^ One of these sequences, occurring in the V3 domain, has been shown to dominate the neutralizing antibody response to infection or immunization. Thus, although infected individuals mount responses to a number of different regions of the env glycoprotein, each responds to the V3 domain and it is this response which is predominant in their antibody repertoire. The sequence is defined by a cysteine-cysteine bridge formed between amino acids 296-309 and 331-343,^ depending on the strain of virus, and occurs in a region exhibiting the exceptionally high rate of over 10"^ non-silent substitutions/site per year. Despite this enormous variation, one consensus V3 sequence predominates in viruses circulating during the current AIDS epidemic in the United States and Europe.^°'"'^^ This amino acid conservation is most pronounced in the central tetrapeptide, GPGR, which is found on the tip of the V3 loop in a position exposed to antibody. The great majority of isolates from both continents display this motif. Evidence from several laboratories suggests that this site, while not influencing the ability of the envelope to bind to the virus receptor, CD4, is a substrate for hydrolysis by cell surface enzymes.'^"'^ The V3 loop may, therefore, represent a fusion domain, playing a direct role in virus infectivity and the potent neutralizing effect of V3-speciflc antibody may reflect blocking of this activity.^ Indeed, it has become clear that this region of the envelope represents a multifunctional molecule and a number of demonstrated structural and functional properties, together with some proposed roles, are outlined in Table 1.
y3 mediates escape from humoral immunity Initially, it was thought that the V3 region represented a simple, linear epitope and that
Antigenic variation and immunity to HIV
Table 1. Structural and functional properties ofthe V3 domain. Established properties Immunodominant B cell epitope'^''"* Cross-reactive epitope^''^" Flexible contiguous neutralization epitope^ CD8 CTL/CD4 T helper cell epitope^' Component of an ordered oligomeric Proposed roles Determinant of cellular tropism' Functional proteolytic substrate'^ Protease inhibitor"
knowledge of the variable amino acids in V3 loops of different HIV strains would lead to a successful peptide-based vaccine. It has now become clear that this is not the case. Viruses exhibiting mutations from their parental amino acid sequences both within and outside the V3 domain, which also have escaped from the neutralizing antibody, have now been derived following passage of the virus both in vitro and in vivo. First, McKeating et al.^^ passaged cloned HIV-1 in vitro in the presence of an isolate-specific neutralizing antibody against V3 determinants and found that variants resistant to neutralization were rapidly selected. More interesting, however, are recent findings of in vivo antigenic drift of HIV-1. In the first of these studies, the emergence of escape mutants was found in sequential viral isolates recovered from chimpanzees up to 32 weeks after infection.^'* Mutants were defined by their relative resistance to neutralization by V3 strain-specific neutralizing antibodies. Surprisingly, there was complete conservation of the parental V3 primary sequence in all cases, although sequence analysis revealed multiple amino acid substitutions in the 32 week isolate, especially in the fourth constant and variable regions. It appeared, therefore, that non-silent mutations, distant from the V3 neutralizing antibody-binding site, were conferring resistance to neutralization. Similar results have been reported from studies of acute seroconversions in humans.^^ Here, neutralizing antibody titres against the homotypic viral strain first isolated from an
217
infected individual were high in all sequential serum samples tested. In contrast, the appearance of a neutralizing antibody response to a particular viral variant occurred only around the time of its isolation. These findings suggest that viral variants in infected individuals may not be effectively neutralized by antibodies generated against previously dominant viral strains. This presents a scenario in which the antibody response follows emerging variants without ever affecting them at the point of emergence.^ Antigenic drift occurring in this manner, within infected individuals, contrasts with that exhibited by influenza virus, which occurs at the population level, though the capacity of these variants to infect individuals with circulating antibody to earlier strains requires further study. In the light of these data, Nara and Goudsmit have recently proposed a scheme whereby the envelope, and the V3 loop in particular, controls host-directed antibody responses to the benefit of the virus.^•'^ Briefiy, they suggest that rapid replication of the virus upon infection leads to the production of neutralizing antibody and subsequent control of primary viraemia (as in other lentiviral infections). The control of acute viral replication in this manner, however, may be viewed as a survival property of the virus, in that the neutralizing antibody selects for viruses which have optimized transmission and longevity. It is proposed that this paradoxical situation is mediated by the V3 loop. Thus, antibody to this region dominates the repertoire, rendering the host less able to respond to emerging viruses with closely related V3 structures and resulting in a relatively restricted dominance of the neutralization response. These authors suggest that the demonstrated flexibility of the V3 domain (involving distant-site amino acid mutations) provides for subtle conformational variability of this region which does not infiuence its critical functional role in infeetivity, but provides escape from host immunity. Thus, molecular evolution may have allowed for the selection of this multifunction region, capable of the kind of conformational flexibility necessary to allow escape from neutralization, while preserving functional integrity.
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HIV may evade cellular immune responses Clearly, humoral immunity is unable to control HIV infection, although it remains unclear exactly which immune responses are important in this regard; at this stage there are no individuals known to have recovered who are available for study. It is apparent, however, that HIV elicits a vigorous, sustained cytotoxic T cell (CTL) response soon after infection and that this response is greatly diminished in individuals who have developed AIDS.^^ In general, CTL responses are directed against only a few short sequences of different proteins and each is apparently restricted to recognition by effector cells from individuals of one or two baplotypes. Several HIV proteins have now been shown to be immunogenic for CTL and some details are presented in Table 2. Comparatively little is known of the role played by CTL reactivity in HIV infection or of the evolutionary pressure which it may exert on the virus. In general, viruses appear to have two potential mechanisms of escape from CTL immunity, that is by mutating to evade either tbe antiviral T cell receptor or host class
I major histocompatibility complex (MHC) molecules. T cell repertoires have been widely considered to be broad enough to allow recognition of mutated viral proteins, providing that antigen processing and MHC association and presentation can occur.^^ Mutations at sites critical for binding to MHC molecules, however, would be expected to render proteins invisible to existing antiviral CTL, as the repertoire of class I MHC molecules of each individual is very small and cannot be expanded. A number of recent studies have focused on the relationship between sequence variation and CTL immunogenicity of HIV. Data from the first of these suggest that a single amino acid residue can contribute most of the binding energy which defines the specificity of the T cell receptor and show that mutations at such a critical site can abrogate recognition of an immunogenic sequence by CTL. Briefiy, murine CTL clones were raised against an H-2D''-restricted immunogenic sequence of env gpl60 (Table 2) in two different strains of HIV-1 (i.e. III-B and MN) and were found to be totally non-cross-reactive. However, reciprocal changes in a single amino acid residue
Table 2. CTL target sites of HIV proteins Protein
aa sequence
env
315-329 112-124 315-329 428-443 834-848 5 sequences 265-279 193-203 219-233 418-433 446-460 172-196 325-349 461-485 495-519 203-219 203-219 73-97 113-128
pol
nef
Class I restriction molecule
Reference
Mouse Human
H-2D'' HLA-A2
(21) (27)
Human Human Human
not determined HLA-B27 HLA-A2
(28) (29) (30)
Human
HLA-B8 HLA-All HLA-B8 HLA-All H-2^ HLA-B17, HLA-B37 HLA-A3 HLA-B17, HLA-B37
(31)
Mouse Human Human Human
(32) (33) (34)
Antigenic variation and immunity to HIV
219
(at the hypervariable position 325) of the respective sequences was sufficient to completely reverse the specificity of CTL recognition, despite five other sequence differences. When considered together with earlier studies by the same group,'^^ these findings have established that as few as two to four amino acid residues in a minimal immunogenic sequence are crucial for the specificity of binding to MHC molecules and T cell recognition. In light of the established potential for change in HIV, even within relatively conserved regions, it seems that mutation at a few critical sites could indeed be an important mechanism whereby HIV might evade cellular immunity. Indeed, the persistence of HIV in infected individuals may rely, at least in part, upon such processes.^^ Evidence that HIV mutants which have escaped CTL immune control may arise during natural infection has now been found.^^ In these studies, fluctuations in the specificity of CTL specific for sequences of the structural protein gag in HIV-seropositive individuals were shown to be matched by variability in proviral gag DNA immunogenic sequences found in the patients' peripheral blood lymphocytes. In vitro analysis, using peptides synthesized to represent each of the proviral variants, confirmed that some of the naturally occurring amino acid substitutions had abolished sequence recognition by circulating CTL, although the degree of variability differed in individuals of differing haplotypes. Thus, variation in immunogenic viral protein sequences may be selected by cellular immune responses, while the accumulation of changes in CTL targets may eventually lead to escape from immune control. Should the number of possible viral target sequences be low, it is conceivable that such variation may eventually outstrip the capacity of the antiviral CTL response to control iniection.
ciation with different class I MHC molecules from the repertoire of that individual.'''"' In addition, uncloned CTL from a single individual may recognize multiple sequences ofgag,''''^ of the pol gene product, reverse transcriptase'*' and of the «e/^ regulatory protein.'''' Our own studies of conserved regions of the env glycoprotein, in this case in the murine system, have also revealed overlapping immunogenic sequences which are recognized by distinct CTL populations in association with different class I alleles of the same inbred strain (Ramsay, Blanden and Cowden, unpubl-data).
Notwithstanding these important findings, it now appears that there may be greater heterogeneity of CTL responses to HIV proteins than previously thought. Using histoeompatibility locus antigen-matched allogeneic target cells, Johnson et al. have recently shown that distinct CTL clones, derived from an infected individual, recognize short, overlapping immunogenic sequences ofgag in asso-
This work was supported by grants from the National Health and Medical Research Council of Australia and the Commonwealth AIDS Research Grants Committee.
Conclusions There is now clear evidence that both humoral and cell-mediated immune responses exert pressure for mutational change in HIV and that escape mutants, resistant to existing immunity, may arise within infected individuals. Data have been presented which suggest that the virus, through its hypervariable envelope glycoprotein, has evolved the capacity to direct host antibody responses and to accommodate mutational changes while retaining infectivity. Recognition by antiviral CTL may be abrogated by even single amino acid changes in relatively conserved immunogenic sequences. The accumulation of variants harbouring such altered sequences may be an important factor in the ultimate progression to AIDS. Despite increasing evidence of heterogeneity in the immune response of infected individuals to various HIV proteins, these findings also have profound implications for vaccination. Protective immunization will almost certainly require immunogens representing a wide diversity of antigenic variants of HIV. Acknowledgements
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