Thus, immunodominance against poorly conserved epitopes is a single flaw that could trigger a cascade of events leading to the multiple mechanisms for CTL failure. Indirect evidence for this hypothesis is the observation that HLA B*57 is strongly associated with better immune control of HIV-1 during early and chronic infection . CTL responses against highly conserved B*57 epitopes are immunodominant even in early infection  as opposed to the shifting pattern of epitope recognition observed for other HLA types such as A*02; earlier targeting of conserved epitopes may be a key factor in the protective influence of B*57. Also suggesting the importance of early HIV-1 control, antiretroviral treatment even relatively late in acute infection can help preserve HIV-1-specific CD4 T helper responses and at least temporarily boost immune control of viremia [46,47].
This unifying hypothesis for CTL failure also provides explanations for seemingly inconsistent data regarding epitope escape mutation. Human studies of epitope escape mutation have demonstrated highly variable results; this variability can be explained by the timing of studies in different stages of infection. CTL targeting differs substantially between early (pre-establishment of set-point viremia) and chronic infection (quasi-steady state viremia during the asymptomatic phase) [48–50]. This finding is consistent with early immunodominant CTL targeting of unconstrained viral epitopes followed by decay of those CTL after epitope mutation [17,24,26]. Subsequently during chronic infection, replacement by subdominant CTL responses against more conserved epitopes takes place to reach relative stability during chronic infection, reflected by set-point viremia [18,35]. Accordingly, escape mutations occur in many epitopes concurrently during acute infection, whereas, during chronic infection, mutations occur in single epitopes infrequently if at all (see below).
Another paradox has been that the development of specific epitope escape mutations usually has not been observed to correlate to obvious changes in viremia. This lack of correlation would be consistent with escape mutation occurring en masse during acute infection (observed as concurrent escape within several epitopes [21–26]). Moreover, during chronic infection, an isolated escape mutation in a single epitope can take place (among multiple non-mutating epitopes), but this event occurs infrequently due to CTL targeting of epitopes in conserved regions of the viral proteome [16–18,35,51]. Thus it would be expected that an escape mutation in any single epitope during either phase of infection would not correlate to major changes in immune control.
While some reports have suggested that escape mutation in single epitopes causes increased set-point viremia during chronic infection [16,52,53], such examples are rare and other studies have shown no such association , or disease progression without epitope mutation [54,55]. A disproportionate contribution of different epitopes to control of viremia is a possible explanation for these inconsistent findings. However, an alternative reason is that increased viral replication resulting from global CTL dysfunction (see above, and Fig. 1) allows development of escape mutations that are difficult to generate because they require multiple (compensatory) mutations [51,56].
In summary, widespread escape mutation in multiple epitopes simultaneously during early infection is a key process setting the course for subsequent hypofunction of CTL through early loss of HIV-1-specific helper CD4 T lymphocytes (and original antigenic sin). In contrast, although CTL have re-targeted to conserved epitopes during chronic infection, CTL dysfunction plays the dominant role for failure of immune containment; viral epitope escape mutation would have only a minor role during this phase.
Assuming that an effective vaccine delivery system is developed to target CTL against HIV-1, what viral sequences should be delivered? There has been vigorous debate on this topic recently, almost all of which has centered on the question of which protein genes should be included in a vaccine. It has been argued that early expressed proteins (Tat, Rev, and Nef) should be included because early epitope expression allows better antiviral activity of CTL in vitro[57,58]. Others have argued that Env should be included for its potential to concurrently elicit neutralizing antibody responses. Still others have argued that large studies of patients show statistically significant negative associations of Gag-specific CTL responses and positive associations of Env- and Nef-specific CTL responses with viremia [59,60], and thus a vaccine should deliver Gag but not Env or Nef. Finally, it has been proposed that a vaccine should be inclusive of multiple proteins, to maximize breadth of CTL epitope targeting both to minimize the chance for escape as well as to maximize the chance of generating effective CTL in the absence of a clear understanding of the determinants of CTL efficacy.
Selecting whole proteins for vaccines is a historical approach, reminiscent of strategies applied to Hepatitis B virus, Clostridium tetani, and other pathogens for which successful antibody-based vaccines have been developed. However, from the standpoint of CTL responses, this approach may not be relevant. For a given CTL, antiviral activity is determined by: (i) its efficiency in killing an infected cell [61,62]; (ii) the temporal relationship of this killing to viral replication [57,58]; and (iii) the propensity for the epitope to mutate and allow escape . Beyond expression level, the protein context of the epitope is minimally relevant. For example, it is likely that Gag-targeting is not intrinsically more protective than Env-targeting, but that the observed statistical trends in vivo[59,60] are reflections that epitopes from within these proteins follow overall trends based on their parent proteins. Both Gag and Env are highly expressed proteins, but Gag is relatively conserved in sequence relative to Env. Accordingly, Gag epitopes will be more conserved than Env epitopes on average, and less prone to escape on average, explaining the association of Gag-specific CTL to reduced viremia across large numbers of infected persons. However, CTL targeting a highly conserved epitope in Env would be less prone to escape than CTL targeting a poorly conserved epitope in Gag, and therefore potentially more effective. Thus, it is important to consider CTL targeting in terms of epitope properties, rather than whole proteins, and vaccine design should not be constrained by thinking of viral proteins as antigenic units.
If early immunodominance against variable epitopes is the central process that leads to CTL failure, then avoiding this process with a vaccine could be the solution to promoting effective responses that delay or prevent disease. A vaccine administered before natural infection could have the opportunity to set CTL memory before exposure to whole HIV-1 and avoid natural immunodominance. Because memory-recall responses in adaptive immunity are rapid, vaccine-primed memory CTL against otherwise subdominant epitopes could respond quickly to HIV-1 challenge, thus having the edge over the usually immunodominant but yet naive CTL responses. By avoiding escape, these vaccine-primed CTL responses could exert earlier control of CD4 T lymphocyte loss, preserving the help that is required for CTL efficacy in chronic infection and avoiding the limitation of original antigenic sin (Fig. 3). Clinical evidence suggests that subdominant CTL responses can be effective in controlling infections .
How might this be implemented in vaccine design? Including whole proteins in a vaccine could recapitulate the problem of natural immunodominance, because variable regions of these proteins would also be available for CTL targeting. The solution may be to include only highly conserved regions of the virus, thus limiting the epitope choices to those falling in very constrained sequences and precluding potentially immunodominant responses against variable regions. Even relatively conserved proteins such as Gag contain variable regions and codons; omitting these from a vaccine could maximize the benefit of general Gag CTL targeting. Similarly, variable proteins such as Env contain conserved regions, offering potential targets for effective CTL responses.
Given that most of the HIV-1 proteome is variable, can this be accomplished? The few regions that are highly conserved may be too short to yield a breadth of CTL responses, and for any given HLA class I profile, there may be few epitopes. However, it is unclear that CTL breadth is crucial for immune control. For other viral infections where CTL responses are effective, these responses can be highly focused on one or two immundominant epitopes [65,66]. Breadth may be less important if CTL targeting is optimized, and having a few responses against conserved epitopes may suffice. Another caveat is that conserved sequences vary between HIV-1 clades. The design of a vaccine that will be immunogenic for CTL against selectively highly conserved epitopes across varying human populations in areas where HIV-1 clades differ will be a substantial challenge for implementing this strategy.
HIV-1 presents a challenge for vaccine development that has bested the historically successful empiric approach of mimicking natural infection. Creating an effective vaccine against HIV-1 will likely require strategies that address the pathophysiologic failure of immunity against the virus. The CTL response is an arm of immunity that appears partially effective during natural infection, and addressing the mechanism of CTL failure will be crucial for design of a vaccine intended to prevent or attenuate disease via CTL. The solution may be to avoid natural patterns of immunodominance that lead to CTL failure during acute infection, by excluding inappropriately variable sequences from the vaccine. Accomplishing this task will require novel approaches that account for the great variations of virus and host.
While this article was in press, Merck and the HIV-1 Vaccine Trials Network announced the early termination of the STEP trial of a rAd5 HIV-1 vaccine, due to lack of protection measured by infection rate or setpoint viremia level. This serious setback in the quest for an effective HIV-1 vaccine underscores the importance of considering mechanisms of CTL failure in vaccine design.
The author has been supported by PHS grants AI043203 and AI051970, and would like to thank countless colleagues for sharing their ideas and discussion.
1. Yang OO. Will we be able to ‘spot’ an effective HIV-1 vaccine? Trends Immunol 2003; 24:67–72.
2. Lieberman J. Tracking the killers: how should we measure CD8 T cells in HIV infection? AIDS 2004; 18:1489–1493.
3. Bennett MS, Ng HL, Dagarag M, Ali A, Yang OO. Epitope-dependent avidity thresholds for cytotoxic T-lymphocyte clearance of virus-infected cells. J Virol 2007; 81:4973–4980.
4. Rosenberg ES, Billingsley JM, Caliendo AM, Boswell SL, Sax PE, Kalams SA, et al
. Vigorous HIV-1-specific CD4+ T cell responses associated with control of viremia. Science 1997; 278:1447–1450.
5. Kalams SA, Buchbinder SP, Rosenberg ES, Billingsley JM, Colbert DS, Jones NG, et al
. Association between virus-specific cytotoxic T-lymphocyte and helper responses in human immunodeficiency virus type 1 infection. J Virol 1999; 73:6715–6720.
6. Champagne P, Ogg GS, King AS, Knabenhans C, Ellefsen K, Nobile M, et al
. Skewed maturation of memory HIV-specific CD8 T lymphocytes. Nature 2001; 410:106–111.
7. Appay V, Dunbar PR, Callan M, Klenerman P, Gillespie GM, Papagno L, et al
. Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections. Nat Med 2002; 8:379–385.
8. Migueles SA, Laborico AC, Shupert WL, Sabbaghian MS, Rabin R, Hallahan CW, et al
. HIV-specific CD8(+) T cell proliferation is coupled to perforin expression and is maintained in nonprogressors. Nat Immunol 2002; 3:1061–1068.
9. Zhang D, Shankar P, Xu Z, Harnisch B, Chen G, Lange C, et al
. Most antiviral CD8 T cells during chronic viral infection do not express high levels of perforin and are not directly cytotoxic. Blood 2003; 101:226–235.
10. Zuniga R, Lucchetti A, Galvan P, Sanchez S, Sanchez C, Hernandez A, et al
. Relative dominance of Gag p24-specific cytotoxic T lymphocytes is associated with human immunodeficiency virus control. J Virol 2006; 80:3122–3125.
11. Dagarag M, Ng H, Lubong R, Effros RB, Yang OO. Differential impairment of lytic and cytokine functions in senescent human immunodeficiency virus type 1-specific cytotoxic T lymphocytes. J Virol 2003; 77:3077–3083.
12. Day CL, Kaufmann DE, Kiepiela P, Brown JA, Moodley ES, Reddy S, et al
. PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Nature 2006; 443:350–354.
13. Harrer T, Harrer E, Kalams SA, Barbosa P, Trocha A, Johnson RP, et al
. Cytotoxic T lymphocytes in asymptomatic long-term nonprogressing HIV-1 infection. Breadth and specificity of the response and relation to in vivo viral quasispecies in a person with prolonged infection and low viral load. J Immunol 1996; 156:2616–2623.
14. Rinaldo C, Huang XL, Fan ZF, Ding M, Beltz L, Logar A, et al
. High levels of antihuman immunodeficiency virus type 1 (HIV-1) memory cytotoxic T-lymphocyte activity and low viral load are associated with lack of disease in HIV-1-infected long-term nonprogressors. J Virol 1995; 69:5838–5842.
15. Yang OO. CTL ontogeny and viral escape: implications for HIV-1 vaccine design. Trends Immunol 2004; 25:138–142.
16. Goulder PJ, Phillips RE, Colbert RA, McAdam S, Ogg G, Nowak MA, et al
. Late escape from an immunodominant cytotoxic T-lymphocyte response associated with progression to AIDS. Nat Med 1997; 3:212–217.
17. Jamieson BD, Yang OO, Hultin L, Hausner MA, Hultin P, Matud J, et al
. Epitope escape mutation and decay of human immunodeficiency virus type 1 specific cytotoxic T lymphocyte responses. J Immunol 2003; 171:5372–5379.
18. Koibuchi T, Allen TM, Lichterfeld M, Mui SK, O'Sullivan KM, Trocha A, et al
. Limited sequence evolution within persistently targeted CD8 epitopes in chronic human immunodeficiency virus type 1 infection. J Virol 2005; 79:8171–8181.
19. Yewdell JW. Confronting complexity: real-world immunodominance in antiviral CD8+ T cell responses. Immunity 2006; 25:533–543.
20. Perelson AS, Essunger P, Ho DD. Dynamics of HIV-1 and CD4+ lymphocytes in vivo. Aids 1997; 11(Suppl A):S17–S24.
21. Borrow P, Lewicki H, Wei X, Horwitz MS, Peffer N, Meyers H, et al
. 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; 3:205–211.
22. Jones NA, Wei X, Flower DR, Wong M, Michor F, Saag MS, et al
. Determinants of human immunodeficiency virus type 1 escape from the primary CD8+ cytotoxic T lymphocyte response. J Exp Med 2004; 200:1243–1256.
23. Price DA, Goulder PJ, Klenerman P, Sewell AK, Easterbrook PJ, Troop M, et al
. Positive selection of HIV-1 cytotoxic T lymphocyte escape variants during primary infection. Proc Natl Acad Sci USA 1997; 94:1890–1895.
24. Cao J, McNevin J, Malhotra U, McElrath MJ. Evolution of CD8+ T cell immunity and viral escape following acute HIV-1 infection. J Immunol 2003; 171:3837–3846.
25. Karlsson AC, Iversen AK, Chapman JM, de Oliviera T, Spotts G, McMichael AJ, et al
. Sequential broadening of CTL responses in early HIV-1 infection is associated with viral escape. PLoS ONE 2007; 2:e225.
26. Oxenius A, Price DA, Trkola A, Edwards C, Gostick E, Zhang HT, et al
. Loss of viral control in early HIV-1 infection is temporally associated with sequential escape from CD8+ T cell responses and decrease in HIV-1-specific CD4+ and CD8+ T cell frequencies. J Infect Dis 2004; 190:713–721.
27. Fazekas de St G, Webster RG. Disquisitions of Original Antigenic Sin. I. Evidence in man. J Exp Med 1966; 124:331–345.
28. Francis T Jr. Influenza: the new acquayantance. Ann Intern Med 1953; 39:203–221.
29. Klenerman P, Zinkernagel RM. Original antigenic sin impairs cytotoxic T lymphocyte responses to viruses bearing variant epitopes. Nature 1998; 394:482–485.
30. Mongkolsapaya J, Dejnirattisai W, Xu XN, Vasanawathana S, Tangthawornchaikul N, Chairunsri A, et al
. Original antigenic sin and apoptosis in the pathogenesis of dengue hemorrhagic fever. Nat Med 2003; 9:921–927.
31. Allen TM, Yu XG, Kalife ET, Reyor LL, Lichterfeld M, John M, et al
. De novo generation of escape variant-specific CD8+ T-cell responses following cytotoxic T-lymphocyte escape in chronic human immunodeficiency virus type 1 infection. J Virol 2005; 79:12952–12960.
32. Yang OO, Daar ES, Jamieson BD, Balamurugan A, Smith DM, Pitt JA, et al
. Human immunodeficiency virus type 1 clade B superinfection: evidence for differential immune containment of distinct clade B strains. J Virol 2005; 79:860–868.
33. Altfeld M, Allen TM, Yu XG, Johnston MN, Agrawal D, Korber BT, et al
. HIV-1 superinfection despite broad CD8+ T-cell responses containing replication of the primary virus. Nature 2002; 420:434–439.
34. Feeney ME, Tang Y, Pfafferott K, Roosevelt KA, Draenert R, Trocha A, et al
. HIV-1 viral escape in infancy followed by emergence of a variant-specific CTL response. J Immunol 2005; 174:7524–7530.
35. Yusim K, Kesmir C, Gaschen B, Addo MM, Altfeld M, Brunak S, et al
. Clustering patterns of cytotoxic T-lymphocyte epitopes in human immunodeficiency virus type 1 (HIV-1) proteins reveal imprints of immune evasion on HIV-1 global variation. J Virol 2002; 76:8757–8768.
36. Mattapallil JJ, Douek DC, Hill B, Nishimura Y, Martin M, Roederer M. Massive infection and loss of memory CD4+ T cells in multiple tissues during acute SIV infection. Nature 2005; 434:1093–1097.
37. Mehandru S, Poles MA, Tenner-Racz K, Horowitz A, Hurley A, Hogan C, et al
. Primary HIV-1 infection is associated with preferential depletion of CD4+ T lymphocytes from effector sites in the gastrointestinal tract. J Exp Med 2004; 200:761–770.
38. Veazey RS, DeMaria M, Chalifoux LV, Shvetz DE, Pauley DR, Knight HL, et al
. Gastrointestinal tract as a major site of CD4+ T cell depletion and viral replication in SIV infection. Science 1998; 280:427–431.
39. Douek DC, Brenchley JM, Betts MR, Ambrozak DR, Hill BJ, Okamoto Y, et al
. HIV preferentially infects HIV-specific CD4+ T cells. Nature 2002; 417:95–98.
40. Matloubian M, Concepcion RJ, Ahmed R. CD4+ T cells are required to sustain CD8+ cytotoxic T-cell responses during chronic viral infection. J Virol 1994; 68:8056–8063.
41. Liu Z, Cumberland WG, Hultin LE, Prince HE, Detels R, Giorgi JV. Elevated CD38 antigen expression on CD8+ T cells is a stronger marker for the risk of chronic HIV disease progression to AIDS and death in the Multicenter AIDS Cohort Study than CD4+ cell count, soluble immune activation markers, or combinations of HLA-DR and CD38 expression. J Acquir Immune Defic Syndr Hum Retrovirol 1997; 16:83–92.
42. Liu Z, Hultin LE, Cumberland WG, Hultin P, Schmid I, Matud JL, et al
. Elevated relative fluorescence intensity of CD38 antigen expression on CD8+ T cells is a marker of poor prognosis in HIV infection: results of 6 years of follow-up. Cytometry 1996; 26:1–7.
43. Yang OO, Lin H, Dagarag M, Ng HL, Effros RB, Uittenbogaart CH. Decreased perforin and granzyme B expression in senescent HIV-1-specific cytotoxic T lymphocytes. Virology 2005; 332:16–19.
44. Saah AJ, Hoover DR, Weng S, Carrington M, Mellors J, Rinaldo CR Jr, et al
. Association of HLA profiles with early plasma viral load, CD4+ cell count and rate of progression to AIDS following acute HIV-1 infection. Multicenter AIDS Cohort Study. AIDS 1998; 12:2107–2113.
45. Altfeld M, Addo MM, Rosenberg ES, Hecht FM, Lee PK, Vogel M, et al
. Influence of HLA-B57 on clinical presentation and viral control during acute HIV-1 infection. AIDS 2003; 17:2581–2591.
46. Oxenius A, Price DA, Easterbrook PJ, O'Callaghan CA, Kelleher AD, Whelan JA, et al
. Early highly active antiretroviral therapy for acute HIV-1 infection preserves immune function of CD8+ and CD4+ T lymphocytes. Proc Natl Acad Sci USA 2000; 97:3382–3387.
47. Rosenberg ES, Altfeld M, Poon SH, Phillips MN, Wilkes BM, Eldridge RL, et al
. Immune control of HIV-1 after early treatment of acute infection. Nature 2000; 407:523–526.
48. Brander C, Riviere Y. Early and late cytotoxic T lymphocyte responses in HIV infection. AIDS 2002; 16(Suppl 4):S97–S103.
49. Goulder PJ, Altfeld MA, Rosenberg ES, Nguyen T, Tang Y, Eldridge RL, et al
. Substantial differences in specificity of HIV-specific cytotoxic T cells in acute and chronic HIV infection. J Exp Med 2001; 193:181–194.
50. Lichterfeld M, Yu XG, Cohen D, Addo MM, Malenfant J, Perkins B, et al
. HIV-1 Nef is preferentially recognized by CD8 T cells in primary HIV-1 infection despite a relatively high degree of genetic diversity. AIDS 2004; 18:1383–1392.
51. Kelleher AD, Long C, Holmes EC, Allen RL, Wilson J, Conlon C, et al
. Clustered mutations in HIV-1 gag are consistently required for escape from HLA-B27-restricted cytotoxic T lymphocyte responses. J Exp Med 2001; 193:375–386.
52. Feeney ME, Tang Y, Roosevelt KA, Leslie AJ, McIntosh K, Karthas N, et al
. Immune escape precedes breakthrough human immunodeficiency virus type 1 viremia and broadening of the cytotoxic T-lymphocyte response in an HLA-B27-positive long-term-nonprogressing child. J Virol 2004; 78:8927–8930.
53. Geels MJ, Jansen CA, Baan E, De Cuyper IM, van Schijndel GJ, Schuitemaker H, et al
. CTL escape and increased viremia irrespective of HIV-specific CD4+ T-helper responses in two HIV-infected individuals. Virology 2006; 345:209–219.
54. Draenert R, Verrill CL, Tang Y, Allen TM, Wurcel AG, Boczanowski M, et al
. Persistent recognition of autologous virus by high-avidity CD8 T cells in chronic, progressive human immunodeficiency virus type 1 infection. J Virol 2004; 78:630–641.
55. Kostense S, Ogg GS, Manting EH, Gillespie G, Joling J, Vandenberghe K, et al
. High viral burden in the presence of major HIV-specific CD8(+) T cell expansions: evidence for impaired CTL effector function. Eur J Immunol 2001; 31:677–686.
56. Crawford H, Prado JG, Leslie A, Hue S, Honeyborne I, Reddy S, et al
. Compensatory mutation partially restores fitness and delays reversion of escape mutation within the immunodominant HLA-B*5703-restricted Gag epitope in chronic human immunodeficiency virus type 1 infection. J Virol 2007; 81:8346–8351.
57. Ali A, Lubong R, Ng H, Brooks DG, Zack JA, Yang OO. Impacts of epitope expression kinetics and class I downregulation on the antiviral activity of HIV-1-specific CTL. J Virol 2003; 78:561–567.
58. van Baalen CA, Guillon C, van Baalen M, Verschuren EJ, Boers PHM, Osterhaus ADME, et al
. Impact of antigen expression kinetics on the effectiveness of HIV-specific cytotoxic T lymphocytes. Eur J Immunol 2002; 32:2644–2652.
59. Kiepiela P, Ngumbela K, Thobakgale C, Ramduth D, Honeyborne I, Moodley E, et al
. CD8+ T-cell responses to different HIV proteins have discordant associations with viral load. Nat Med 2007; 13:46–53.
60. Masemola A, Mashishi T, Khoury G, Mohube P, Mokgotho P, Vardas E, et al
. Hierarchical targeting of subtype C human immunodeficiency virus type 1 proteins by CD8+ T cells: correlation with viral load. J Virol 2004; 78:3233–3243.
61. Yang OO, Kalams SA, Rosenzweig M, Trocha A, Jones N, Koziel M, et al
. Efficient lysis of human immunodeficiency virus type 1-infected cells by cytotoxic T lymphocytes. J Virol 1996; 70:5799–5806.
62. Yang OO, Kalams SA, Trocha A, Cao H, Luster A, Johnson RP, et al
. Suppression of human immunodeficiency virus type 1 replication by CD8+ cells: evidence for HLA class I-restricted triggering of cytolytic and noncytolytic mechanisms. J Virol 1997; 71:3120–3128.
63. Yang OO, Sarkis PT, Ali A, Harlow JD, Brander C, Kalams SA, et al
. Determinants of HIV-1 mutational escape from cytotoxic T lymphocytes. J Exp Med 2003; 197:1365–1375.
64. Frahm N, Kiepiela P, Adams S, Linde CH, Hewitt HS, Sango K, et al
. Control of human immunodeficiency virus replication by cytotoxic T lymphocytes targeting subdominant epitopes. Nat Immunol 2006; 7:173–178.
65. Callan MF, Fazou C, Yang H, Rostron T, Poon K, Hatton C, et al
. CD8(+) T-cell selection, function, and death in the primary immune response in vivo. J Clin Invest 2000; 106:1251–1261.
66. Hoshino Y, Morishima T, Kimura H, Nishikawa K, Tsurumi T, Kuzushima K. Antigen-driven expansion and contraction of CD8+-activated T cells in primary EBV infection. J Immunol 1999; 163:5735–5740.