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Viral suppression of multiple escape mutants by de novo CD8+T cell responses in a human immunodeficiency virus-1 Infected elite suppressor
© O'Connell et al; licensee BioMed Central Ltd. 2011
- Received: 3 June 2011
- Accepted: 3 August 2011
- Published: 3 August 2011
Elite suppressors or controllers (ES) are HIV-1 infected patients who maintain undetectable viral loads without treatment. While HLA-B*57-positive ES are usually infected with virus that is unmutated at CTL epitopes, a single, dominant variant containing CTL escape mutations is typically seen in plasma during chronic infection. We describe an ES who developed seven distinct and rare escape variants at an HLA-B*57-restricted Gag epitope over a five year period. Interestingly, he developed proliferative, de novo CTL responses that suppressed replication of each of these variants. These responses, in combination with low viral fitness of each variant, may contribute to sustained elite control in this ES.
- Escape Mutation
- Plasma Variant
- Viral Fitness
- Tetramer Staining
- Plasma Virus
Elite suppressors (ES) are HIV-1 infected individuals who control viremia naturally, maintaining clinically undetectable levels of virus in the plasma (< 50 copies of HIV-1 RNA/ml) without antiretroviral therapy[1–3]. We have previously shown that HIV-1 sequences amplified from resting CD4+T cells of these patients are distinct from those amplified from free virus in the plasma, with CTL escape mutations apparent in the plasma virus but largely absent in proviral DNA [4–6]. We have also shown that the escape mutations present in plasma virus generally do not vary over time. This suggests that virus in ES achieves a balance between fitness and CTL escape which eliminates the development of further nonsynonymous mutations during chronic infection, despite ongoing viral replication [7–9].
The HLA-B*57 and B*5801 alleles are overrepresented in ES and are thought to play a causative role in control of viremia in these patients [1–3]. The TW10 epitope in Gag (amino acids 240-249) is a well-characterized, immunodominant epitope restricted by these alleles. Escape via a T242N mutation frequently occurs early in the majority of HLA-B*57 chronic progressors (CP)  and ES [4, 11–14]. Compensatory mutations upstream of TW10 in the CypA binding site partially or completely compensate for the negative fitness impact of the T242N mutation . Interestingly, some ES develop rare, alternative mutations in TW10 [4, 13], some of which also have dramatic fitness defects .
Here we describe an HLA-B*57-positive ES who displayed unprecedented sequence variation at the TW10 epitope while never acquiring the T242N mutation. Seven different TW10 variants were observed in the plasma sequences from this patient over five years, all of which otherwise shared the same gag backbone. Only two of the seven variants have been previously documented in the LANL database, and together these two variants were only present in 5 of the 1510 Clade B HIV-1 sequences in the database http://www.hiv.lanl.gov/content/sequence/HIV/COMPENDIUM/2010compendium.html. Using IFN-γ ELISPOT analysis, we previously showed that this patient, ES8, was able to mount a CTL response against each of the variants . To understand the presence of this atypical ongoing evolution, we also determined the impact of these mutations on viral replication capacity and multiple aspects of the CTL response.
Autologous plasma variants from ES8 contain rare mutations in TW10 and are significantly less fit than proviral variants or laboratory isolates
As shown previously, the T242N mutation slightly reduced fitness of otherwise wild type NL43nGFP [15, 17]. Viruses expressing the I147M mutation in the IW9 epitope and any of the ES8 plasma mutations in TW10, however, showed considerably less infection of donor cells than the control viruses. Virus containing the T242N mutation in the patient's plasma gag backbone also showed little replication. Interestingly, reversion of the TW10 mutations seen in the plasma variants to a wild type TW10 motif (ES8WT) did not rescue viral fitness and, if anything, decreased viral fitness further. This may suggest that compensatory mutations exist in the plasma variants which partially rescue fitness defects caused by the TW10 mutations, but that these compensatory mutations reduce the fitness of a virus with wild type TW10. Virus with Gag derived from provirus of ES8 (ES8WTProv) had an intermediate level of fitness (Figure 2b). The proviral-derived Gag had no mutations in either the IW9 or TW10 epitopes, suggesting that mutations in the HLA-B*57 restricted epitopes are not the only factors impacting the fitness of virus from ES8.
To elucidate the relevance of the TW10 mutations in virus obtained directly from the patient, we compared the fitness of two viral variants which we cultured from ES8. ES8-1A and ES8-17 are variants cultured from activated CD4+T cells and resting CD4+T cells from the patient, respectively . The ES8-1A variant expresses mutant IW9 and TW10 epitopes, specifically the TSTLAEQVAW variant, and is thus identical to some plasma viral clones. The ES8-17 virus is unmutated at the TW10 and IW9 epitopes. We infected activated CD4+T cells from uninfected donors with equal amounts of each virus via spinoculation  in triplicate, as well as the laboratory strain Ba-L (virus quantified using p24 assay), and found that the ES8-17 variant showed similar replication kinetics to the laboratory strain, whereas ES8-1A had reduced replication kinetics (Figure 2c). Together, these studies examining the fitness of viral variants from this patient show that plasma variants were less fit than the ancestral virus which was retained in the proviral compartment and less fit than wild type NL43. The low fitness of the plasma variants relative to proviral suggests that viral fitness is a consequence rather than a cause of elite control in this patient.
The CD8+T cell response to autologous plasma virus is proliferative and de novo
While IFN-γ provides a measure of whether an antigen is recognized by CTL, IFN-γ is not a good measure of an effective CTL response and does not correlate with control of viral replication [20–22]. The capacity of CD8+T cells to proliferate in response to HIV-1 antigen, however, is associated with control of HIV-1 viremia [23, 24]. CFSE analysis was therefore used to determine whether CTL proliferated in response to the escape mutants. In addition, we looked at CFSE in conjunction with IFN-γ and HLA-B*57 TW10 tetramer staining to elucidate the population of CD8+T cells responding to peptide. We found that the vast majority of IFN-γ producing cells were also CFSElow, suggesting that cells producing cytokines in response to peptides were also proliferating (Figure 3b). Interestingly, we found that cells responding to the patient's autologous peptides did not react to the tetramer, and only wild type TW10 elicited a response from tetramer staining cells (Figure 3c). This implies that the response to the patient's autologous peptides was the result of an entirely de novo response rather than cross-reactivity by T cells specific for wild type TW10 peptides.
CTL from ES8 suppress viral replication
While cross-reactivity between wild type and mutant epitopes has been documented, de novo responses to mutated HIV-1 epitopes are rare. With regard to HLA-B*57-restricted epitopes, one study suggested that de novo CTL responses to autologous epitope variants emerged more frequently in children than in adults . However, the emergence of de novo responses to mutant TW10 variants has been observed by one group in both CP and LTNPs. We  and others  have reported de novo responses to escape mutants in ES, but these studies have looked only at IFN-γ secretion. IFN-γ is not the ideal measure of an effective CTL response and does not correlate with immune protection [20–22]. In contrast, HIV-specific CD8+ T cells that proliferate in response to antigen have been associated with perforin secretion and effective immune control of viral replication . Furthermore the ability of primary CD8+ T cells to inhibit HIV replication has been shown to be a correlate of immunity [26, 27]. We show here that CD8+ T cells from this patient proliferate effectively and inhibit replication of multiple autologous escape variants. This may explain his continued elite control.
In this study we describe an ES whose immune system has selected for seven rare TW10 variants in Gag. The development of these variants was apparently due to the selective pressure placed on the virus by the CTL response to the wild type TW10 epitope, as shown in Figure 3c. These mutants have dramatically reduced viral fitness and are continuing to evolve over time. It is not clear why the T242N mutation never developed in this patient, analysis of TW10-specific T cell receptors in patients who develop rare alternate mutations in this epitope may be revealing. The continued evolution in this patient is striking as the typical ES has one epitope variant present in the plasma which remains constant , but the presence of multiple variants may be partially due to the fact that this patient has maintained higher viral loads (mean of 26 copies/ml) than the other ES in our cohort . ES8 has developed de novo CTL responses to the variants which proliferate in response to antigen and suppress viral replication. Further studies will be needed to determine whether the induction of de novo proliferative suppressive CTL responses is feasible and protective.
Acknowledgements and funding
This work was supported by NIH Grant R01 AI080328 (JNB) and the Howard Hughes Medical Institute (RFS).
- O'Connell KA, Bailey JR, Blankson JN: Elucidating the elite: mechanisms of control in HIV-1 infection. Trends Pharmacol Sci. 2009, 30 (12): 631-637. 10.1016/j.tips.2009.09.005.View ArticlePubMedGoogle Scholar
- Migueles SA, Connors M: Long-term nonprogressive disease among untreated HIV-infected individuals: clinical implications of understanding immune control of HIV. JAMA. 2010, 304 (2): 194-201. 10.1001/jama.2010.925.View ArticlePubMedGoogle Scholar
- Okulicz JF, Lambotte O: Epidemiology and clinical characteristics of elite controllers. Curr Opin HIV AIDS. 2011, 6 (3): 163-168. 10.1097/COH.0b013e328344f35e.View ArticlePubMedGoogle Scholar
- Bailey JR, Williams TM, Siliciano RF, Blankson JN: Maintenance of viral suppression in HIV-1-infected HLA-B*57+ elite suppressors despite CTL escape mutations. J Exp Med. 2006, 203 (5): 1357-1369. 10.1084/jem.20052319.PubMed CentralView ArticlePubMedGoogle Scholar
- Bailey JR, Brennan TP, O'Connell KA, Siliciano RF, Blankson JN: Evidence of CD8+ T-cell-mediated selective pressure on human immunodeficiency virus type 1 nef in HLA-B*57+ elite suppressors. J Virol. 2009, 83 (1): 88-97. 10.1128/JVI.01958-08.PubMed CentralView ArticlePubMedGoogle Scholar
- O'Connell KA, Brennan TP, Bailey JR, Ray SC, Siliciano RF, Blankson JN: Control of HIV-1 in elite suppressors despite ongoing replication and evolution in plasma virus. J Virol. 2010, 84 (14): 7018-7028. 10.1128/JVI.00548-10.PubMed CentralView ArticlePubMedGoogle Scholar
- Mens H, Kearney M, Wiegand A, Shao W, Schonning K, Gerstoft J, Obel N, Maldarelli F, Mellors JW, Benfield T, Coffin JM: HIV-1 continues to replicate and evolve in patients with natural control of HIV infection. J Virol. 2010, 84 (24): 12971-12981. 10.1128/JVI.00387-10.PubMed CentralView ArticlePubMedGoogle Scholar
- O'Connell KA, Xu J, Durbin AP, Apuzzo LG, Imteyaz H, Williams TM, Ray SC, Margolick JB, Siliciano RF, Blankson JN: HIV-1 evolution following transmission to an HLA-B*5801-positive patient. J Infect Dis. 2009, 200 (12): 1820-1824. 10.1086/648377.PubMed CentralView ArticlePubMedGoogle Scholar
- Salgado M, Brennan TP, O'Connell KA, Bailey JR, Ray SC, Siliciano RF, Blankson JN: Evolution of the HIV-1 nef gene in HLA-B*57 positive elite suppressors. Retrovirology. 2010, 7: 94-10.1186/1742-4690-7-94.PubMed CentralView ArticlePubMedGoogle Scholar
- Leslie AJ, Pfafferott KJ, Chetty P, Draenert R, Addo MM, Feeney M, Tang Y, Holmes EC, Allen T, Prado JG, Altfeld M, Brander C, Dixon C, Ramduth D, Jeena P, Thomas SA, St John A, Roach TA, Kupfer B, Luzzi G, Edwards A, Taylor G, Lyall H, Tudor-Williams G, Novelli V, Martinez-Picado J, Kiepiela P, Walker BD, Goulder PJ: HIV evolution: CTL escape mutation and reversion after transmission. Nat Med. 2004, 10 (3): 282-289. 10.1038/nm992.View ArticlePubMedGoogle Scholar
- Migueles SA, Laborico AC, Imamichi H, Shupert WL, Royce C, McLaughlin M, Ehler L, Metcalf J, Liu S, Hallahan CW, Connors M: The differential ability of HLA B*5701+ long-term nonprogressors and progressors to restrict human immunodeficiency virus replication is not caused by loss of recognition of autologous viral gag sequences. J Virol. 2003, 77 (12): 6889-6898. 10.1128/JVI.77.12.6889-6898.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Navis M, Schellens I, van Baarle D, Borghans J, van Swieten P, Miedema F, Kootstra N, Schuitemaker H: Viral replication capacity as a correlate of HLA B57/B5801-associated nonprogressive HIV-1 infection. J Immunol. 2007, 179 (5): 3133-3143.View ArticlePubMedGoogle Scholar
- Miura T, Brockman MA, Schneidewind A, Lobritz M, Pereyra F, Rathod A, Block BL, Brumme ZL, Brumme CJ, Baker B, Rothchild AC, Li B, Trocha A, Cutrell E, Frahm N, Brander C, Toth I, Arts EJ, Allen TM, Walker BD: HLA-B57/B*5801 human immunodeficiency virus type 1 elite controllers select for rare gag variants associated with reduced viral replication capacity and strong cytotoxic T-lymphocyte [corrected] recognition. J Virol. 2009, 83 (6): 2743-2755. 10.1128/JVI.02265-08.PubMed CentralView ArticlePubMedGoogle Scholar
- Miura T, Brumme CJ, Brockman MA, Brumme ZL, Pereyra F, Block BL, Trocha A, John M, Mallal S, Harrigan PR, Walker BD: HLA-associated viral mutations are common in human immunodeficiency virus type 1 elite controllers. J Virol. 2009, 83 (7): 3407-3412. 10.1128/JVI.02459-08.PubMed CentralView ArticlePubMedGoogle Scholar
- Brockman MA, Schneidewind A, Lahaie M, Schmidt A, Miura T, Desouza I, Ryvkin F, Derdeyn CA, Allen S, Hunter E, Mulenga J, Goepfert PA, Walker BD, Allen TM: Escape and compensation from early HLA-B57-mediated cytotoxic T-lymphocyte pressure on human immunodeficiency virus type 1 Gag alter capsid interactions with cyclophilin A. J Virol. 2007, 81 (22): 12608-12618. 10.1128/JVI.01369-07.PubMed CentralView ArticlePubMedGoogle Scholar
- O'Connell KA, Pelz RK, Dinoso JB, Dunlop E, Paik-Tesch J, Williams TM, Blankson JN: Prolonged control of an HIV type 1 escape variant following treatment interruption in an HLA-B*27-positive patient. AIDS Res Hum Retroviruses. 2010, 26 (12): 1307-1311. 10.1089/aid.2010.0135.PubMed CentralView ArticlePubMedGoogle Scholar
- Martinez-Picado J, Prado JG, Fry EE, Pfafferott K, Leslie A, Chetty S, Thobakgale C, Honeyborne I, Crawford H, Matthews P, Pillay T, Rousseau C, Mullins JI, Brander C, Walker BD, Stuart DI, Kiepiela P, Goulder P: Fitness cost of escape mutations in p24 Gag in association with control of human immunodeficiency virus type 1. J Virol. 2006, 80 (7): 3617-3623. 10.1128/JVI.80.7.3617-3623.2006.PubMed CentralView ArticlePubMedGoogle Scholar
- Blankson JN, Bailey JR, Thayil S, Yang HC, Lassen K, Lai J, Gandhi SK, Siliciano JD, Williams TM, Siliciano RF: Isolation and characterization of replication-competent human immunodeficiency virus type 1 from a subset of elite suppressors. J Virol. 2007, 81 (5): 2508-2518. 10.1128/JVI.02165-06.PubMed CentralView ArticlePubMedGoogle Scholar
- O'Doherty U, Swiggard WJ, Malim MH: Human immunodeficiency virus type 1 spinoculation enhances infection through virus binding. J Virol. 2000, 74 (21): 10074-10080. 10.1128/JVI.74.21.10074-10080.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Addo MM, Yu XG, Rathod A, Cohen D, Eldridge RL, Strick D, Johnston MN, Corcoran C, Wurcel AG, Fitzpatrick CA, Feeney ME, Rodriguez WR, Basgoz N, Draenert R, Stone DR, Brander C, Goulder PJ, Rosenberg ES, Altfeld M, Walker BD: Comprehensive epitope analysis of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses, but no correlation to viral load. J Virol. 2003, 77 (3): 2081-2092. 10.1128/JVI.77.3.2081-2092.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Betts MR, Ambrozak DR, Douek DC, Bonhoeffer S, Brenchley JM, Casazza JP, Koup RA, Picker LJ: Analysis of total human immunodeficiency virus (HIV)-specific CD4(+) and CD8(+) T-cell responses: relationship to viral load in untreated HIV infection. J Virol. 2001, 75 (24): 11983-11991. 10.1128/JVI.75.24.11983-11991.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Betts MR, Nason MC, West SM, De Rosa SC, Migueles SA, Abraham J, Lederman MM, Benito JM, Goepfert PA, Connors M, Roederer M, Koup RA: HIV nonprogressors preferentially maintain highly functional HIV-specific CD8+ T cells. Blood. 2006, 107 (12): 4781-4789. 10.1182/blood-2005-12-4818.PubMed CentralView ArticlePubMedGoogle Scholar
- Zimmerli SC, Harari A, Cellerai C, Vallelian F, Bart PA, Pantaleo G: HIV-1-specific IFN-gamma/IL-2-secreting CD8 T cells support CD4-independent proliferation of HIV-1-specific CD8 T cells. Proc Natl Acad Sci USA. 2005, 102 (20): 7239-7244. 10.1073/pnas.0502393102.PubMed CentralView ArticlePubMedGoogle Scholar
- Migueles SA, Laborico AC, Shupert WL, Sabbaghian MS, Rabin R, Hallahan CW, Van Baarle D, Kostense S, Miedema F, McLaughlin M, Ehler L, Metcalf J, Liu S, Connors M: HIV-specific CD8+ T cell proliferation is coupled to perforin expression and is maintained in nonprogressors. Nat Immunol. 2002, 3 (11): 1061-1068. 10.1038/ni845.View ArticlePubMedGoogle Scholar
- Feeney ME, Tang Y, Pfafferott K, Roosevelt KA, Draenert R, Trocha A, Yu XG, Verrill C, Allen T, Moore C, Mallal S, Burchett S, McIntosh K, Pelton SI, St John MA, Hazra R, Klenerman P, Altfeld M, Walker BD, Goulder PJ: HIV-1 viral escape in infancy followed by emergence of a variant-specific CTL response. J Immunol. 2005, 174 (12): 7524-7530.View ArticlePubMedGoogle Scholar
- Saez-Cirion A, Lacabaratz C, Lambotte O, Versmisse P, Urrutia A, Boufassa F, Barre-Sinoussi F, Delfraissy JF, Sinet M, Pancino G, Venet A, Agence Nationale de Recherches sur le Sida EP36 HIV Controllers Study Group: HIV controllers exhibit potent CD8 T cell capacity to suppress HIV infection ex vivo and peculiar cytotoxic T lymphocyte activation phenotype. Proc Natl Acad Sci USA. 2007, 104 (16): 6776-6781. 10.1073/pnas.0611244104.PubMed CentralView ArticlePubMedGoogle Scholar
- Saez-Cirion A, Sinet M, Shin SY, Urrutia A, Versmisse P, Lacabaratz C, Boufassa F, Avettand-Fenoel V, Rouzioux C, Delfraissy JF, Barre-Sinoussi F, Lambotte O, Venet A, Pancino G, ANRS EP36 HIV Controllers Study Group: Heterogeneity in HIV suppression by CD8 T cells from HIV controllers: association with Gag-specific CD8 T cell responses. J Immunol. 2009, 182 (12): 7828-7837. 10.4049/jimmunol.0803928.View ArticlePubMedGoogle Scholar
- Dinoso JB, Kim SY, Siliciano RF, Blankson JN: A comparison of viral loads between HIV-1-infected elite suppressors and individuals who receive suppressive highly active antiretroviral therapy. Clin Infect Dis. 2008, 47 (1): 102-104. 10.1086/588791.PubMed CentralView ArticlePubMedGoogle Scholar
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