The spread of HIV-1 is one of the most challenging problems by which the global health community is currently faced. Despite the continuing improvement in antiretroviral therapy, a safe and effective vaccine remains the best hope of controlling the HIV-1 epidemic. The rarity of broadly neutralizing antibodies1,2 combined with several lines of evidence implicating CD8+ T cells in the control of HIV-1 replication3–14 has led much vaccine research to be focused on generating virus-specific cytotoxic T lymphocytes (CTL).15 However, determining which CTL epitopes represent good targets for an HIV-1 vaccine is a complex undertaking.
The error-prone nature of HIV-1 reverse transcription16,17 combined with its high replicative ability has resulted in the virus possessing a vast amount of genetic diversity. The most common subgroup of HIV-1, group M, consists of several clades, subclades, and circulating recombinant forms, which can possess sequence variation of up to 30%. In addition, viruses from within the same clade can also show substantial sequence differences, sometimes up to 15%.18 This confronts those attempting to design a vaccine that will be effective against a wide range of HIV-1 subtypes and within several infected populations with a difficult task.
Several studies advocate the use of conserved regions of HIV-1 as vaccine targets, and many utilize exogenously loaded peptide to test such theories.19–21 However, sequence conservation of the epitope itself and recognition of externally administered optimal peptide epitopes does not ensure that a response will be generated toward a given antigen in the context of natural infection with potentially diverse viral isolates. It has been demonstrated that sequence variation in the flanking regions of T-cell epitopes can abrogate their processing and presentation.22–24 Work from our group has shown that the flanking sequence of a conserved HLA-B*08 restricted nef epitope FL8 affects the processing and presentation of the peptide and that certain amino acids upstream of the epitope are important in determining if the epitope is presented at the cell surfaces.25
In this article, we further develop these findings by investigating the processing and presentation of an HLA-B*40-restricted nef epitope KL9 that overlaps with the HLA-B*08-restricted epitope studied by Ranasinghe et al.25 Recombinant vaccinia viruses (rVV) expressing nef from several viral isolates were used to infect an HLA-B*40+ B cell line that was then assayed for the presence of KL9 at the cell surface. The results showed that the pattern of antigen processing can differ substantially between epitopes that overlap by several amino acids. A shift of just 2 amino acids can lead to both a reduction and an increase in the efficiency of processing of a peptide epitope compared with the epitope beginning 2 amino acids previously. These results emphasize the complexity of antigen processing and presentation and represent further evidence that relying on exogenously loaded peptide to evaluate vaccine targets may substantially overestimate recognition of antigenic epitopes.
MATERIALS AND METHODS
B Cell Line and CTL Clone Generation
To produce transformed B-cell lines, peripheral blood mononuclear cells (PBMCs) were incubated in Epstein–Barr virus culture supernatant for 3 hours. One hundred microliters of R15 [RPMI supplemented with 15% fetal calf serum (Sigma, Gillingham, United Kingdom), 2 mM L-glutamine, and 50 U/mL penicillin/streptomycin] containing 2 μg/mL cyclosporin A (Sandimmun, Novartis, Frimley, United Kingdom) was then added, and the cells were incubated for 2 weeks. The media were exchanged when necessary. After 2 weeks, the cyclosporin A was omitted.
To generate CD8+ T-cell clones, PBMCs from an HLA-B*40+ HIV+ patient were stained with major histocompatibility complex (MHC) class I tetramer and anti-CD8-allophycocyanin (APC; BD Biosciences, San Jose, CA) and then resuspended in 500 μL of H10 [RPMI supplemented with 10% human AB serum (NBS, Bristol, United Kingdom), 2 mM L-glutamine, and 50 U/mL penicillin/streptomycin] for fluorescence activated cell sorting. Sorted tetramer+ cells were expanded using irradiated allogeneic PBMCs containing 50 μg/mL phytohemagutinin. Cells were cloned using limited dilution. On day 3 post restimulation, interleukin 2 (final concentration: 200 U/mL) was added to the culture media. CD8+ T-cell clones were also generated from 2 HIV-1+ patients attending an outpatient clinic in Oxford, one of whom was HLA-A*24+, whereas the other expressed HLA-B*08. Ethical approval was obtained from Oxford Tropical Ethics Committee, and all patients gave informed consent.
Infection of B Cells With rVV
The rVVs expressing HIV-1 Nef protein (rVV-Nef) from 7 group M subtypes of HIV-1 were acquired from the NIH AIDS Research and Reference Reagent Program: HIV-192UG037.1(A), HIV-1MN (B), HIV-196ZM651 (C), HIV-194UG114.1 (D), HIV-193BR020 (F), HIV-192NG83.2 (G), and HIV-190CF056 (H). Two plaque-forming units per target cell of rVV were added to B-cell lines previously washed in RPMI. The cells were then incubated for 1 hour. One milliliter of R10 was added before incubating for further 2 hours. The cells were then washed and resuspended at the required concentration.
Interferon γ ELISpot Assays
Standard human interferon γ (IFN-γ) enzyme-linked immunospot assays were performed as described elsewhere.26 In brief, CD8+ T-cell clones were added to precoated IFN-γ enzyme-linked immunospot assay plates with either peptide pulsed or rVV-infected target cells at an E:T ratio of 1:50, in duplicate, in a final volume of 100 microliters per well. Assays were incubated for 18 hours at 37°C, 5% CO2, and developed as normal.
CD8+ T-cell clones at a concentration of 5 × 105/mL were incubated with 2 microliters per well of anti-CD107a-phycoerythrin (PE; BD Biosciences) at 4°C for 30 minutes. Peptide-pulsed or rVV-infected B-cell targets (106/mL) were then added followed by incubation for 1 hour. Brefeldin A at a concentration of 10 μg/mL was added before 3-hour incubation. Cells were stained with anti-CD8 APC (BD Biosciences), washed twice, and fixed before being read on a CyAn adenosine diphosphate flow cytometer. Analysis was performed using FlowJo software (Tree Star Inc).
Two Overlapping HIV-1 CTL Epitopes Rely on Different Amino Acids in the Flanking Region for Efficient Processing
An HLA-B*40-restricted CD8+ T-cell clone specific for a nef epitope (KL9), which overlaps with the HLA-B*08 restricted FL8 epitope by 6 amino acids (Table 1), was used to determine whether this epitope is processed and presented when an HLA-B*40+ B cell line was infected with several rVV containing HIV-1 nef proteins of varying sequences (Table 1). The infected cells were tested for recognition by the KL9-specific clone in an IFN-γ ELISpot assay (Fig. 1A). In addition, the presence of the KL9 epitope on the surface of the B cell line was determined by measuring the percentage of clones expressing the degranulation marker CD107a after incubation with the infected B cells (Fig. 1B). Peptides matching the sequences of the KL9 epitope contained with the rVV were used to pulse the HLA-B*40+ B cells (WT = KEKGGLEGL, within clade C, D, and F rVV; 7D = KEKGGLDGL, within clade A, B, G, and H rVV) to determine recognition of the epitope sequence by the KL9-specific clone.
The rVVs that were not recognized by the KL9-specific clone were assayed for infectivity by determining the presence of either the FL8 epitope or an HLA-A*24-restricted nef epitope (RW8) on the surface of infected B cells (either HLA-B*40+/HLA-A*24+ or HLA-B*08+) using specific clones (Fig. 1C). This demonstrates that the lack of the KL9 epitope on the B-cell surface after infection with rVV A, B, and H does not stem from a lack of rVV infectivity and results from a decrease in processing efficiency. The results of these experiments, summarized in Table 1, show that depending on the nef sequence inserted into the rVV one, both or neither of the KL9 and FL8 epitopes were processed and presented.
The KL9-Specific CTL Clone Recognizes Both the Wild-Type and the 7D Variant Peptide With the Same Functional Avidity
The nef sequences contained within the 3 rVV that were not recognized by the KL9-specific clone (A, B, and H) all possess a glutamic acid to an aspartic acid substitution at position 7 within the KL9 epitope (Table 1). The KL9-specific clone is able to recognize this 7D variant epitope as illustrated by the generation of effector functions after incubation with variant peptide-pulsed B cells and target cells infected with clade G rVV (1A, 1B and Table 1).
It is however possible that the KL9-specific clone recognizes this mutant epitope with a reduced functional avidity compared with the wild-type (WT) peptide, requiring a greater amount of peptide and in turn more efficient antigen processing to induce a response. The functional avidity of the KL9-specific clone for the WT and the 7D mutant peptide was determined using B cells pulsed with various concentrations of both peptides in an IFN-γ ELISpot assay. The peptide concentration that elicited the half-maximal IFN-γ release from the clone was the same for both peptides (Fig. 1D).
Many studies aimed at characterizing HIV-1–specific CD8+ T cells make use of exogenously loaded peptide antigen. However, such assays do not take into account the multiple steps that must occur to generate a peptide–MHC complex at the cell surface from a cytosolic viral protein. Numerous variables impact on the efficacy of this process including the viral sequence of the epitope and its flanking regions and features of the antigen-presenting cell such as polymorphisms in the proteins involved in antigen processing.
These results demonstrate that infection with 3 of 7 rVV-containing HIV-1 nef sequences did not result in the processing and presentation of an HLA-B*40-restricted epitope. It is worth noting that infection with one particular rVV, containing a clade C nef sequence, gave a positive response from the KL9-specific clone in an IFN-γ ELISpot assay but not a CD107a assay. It is unclear why this might be but these 2 assays measure different effector functions, cytokine secretion, and degranulation, which may require different levels of peptide stimulation. The discrepancies shown highlight the importance of using more than 1 readout to determine the efficient processing and presentation of the KL9 epitope.
This study demonstrates that the KL9 epitope relies on different amino acids for effective processing compared with an HLA-B*08-restricted epitope with which it overlaps by 6 amino acids. The processing of the HLA-B*08-restricted epitope FL8 is heavily influenced by the amino acid directly preceding its N-terminus. Efficient processing does not occur when a phenylalanine is present at this position. As the HLA-B*40-restricted KL9 epitope is situated 2 amino acids further along the nef sequence and has a conserved leucine directly preceding its N-terminus, it could be hypothesized that more efficient processing of the epitope occurs. However, as outlined in Table 1, a conserved leucine instead of a phenylalanine at position −1 does not ensure processing and presentation of the KL9 epitope.
It is perhaps unsurprising that the efficient processing and presentation of an epitope cannot be ensured by the presence of one specific amino acid. Only the generation of particular peptide fragments will result in the presentation of a given antigen. The correct C-terminus of the antigen must be produced before transporter-associated-with-antigen-processing transport occurs as only N-terminal peptidases are present in the endoplasmic reticulum.26–28 Therefore, subtle changes in peptide sequence that lead to even minor alterations in the fragments produced by the proteasome and other cytosolic proteases could result in the abrogation of processing of a given epitope.
Although the presence of a phenylalanine directly preceding the FL8 epitope was highlighted as reducing the efficiency of the processing of the epitope, experiments using laboratory strain HIV-1 isolates revealed that even when a histidine is present at this position effective processing of the antigen is not guaranteed.25 Similarly, comparing the peptide sequences of the nef genes within the rVV whereby infection results in presentation of the KL9 epitope with those that were not recognized by the KL9-specific clone reveal that no one particular amino acid can be used to segregate the 2 groups.
The experiments outlined in Figure 1 were carried out using different target cells to those used by Ranasinghe et al.25 Therefore, it is possible that factors other than sequence variation of nef within the rVV could account for the differential pattern of processing displayed by the FL8 and KL9 epitopes. Polymorphisms exist in several of the proteins involved in antigen processing and presentation.29 Differences between the 2 target cell lines are known to be present at the MHC class I locus and could potentially be present within other molecules such as the proteasome, TAP, or chaperone molecules such as tapasin. In addition, different MHC class I molecules vary in their interactions with such molecules. For example, certain HLA molecules are more dependent on tapasin for efficient peptide loading than others.29
The complexity of the antigen-processing pathway makes it difficult to draw definitive conclusions about the mechanisms responsible for the differences in epitope recognition observed. It is however apparent that even subtle changes affecting either viral or host cell factors can have a substantial impact on the process of antigen generation. Furthermore, the effects of flanking region mutations on CD8+ T-cell epitope generation do not simply manifest during HIV-1 infection but have been found to influence responses to other viral infections30 and tumors,31 suggesting that this is a common phenomenon with wide-ranging implications.
Antigen processing and presentation is a complex process that is susceptible to alteration by many factors. The effective generation of a particular epitope does not ensure the efficient production of another epitope with which it substantially overlaps. Thus, the exact position of any amino acid substitutions involved in modulating antigen processing is crucial. Sensitivity to flanking region mutations could be a common trait of many HIV-1 and indeed other viral epitopes, allowing such pathogens an increased opportunity for immune escape. This makes the selection of potential HIV-1 vaccine targets a difficult task.
1. Alter G, Moody MA. The humoral response to HIV-1: new insights, renewed focus. J Infect Dis. 2010;202(suppl 2):S315–S322.
2. Gray ES, Madiga MC, Moore PL, et al.. Broad neutralization of human immunodeficiency virus type 1 mediated by plasma antibodies against the gp41 membrane proximal external region. J Virol. 2009;83:11265–11274.
3. Pereyra F, Jia X, McLaren PJ, et al.. The major genetic determinants of HIV-1 control affect HLA class I peptide presentation. Science. 2010;330:1551–1557.
4. Fellay J, Shianna KV, Ge D, et al.. A whole-genome association study of major determinants for host control of HIV-1. Science. 2007;317:944–947.
5. Moore CB, John M, James IR, et al.. Evidence of HIV-1 adaptation to HLA-restricted immune responses at a population level. Science. 2002;296:1439–1443.
6. Gao X, Nelson GW, Karacki P, et al.. Effect of a single amino acid change in MHC class I molecules on the rate of progression to AIDS. N Engl J Med. 2001;344:1668–1675.
7. Schmitz JE, Kuroda MJ, Santra S, et al.. Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes. Science. 1999;283:857–860.
8. Jin X, Bauer DE, Tuttleton SE, et al.. Dramatic rise in plasma viremia after CD8(+) T cell depletion in simian immunodeficiency virus-infected macaques. J Exp Med. 1999;189:991–998.
9. Carrington M, Nelson GW, Martin MP, et al.. HLA and HIV-1: heterozygote advantage and B*35-Cw*04 disadvantage. Science. 1999;283:1748–1752.
10. Price DA, Goulder PJ, Klenerman P, et al.. Positive selection of HIV-1 cytotoxic T lymphocyte escape variants during primary infection. Proc Natl Acad Sci U S A. 1997;94:1890–1895.
11. Haynes BF, Pantaleo G, Fauci AS. Toward an understanding of the correlates of protective immunity to HIV infection. Science. 1996;271:324–328.
12. Koup RA, Safrit JT, Cao Y, et al.. Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome. J Virol. 1994;68:4650–4655.
13. Borrow P, Lewicki H, Hahn BH, et al.. Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection. J Virol. 1994;68:6103–6110.
14. Phillips RE, Rowland-Jones S, Nixon DF, et al.. Human immunodeficiency virus genetic variation that can escape cytotoxic T cell recognition. Nature. 1991;354:453–459.
15. McMichael AJ. HIV vaccines. Annu Rev Immunol. 2006;24:227–255.
16. Roberts JD, Bebenek K, Kunkel TA. The accuracy of reverse transcriptase from HIV-1. Science. 1988;242:1171–1173.
17. Boyer JC, Bebenek K, Kunkel TA. Unequal human immunodeficiency virus type 1 reverse transcriptase error rates with RNA and DNA templates. Proc Natl Acad Sci U S A. 1992;89:6919–6923.
18. Korber B, Gaschen B, Yusim K, et al.. Evolutionary and immunological implications of contemporary HIV-1 variation. Br Med Bull. 2001;58:19–42.
19. Letourneau S, Im EJ, Mashishi T, et al.. Design and pre-clinical evaluation of a universal HIV-1 vaccine. PLoS One. 2007;2:e984.
20. Frahm N, Nickle DC, Linde CH, et al.. Increased detection of HIV-specific T cell responses by combination of central sequences with comparable immunogenicity. AIDS. 2008;22:447–456.
21. Santra S, Korber BT, Muldoon M, et al.. A centralized gene-based HIV-1 vaccine elicits broad cross-clade cellular immune responses in rhesus monkeys. Proc Natl Acad Sci U S A. 2008;105:10489–10494.
22. Draenert R, Le Gall S, Pfafferott KJ, et al.. Immune selection for altered antigen processing leads to cytotoxic T lymphocyte escape in chronic HIV-1 infection. J Exp Med. 2004;199:905–915.
23. Milicic A, Price DA, Zimbwa P, et al.. CD8+ T cell epitope-flanking mutations disrupt proteasomal processing of HIV-1 Nef. J Immunol. 2005;175:4618–4626.
24. Zimbwa P, Milicic A, Frater J, et al.. Precise identification of a human immunodeficiency virus type 1 antigen processing mutant. J Virol. 2007;81:2031–2038.
25. Ranasinghe SR, Kramer HB, Wright C, et al.. The antiviral efficacy of HIV-specific CD8 T-cells to a conserved epitope is heavily dependent on the infecting HIV-1 isolate. PLoS Pathog. 2011;7:e1001341.
26. Dong T, Stewart-Jones G, Chen N, et al.. HIV-specific cytotoxic T cells from long-term survivors select a unique T cell receptor. J Exp Med. 2004;200:1547–1557.
27. Cascio P, Hilton C, Kisselev AF, et al.. 26S proteasomes and immunoproteasomes produce mainly N-extended versions of an antigenic peptide. EMBO J. 2001;20:2357–2366.
28. Saric T, Chang SC, Hattori A, et al.. An IFN-gamma-induced aminopeptidase in the ER, ERAP1, trims precursors to MHC class I-presented peptides. Nat Immunol. 2002;3:1169–1176.
29. McCluskey J, Rossjohn J, Purcell AW. TAP genes and immunity. Curr Opin Immunol. 2004;16:651–659.
30. Seifert U, Liermann H, Racanelli V, et al.. Hepatitis C virus mutation affects proteasomal epitope processing. J Clin Invest. 2004;114:250–259.
31. Theobald M, Ruppert T, Kuckelkorn U, et al.. The sequence alteration associated with a mutational hotspot in p53 protects cells from lysis by cytotoxic T lymphocytes specific for a flanking peptide epitope. J Exp Med. 1998;188:1017–1028.
Keywords:© 2012 Lippincott Williams & Wilkins, Inc.
HIV-1; CD8+ T cells; antigen processing