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Viral adaptation to immune selection pressure by HLA class I-restricted CTL responses targeting epitopes in HIV frameshift sequences.

Berger CT, Carlson JM, Brumme CJ, Hartman KL, Brumme ZL, Henry LM, Rosato PC, Piechocka-Trocha A, Brockman MA, Harrigan PR, Heckerman D, Kaufmann DE, Brander C - J. Exp. Med. (2010)

Bottom Line: Using host genetic (human leukocyte antigen [HLA]) and plasma viral sequence information from 765 HIV-infected subjects, we identified 64 statistically significant (q<0.2) associations between specific HLA alleles and sequence polymorphisms in alternate reading frames of gag, pol, and nef that did not affect the regular frame protein sequence.Peptides spanning the top 20 HLA-associated imprints were used to test for ex vivo immune responses in 85 HIV-infected subjects and showed responses to 10 of these ARF peptides.The most frequent response recognized an HLA-A*03-restricted +2 frame-encoded epitope containing a unique A*03-associated polymorphism at position 6.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, Boston, MA 02129, USA.

ABSTRACT
CD8+ cytotoxic T lymphocyte (CTL)-mediated immune responses to HIV contribute to viral control in vivo. Epitopes encoded by alternative reading frame (ARF) peptides may be targeted by CTLs as well, but their frequency and in vivo relevance are unknown. Using host genetic (human leukocyte antigen [HLA]) and plasma viral sequence information from 765 HIV-infected subjects, we identified 64 statistically significant (q<0.2) associations between specific HLA alleles and sequence polymorphisms in alternate reading frames of gag, pol, and nef that did not affect the regular frame protein sequence. Peptides spanning the top 20 HLA-associated imprints were used to test for ex vivo immune responses in 85 HIV-infected subjects and showed responses to 10 of these ARF peptides. The most frequent response recognized an HLA-A*03-restricted +2 frame-encoded epitope containing a unique A*03-associated polymorphism at position 6. Epitope-specific CTLs efficiently inhibited viral replication in vitro when viruses containing the wild-type sequence but not the observed polymorphism were tested. Mutating alternative internal start codons abrogated the CTL-mediated inhibition of viral replication. These data indicate that responses to ARF-encoded HIV epitopes are induced during natural infection, can contribute to viral control in vivo, and drive viral evolution on a population level.

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Magnitude and breadth of CTL responses to frameshift-derived peptides. (A–C) 85 HIV-infected individuals and 32 HIV-negative controls were tested for CTL responses in ex vivo IFN-γ ELISPOT using fresh PBMCs. 21 out of 85 patients displayed a CTL response to at least one of the peptides tested. Peptides were exclusively recognized in HIV-infected individuals. (A) The number of peptides targeted by each individual tested is shown (A). Magnitude of responses in the IFN-γ ELISPOT is expressed as SFCs/106 PBMCs. The horizontal bar represents the median magnitude (B). The frequency and sequence of the responses to individual peptides are shown. All peptides tested and the respective number of patients mounting a CTL response are indicated. The 17mer GQQRSTLERTSKASLER (GR17) was recognized by 10 out of the 85 individuals tested (C).
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fig1: Magnitude and breadth of CTL responses to frameshift-derived peptides. (A–C) 85 HIV-infected individuals and 32 HIV-negative controls were tested for CTL responses in ex vivo IFN-γ ELISPOT using fresh PBMCs. 21 out of 85 patients displayed a CTL response to at least one of the peptides tested. Peptides were exclusively recognized in HIV-infected individuals. (A) The number of peptides targeted by each individual tested is shown (A). Magnitude of responses in the IFN-γ ELISPOT is expressed as SFCs/106 PBMCs. The horizontal bar represents the median magnitude (B). The frequency and sequence of the responses to individual peptides are shown. All peptides tested and the respective number of patients mounting a CTL response are indicated. The 17mer GQQRSTLERTSKASLER (GR17) was recognized by 10 out of the 85 individuals tested (C).

Mentions: To investigate if the observed HLA-associated polymorphisms could in fact be the result of viral adaptation to CTL-mediated immune pressure on alternative-frame HIV peptides, in vitro T cell responses to ARF peptides spanning the region of the associations were assessed. Peptides were synthesized to either contain the identified imprint in the center of two overlapping peptide (OLP) sequences (between 13 and 20 amino acids in length, and overlapping by 9–10 amino acids) or as short (9–12 amino acids long) predicted CTL epitopes using a recently developed epitope prediction algorithm (Heckerman et al., 2007), and were then tested by ex vivo IFN-γ ELISPOT assay using PBMCs from untreated, chronically HIV-infected individuals. Peptides included 9 sequences from the +1 ORF and 12 from the +2 ORF, covering 10, 8, and 2 regions of imprints in gag, pol, and nef, respectively (Table II). Responses were observed in 21 out of 85 HIV-infected individuals and 0 out of 32 HIV-negative individuals tested (P = 0.001 using Fisher’s exact test), indicating that responses are HIV specific (Fig. 1 A). Of note, there was no statistical difference in HLA class I allele representation between the HIV-infected and -uninfected individuals, indicating that the HIV-negative group was a suitable control population (Tables S2 and S3).


Viral adaptation to immune selection pressure by HLA class I-restricted CTL responses targeting epitopes in HIV frameshift sequences.

Berger CT, Carlson JM, Brumme CJ, Hartman KL, Brumme ZL, Henry LM, Rosato PC, Piechocka-Trocha A, Brockman MA, Harrigan PR, Heckerman D, Kaufmann DE, Brander C - J. Exp. Med. (2010)

Magnitude and breadth of CTL responses to frameshift-derived peptides. (A–C) 85 HIV-infected individuals and 32 HIV-negative controls were tested for CTL responses in ex vivo IFN-γ ELISPOT using fresh PBMCs. 21 out of 85 patients displayed a CTL response to at least one of the peptides tested. Peptides were exclusively recognized in HIV-infected individuals. (A) The number of peptides targeted by each individual tested is shown (A). Magnitude of responses in the IFN-γ ELISPOT is expressed as SFCs/106 PBMCs. The horizontal bar represents the median magnitude (B). The frequency and sequence of the responses to individual peptides are shown. All peptides tested and the respective number of patients mounting a CTL response are indicated. The 17mer GQQRSTLERTSKASLER (GR17) was recognized by 10 out of the 85 individuals tested (C).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2812535&req=5

fig1: Magnitude and breadth of CTL responses to frameshift-derived peptides. (A–C) 85 HIV-infected individuals and 32 HIV-negative controls were tested for CTL responses in ex vivo IFN-γ ELISPOT using fresh PBMCs. 21 out of 85 patients displayed a CTL response to at least one of the peptides tested. Peptides were exclusively recognized in HIV-infected individuals. (A) The number of peptides targeted by each individual tested is shown (A). Magnitude of responses in the IFN-γ ELISPOT is expressed as SFCs/106 PBMCs. The horizontal bar represents the median magnitude (B). The frequency and sequence of the responses to individual peptides are shown. All peptides tested and the respective number of patients mounting a CTL response are indicated. The 17mer GQQRSTLERTSKASLER (GR17) was recognized by 10 out of the 85 individuals tested (C).
Mentions: To investigate if the observed HLA-associated polymorphisms could in fact be the result of viral adaptation to CTL-mediated immune pressure on alternative-frame HIV peptides, in vitro T cell responses to ARF peptides spanning the region of the associations were assessed. Peptides were synthesized to either contain the identified imprint in the center of two overlapping peptide (OLP) sequences (between 13 and 20 amino acids in length, and overlapping by 9–10 amino acids) or as short (9–12 amino acids long) predicted CTL epitopes using a recently developed epitope prediction algorithm (Heckerman et al., 2007), and were then tested by ex vivo IFN-γ ELISPOT assay using PBMCs from untreated, chronically HIV-infected individuals. Peptides included 9 sequences from the +1 ORF and 12 from the +2 ORF, covering 10, 8, and 2 regions of imprints in gag, pol, and nef, respectively (Table II). Responses were observed in 21 out of 85 HIV-infected individuals and 0 out of 32 HIV-negative individuals tested (P = 0.001 using Fisher’s exact test), indicating that responses are HIV specific (Fig. 1 A). Of note, there was no statistical difference in HLA class I allele representation between the HIV-infected and -uninfected individuals, indicating that the HIV-negative group was a suitable control population (Tables S2 and S3).

Bottom Line: Using host genetic (human leukocyte antigen [HLA]) and plasma viral sequence information from 765 HIV-infected subjects, we identified 64 statistically significant (q<0.2) associations between specific HLA alleles and sequence polymorphisms in alternate reading frames of gag, pol, and nef that did not affect the regular frame protein sequence.Peptides spanning the top 20 HLA-associated imprints were used to test for ex vivo immune responses in 85 HIV-infected subjects and showed responses to 10 of these ARF peptides.The most frequent response recognized an HLA-A*03-restricted +2 frame-encoded epitope containing a unique A*03-associated polymorphism at position 6.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, Boston, MA 02129, USA.

ABSTRACT
CD8+ cytotoxic T lymphocyte (CTL)-mediated immune responses to HIV contribute to viral control in vivo. Epitopes encoded by alternative reading frame (ARF) peptides may be targeted by CTLs as well, but their frequency and in vivo relevance are unknown. Using host genetic (human leukocyte antigen [HLA]) and plasma viral sequence information from 765 HIV-infected subjects, we identified 64 statistically significant (q<0.2) associations between specific HLA alleles and sequence polymorphisms in alternate reading frames of gag, pol, and nef that did not affect the regular frame protein sequence. Peptides spanning the top 20 HLA-associated imprints were used to test for ex vivo immune responses in 85 HIV-infected subjects and showed responses to 10 of these ARF peptides. The most frequent response recognized an HLA-A*03-restricted +2 frame-encoded epitope containing a unique A*03-associated polymorphism at position 6. Epitope-specific CTLs efficiently inhibited viral replication in vitro when viruses containing the wild-type sequence but not the observed polymorphism were tested. Mutating alternative internal start codons abrogated the CTL-mediated inhibition of viral replication. These data indicate that responses to ARF-encoded HIV epitopes are induced during natural infection, can contribute to viral control in vivo, and drive viral evolution on a population level.

Show MeSH
Related in: MedlinePlus