The translation of an RNA or DNA template into proteins is a complex process that can produce a wide variety of functional and non-functional protein products. Translation involves “reading” the genetic code, and the nature of the protein produced depends on where the reading of the code starts and stops (called the “reading frame”) and also which direction the code is read in (forwards is called “sense” while backwards is called “antisense”). HIV’s RNA genome contains nine reading frames encoding functional proteins, but it is also known that alternate reading frames exist which can be translated into proteins that are non-functional (or have unknown functions). Antisense translation can also lead to the production of such proteins. The extent to which these proteins are targeted by the immune system in HIV-infected people has not been well characterized.
Two new papers in the Journal of Experimental Medicine address this knowledge gap by evaluating CD8 T cell response to “cryptic” epitopes derived from HIV proteins produced from both alternate reading frames and antisense transcription. The reported results indicate that targeting of cryptic HIV epitopes by CD8 T cells is surprisingly common. Both papers also present evidence that CD8 T cell responses against cryptic epitopes can result in mutations in the RNA sequences encoding these epitopes that abrogate CD8 T cell recognition (immune escape).
The implication of these findings is that there is an unexplored plethora of potential targets for HIV-specific T cell responses that could conceivably be exploited to improve vaccine design. Anju Bansal and colleagues note that the process of codon optimization, which is commonly used to try and improve expression of HIV proteins by vaccine candidates, alters the proteins produced from alternate reading frames so that they differ (by as much as 80%) from those generated by natural infection. So counter-intuitively, it is possible that codon optimization – which was used in the Merck Ad5 vaccine – actually reduces the breadth of the resultant HIV-specific CD8 T cell response. Bansal et al also point out that that the ALVAC-HIV vaccine used in the recently reported RV-144 vaccine trial is not codon optimized and could potentially induce responses to cryptic epitopes (CE); they conclude that "it may be important to take into account the contribution of CE-induced responses in the overall CD8 T cell repertoire of future vaccine constructs."
Published online January 11, 2010
The Journal of Experimental Medicine
Brief Definitive Report
Anju Bansal1, Jonathan Carlson4, Jiyu Yan1, Olusimidele T. Akinsiku2, Malinda Schaefer5,6, Steffanie Sabbaj1, Anne Bet2, David N. Levy7, Sonya Heath1, Jianming Tang1,2, Richard A. Kaslow3, Bruce D. Walker8,9, Thumbi Ndung’u8,9, Philip J. Goulder8,9,10, David Heckerman4, Eric Hunter5,6, and Paul A. Goepfert1,2
1 Department of Medicine, 2 Department of Microbiology, and 3 Department of Epidemiology, University of Alabama at Birmingham, Birmingham, AL 35294. 4 Microsoft Research, Redmond, WA 98052. 5 Emory Vaccine Center at Yerkes National Primate Research Center and 6 Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30329. 7 Department of Basic Science, New York University College of Dentistry, New York, NY 10010. 8 Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard, Boston, MA 02129. 9 HIV Pathogenesis Program, Doris Duke Medical Research Institute, University of KwaZulu-Natal, Durban 4013, South Africa. 10 Department of Pediatrics, University of Oxford, Oxford OX1 3SY, England, UK
Retroviruses pack multiple genes into relatively small genomes by encoding several genes in the same genomic region with overlapping reading frames. Both sense and antisense HIV-1 transcripts contain open reading frames for known functional proteins as well as numerous alternative reading frames (ARFs). At least some ARFs have the potential to encode proteins of unknown function, and their antigenic properties can be considered as cryptic epitopes (CEs). To examine the extent of active immune response to virally encoded CEs, we analyzed human leukocyte antigen class I–associated polymorphisms in HIV-1 gag, pol, and nef genes from a large cohort of South Africans with chronic infection. In all, 391 CEs and 168 conventional epitopes were predicted, with the majority (307; 79%) of CEs derived from antisense transcripts. In further evaluation of CD8 T cell responses to a subset of the predicted CEs in patients with primary or chronic infection, both sense- and antisense-encoded CEs were immunogenic at both stages of infection. In addition, CEs often mutated during the first year of infection, which was consistent with immune selection for escape variants. These findings indicate that the HIV-1 genome might encode and deploy a large potential repertoire of unconventional epitopes to enhance vaccine-induced antiviral immunity.
Published online January 11, 2010
The Journal of Experimental Medicine
Christoph T. Berger1, Jonathan M. Carlson2, Chanson J. Brumme1, Kari L. Hartman1, Zabrina L. Brumme1,3,4, Leah M. Henry1, Pamela C. Rosato1, Alicja Piechocka-Trocha1, Mark A. Brockman1,3,4, P. Richard Harrigan3,5, David Heckerman2, Daniel E. Kaufmann1, and Christian Brander1,6,7
1 Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard, Boston, MA 02129. 2 Microsoft Research, Seattle, WA 98033. 3 British Columbia Centre for Excellence in HIV/AIDS, Vancouver, BC V6B 5S8, Canada. 4 Simon Fraser University, Burnaby, BC V5A 4Y7, Canada. 5 Division of AIDS, University of British Columbia, Vancouver, BC V6Z 1Y6, Canada. 6 Institucio Catalana de Recerca i Estudis Avancats, 08010 Barcelona, Spain. 7 Irsicaixa HIV Research Institute–HIVACAT, Hospital Germans Trias i Pujol, Badalona, 08916 Barcelona, Spain
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.