Modification of One Epitope-Flanking Amino Acid Allows for the Induction of Friend Retrovirus-Specific CD8⁺T Cells by Adenovirus-Based Immunization

Abstract

While Friend retrovirus-infected mice readily mount a vigorous CD8؉ T cell response to the leader-gag-derived peptide GagL 85-93 , no GagL 85-93 -specific T cells were detectable in mice immunized against Friend virus (FV) with viral vectors or DNA vaccines. By exchanging one epitope-flanking amino acid or using a scaffold protein we were able to demonstrate for the first time the induction of GagL 85-93 -specific CD8 ؉ T cells by genetic vaccination and show their high protective effect against FV challenge infection. The Gag polyprotein p65 of Friend murine leukemia virus (FMuLV) consists of the structural proteins p15, p12, p30, and p10, which are the main components of the viral capsid. Usage of an alternative start codon upstream of the gag open reading frame adds a leader region to the Gag polyprotein, resulting in the glycosylated protein gp85 gag (12). The leader region of gp85 gag contains an immunodominant CD8 ϩ T cell epitope of Friend virus (FV) (9), and in acutely FV-infected mice up to 15% of the activated CD8 ϩ T cells are GagL 85-93 specific (26). Despite the immunodominance of this epitope in natural FV infection, no induction of GagL 85-93 -specific CD8 ϩ T cells could be detected after immunization of mice with adenovirus (Ad)-based vectors encoding leader-gag (3). Similarly, in mice genetically immunized by plasmid DNA encoding leader-gag, no GagL 85-93 -specific CD8 ϩ T cells could be detected before FV challenge (11), and in vaccination studies with vaccinia virus-based vectors encoding different parts of p65 gag or gp85 gag , no difference in protection was seen whether or not the leader region was included in the vaccine (19). Thus, although the FV model has been used extensively for the development of new concepts for vaccination against retroviruses, none of the genetic vaccines utilized in the past were able to induce CD8 ϩ T cell responses against the immunodominant GagL 85-93 epitope of FV. Therefore, the goal of this study was to develop a genetic vaccine capable of inducing GagL 85-93 -specific immunity.Two factors critically influence if a peptide sequence can be presented as an epitope on a major histocompatibility complex (MHC): the ability to bind to a certain MHC allele and the efficiency of proteasomal degradation resulting in that peptide. Interestingly, neither property is predicted for the immunodominant GagL 85-93 epitope in H-2D b mice using the prediction tools Net-MHC (17) and NetChop (20). As the tyrosine flanking the GagL 85-93 epitope C terminally (C ϩ 1) in the native sequence has been described to be unfavorable for proteasomal degradation (16), inefficiency of processing might be an explanation for the lack of CD8 ϩ T cell immunity after genetic immunization. To overcome this problem, we exchanged the Cϩ1 tyrosine with lysine, since for the resulting protein, leader-gag C1K , proteasomal cleavage after Leu 93 is predicted (20).As an alternative approach, we constructed an adenoviral vector encoding a fusion protein of the murine cellular protein thioredoxin (Txn)...

Immunodominance of Adenovirus-Derived CD8 + T Cell Epitopes Interferes with the Induction of Transgene-Specific Immunity in Adenovirus-Based Immunization

“…GagL 85-93 -specific CD8 ؉ T cells are efficiently induced by DNA immunization with a plasmid encoding the native leader-Gag sequence. In previous work, we were surprised to find that the native leader-Gag sequence did not induce any GagL 85-93 -specific CD8 ϩ T cell responses in mice if it was delivered by an Ad-based vector (20); this was an unexpected finding because of the apparent immunodominance of the GagL 85-93 epitope in the native FV infection. We hypothesized that either other FV-derived proteins, such as F-MuLV protease, may be necessary for efficient processing of the GagL 85-93 epitope or the GagL 85-93 -specific response is dominated by Ad-derived T cell epitopes.…”

“…However, once the leader-Gag protein is placed in an environment conditioned by a viral vector, the GagL 85-93 epitope seems to lose its dominance. Our work with Ad-based vectors (20) and with the epitope strings presented here clearly demonstrates an impaired induction of GagL 85-93 -specific CD8 ϩ T cells in the presence of Ad-derived epitopes, and results from earlier studies using vaccinia virus-based vectors to deliver leader-Gag indicate a similar situation (32); while those authors did not analyze the CD8 ϩ T cell response to the GagL 85-93 epitope directly, which was described only some years later (15), their experiments indicated that the matrix protein contained the most protective determinant, not the Gag-leader, suggesting that GagL 85-93 -specific CD8 ϩ T cells were not induced by the vaccinia virus-based vaccine.…”

“…The high number of epitopes in the Ad proteins that we already see in mice and that is an even more important problem for use in humans renders the development of epitope-modified Ad vectors hardly feasible. On the other hand, the approach that we employed in the past, i.e., a modification of the epitope-flanking region (20), to increase the efficiency of proteasomal processing and thereby improve the overall presentation rate is a feasible and widely applicable approach that should be considered in vector-based vaccine development.…”

“…Interestingly, we previously showed that the induction of GagL 85-93 -specific CD8 ϩ T cells by immunization with a leader-Gag-encoding Ad vector required the modification of the immunogen, as the immunization with the native sequence did not result in any detectable induction of GagL 85-93 -specific CD8 ϩ T cells (20). The modification of the epitope-flanking amino acid that was introduced to improve proteasomal degradation led to improved stimulation of GagL 85-93 -specific CD8 ϩ T cells in vitro and to their improved induction in vivo in immunization experiments.…”

Identification and Enhancement of HLA-A2.1-Restricted CTL Epitopes in a New Human Cancer Antigen-POTE

“…Epitope enhancement by sequence modification was designed to improve such vaccines by increasing the immunogenicity of cancer epitopes that are often derived from self antigens [3], [6]–[8], [34], [35]. Despite occasional findings that some modified peptides may not always be recognized by tumor-reactive T cells [36], sequence modifications both within the epitope and in flanking residues have been useful in increasing immunogenicity in many cases, by affecting MHC binding or antigen processing [3], [7], [23], [37], [38]. For these reasons, we have undertaken identification and epitope enhancement of epitopes from the novel tumor antigen POTE presented by the most common human HLA class I molecule, HLA-A2 (HLA-A*0201).…”