This study describes the development and use of retroviral vectors to evaluate EIAV-specific CTL responses. Vector constructs expressing EIAV Gag/Pr and SU proteins which were efficient transducers of EK cells and expressed easily detectable levels of protein were identified. These vectors were also shown to be efficient targets for EIAV-specific lysis, in contrast to previous studies using a similar retroviral vector system for evaluation of feline immunodeficiency virus capsid-specific responses (46
). SU-specific CTL responses were demonstrated in EIAV-infected horses, extending previous studies demonstrating Env-specific (SU and/or TM) CTL in both acutely and chronically infected horses on vaccinia virus-infected targets (27
). Furthermore, preliminary studies suggested that these vectors could also induce EIAV-specific CTL in vivo.
To determine which proteins are recognized by CTL and to express truncated genes for preliminary epitope mapping, investigators have used viral vectors, primarily vaccinia virus (12
). Retroviral vectors have been used to transduce CTL target cells in a limited number of reports (14
). We found retroviral vectors easier to develop than recombinant vaccinia viruses, as recombination, selection, and cloning were not necessary for establishing high-titer supernatants for CTL target cell transduction. Retroviral vector-transduced target cells have several other advantages, which include failure to down regulate MHC class I molecules (52
), expression of only the protein of interest and the selectable marker neo
, lack of cytotoxicity of transduced target cells, and failure to co-opt synthesis of host cell proteins (2
). The disadvantages to these vectors include the necessity for in vitro cell line selection, though once expanded target cells can be frozen for future assays; the variability of expression, though again this disadvantage can be overcome by bulk target cell culture of target cells with demonstrated expression; and relative instability when utilizing some promoter-protein combinations as described below.
We constructed a total of six retroviral vectors expressing EIAV proteins which varied in the promoter and the order in which the gene of interest and the selectable marker were expressed. Significant differences among the vectors were found in levels of mRNA expression, protein expression, and cytolysis by EIAV-specific CTLm. EK cells transduced by retroviral vectors using the MoMSV LTR to promote expression of inserted genes upstream of the neo
gene had consistently good EIAV protein expression and were consistently good CTL targets. Those vectors utilizing the CMV immediate-early promoter or SV40 early promoter and expressing the gene of interest downstream of the selectable marker neo
did not express detectable levels of EIAV protein in either cloned EK cells or bulk EK cell cultures following selection with G418 sulfate. However, when these preparations were examined for insert-specific mRNA transcript, variable but occasionally detectable levels of mRNA were evident. Variability of expression in retroviral vector-transduced cells has been recognized in previous studies (46
) and attributed to cell-specific promoter activity (41
), DNA integration into regions of the genome not suitable for efficient transcription (9
), or promoter selection (29
). It is not evident which of these effects was responsible for the lack of protein expression by vectors using CMV and SV40 promoters; however, only vectors vLGSN and vLGP90SN, which used the MoMSV LTR to express EIAV proteins, were evaluated further.
vLGSN and vLGP90SN were both produced in the packaging cell line PG13, demonstrating that the GALV Env could be used to internalize retroviral vector virions into equine cell lines. vLGSN encoded the entire gag
and 5′ pol
gene of EIAV. The gag
gene is expressed as a 55-kDa polyprotein and subsequently cleaved into four major internal proteins designated p15, p26, p11, and p9 by the protease encoded within the 5′ pol
). Gag proteins account for approximately 80% of total EIAV virion structural proteins by weight, and significant sequence homology has been identified between the p26 of EIAV isolates and p24 of human immunodeficiency virus type 1 (13
). This sequence homology is reflected in antigenic cross-reactivity and suggests interspecies conservation of lentiviral core proteins (10
). For these reasons, the gag
gene was included in the vLGSN construct, whereas the protease gene was included to more closely mimic antigen presentation in wild-type EIAV-infected cells. In expression analysis, however, easily detectable levels of the Pr55 were noted, but detectable p26 cleavage was not evident in transduced EK cell lysates. This result does not preclude the possibility that undetectable levels of processing occurred, since in previous studies, processed p26 protein was not evident in vaccinia virus (vGag/Pr)-infected EK lysates without concentrating subviral particles (25
). The EIAV env
gene encodes two glycosylated proteins, SU (gp90) and TM (gp45) (1
). The EIAV env
sequence has insignificant identity with related lentiviruses, but significant structural, and therefore putative functional, similarities do exist (3
). As in other lentivirus systems, significant CTL activity and antibody reactivity against the EIAV env
gene products have been demonstrated, but further characterization is necessary (51
). For these reasons, we constructed the retroviral vector vLGP90SN encoding EIAV SU, which expressed a single reactive 90-kDa protein on immunoblot analysis.
All EIAV-infected horses examined had significant CTL activity to Gag proteins, reflecting the possible immunodominance of these proteins. In contrast, the PBMC isolated from these same horses often had significant, but usually lower, SU-specific CTL activity. This may be due to the relative stability of Gag epitopes, allowing for the continued antigenic stimulation of CTLm in comparison to the more variable SU. Results of assays designed to compare the relative efficiencies of EIAV-specific CTL lysis of recombinant vaccinia virus-infected and retroviral vector-transduced EK cell targets were equivocal in that experimental results varied depending on donor horse (Fig. ). Experimental variation in bulk CTL assays or differences in retroviral vector expression in EK cells from MHC class I-dissimilar donor horses may also explain the experimental results. In some assays, EK cell targets infected with EIAV or vGag/Pr were significantly killed by ELA-A-mismatched effectors, while the same EK cell targets transduced with the retroviral vectors were not killed (Fig. C and C). This may be because retroviral vectors do not perturb host cell machinery and do not cause decreased MHC class I molecule expression, thereby avoiding recognition by natural killer cells activated during the in vitro stimulation of PBMC (21
). MHC class I-unrestricted CTL killing has been observed by other investigators and can be quenched by the addition of cold CTL targets (37
The ability of retroviral vectors to stimulate EIAV-specific CTL in vivo was also examined. There is a paucity of information regarding the use of retroviral vectors in vivo for induction of antiviral immune responses, as only four studies have been reported (14
). A preliminary study utilizing five EIAV-negative horses was undertaken to determine if intramuscular inoculation with a mixture of vLGSN and vLGP90SN could induce CTL. Of five horses inoculated, one, H555, developed EIAV-specific CTL. These results are in contrast to results obtained in murine and nonhuman primate models (14
) but similar to results of a study in humans (52
). Though retroviral vectors have multiple advantages with regard to antigen presentation, hurdles exist for efficient in vivo transduction and immune stimulation. Stable transduction is most efficient when cells enter S phase immediately following vector uptake, allowing for integration into the host cell chromosome (32
). Induction and timing of S phase are difficult in myocytes that normally reside in G0
but were attempted in this study through the use an acidic (pH 4.2) local anesthetic, Bupivicaine. It is possible that lack of detectable CTL induction in four of five animals was due to improper timing of pretreatment of injection sites, resulting in poor transduction efficiency. Another explanation may be that since retroviral vectors do not perturb host cell machinery, transduced cells may not be recognized as infected, evidenced by the lack of NK-like activity in CTL assays. This may result in only the protein of interest being expressed, without stimulation of the cytokines IL-2, IL-12, and gamma interferon, which have been demonstrated as being important for CTL activation. Polycistronic expression vectors encoding for these important costimulatory molecules may help to overcome this possible shortcoming.
In conclusion, these studies have further demonstrated the utility of retroviral vectors in dissection of CTL responses in vitro. Retroviral vectors expressing EIAV Gag/Pr and SU and using the GALV Env for internalization transduced EK cells which were efficiently lysed by EIAV-specific CTL. These results confirmed CTL activity to EIAV Gag/Pr proteins and provided information regarding CTL epitope localization by demonstrating CTL reactivity to the SU protein of EIAV. Finally, preliminary studies confirmed that even though methods to enhance responses to their expressed proteins may be needed, retroviral vectors have the potential of eliciting virus-specific CTL.