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The Journal of Immunology, 2004, 173: 4387-4393.
Copyright © 2004 by The American Association of Immunologists

Repertoire and Immunofocusing of CD8 T Cell Responses Generated by HIV-1 gag-pol and Expression Library Immunization Vaccines1

Rana A. K. Singh* and Michael A. Barry2,*,{dagger},{ddagger}

* Center for Cell and Gene Therapy, {dagger} Departments of Molecular and Human Genetics, and Immunology, Baylor College of Medicine, and {ddagger} Department of Bioengineering, Rice University, Houston, TX 77251


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Several gene-based vaccine approaches are being tested to drive multivalent cellular immune responses to control HIV-1 viral variants. To compare the utility of these approaches, HLA-A*0201 transgenic mice were genetically immunized with plasmids encoding wild-type (wt) gag-pol, codon-optimized (CO) gag-pol, and an expression library immunization (ELI) vaccine genetically re-engineered to express non-CO fragments of gag and pol fused to ubiquitin for proteasome targeting. Equimolar delivery of each vaccine into HLA-A*0201 transgenic mice generated CD8 T cell responses, with the ELI vaccine producing up to 10-fold higher responses than the wt or CO gag-pol plasmids against cognate and mutant epitopes. All three vaccines generated multivalent CD8 responses against varying numbers of epitopes after priming. However, when the animals were immunized again, the wt and CO gag-pol vaccines boosted only the responses against a subset of epitopes and attenuated the responses against all other Ags including epitopes from clade and drug-resistant viral variants. In contrast, the ELI vaccine boosted CD8 responses against all of the gag-pol Ags and against mutant epitopes from clade and drug-resistant variants. These data suggest that HIV-1 vaccines expressing structurally intact gag and pol proteins drive immunofocused CD8 responses that reduce the repertoire of T cell responses. In contrast, the genetically re-engineered ELI vaccine appears to better maintain the multivalent CD8 responses that may be required to control HIV-1 viral variants.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Acquired immunodeficiency syndrome, caused by HIV-1 infection, has become one of the largest infectious disease killers in the world, with more than 24 million deaths to date and more than 40 million people infected worldwide. The development of an effective HIV-1 vaccine has become increasingly important as the only means to arrest this growing epidemic. Recent studies in both humans and nonhuman primates suggest that CTL control virus replication and delay disease progression (1, 2, 3). Host T cell immunity against HIV-1 can be induced by immunizing with a genetic vaccine that can mediate the expression of HIV-1 genes in the appropriate APCs. The immunogenic potential in laboratory animals of several DNA and recombinant viral vectors and combinations thereof has been described in numerous reports.

In most cases, HIV vaccines deliver viral proteins in their intact, active forms. Although this makes sense on one level, the potency and repertoire of immune responses generated by this type of vaccine may be limited by immunoevasive aspects inherent to HIV-1 proteins. This hypothesis is consistent with a variety of recent reports indicating that repetitive boosting with simian-human immunodeficiency virus vaccines results in the narrowing of the repertoire of immune responses, which become focused on only a few immunodominant epitopes (Refs.4 and 5 , and references therein). This immunofocusing phenomenon may pose a serious problem for applying HIV-1 vaccines, because focused responses against one or only a few viral epitopes can be easily evaded by mutation of the targeted epitopes (6).

Given these issues, we have been testing a genetically re-engineered HIV-1 genetic vaccine in which all HIV Ags are expressed as ~150-aa protein fragments to break down protein structure, facilitate proteasome degradation, and reduce the toxicity of the proteins (7, 8, 9). In addition, each fragment is fused to ubiquitin to target the Ags to the proteasome to increase CTL responses (9, 10). This HIV-1 expression library immunization (ELI)3 vaccine delivers these re-engineered HIV Ags in a library of 32 plasmids (see Fig. 1). We have demonstrated in HLA transgenic mice that the ELI vaccine simultaneously provokes robust HLA-A*0201-restricted T cell responses against all 32 of the HIV-1-ELI vaccine library Ags after single immunization by gene gun (11).



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  		FIGURE 1. Schematic of HIV-1 ubiquitin ELI library and locations of known HLA-A*0201-restricted CTL epitopes. HLA-restricted epitopes that were tested in this study are shown in bold and larger font. Other HLA-A2 epitopes that were not tested are shown in smaller font to represent where possible subdominant epitopes may lie in Ags.

 
Although the ELI vaccine appeared potent in HLA transgenic mice, this non-codon-optimized (CO) vaccine has not to date been compared directly to a comparable genetic vaccine expressing wild-type (wt) HIV-1 proteins. Furthermore, we did not compare the ELI vaccine against newer CO HIV-1 Ag plasmids with increased expression. Therefore, in this work, we have tested our hypothesis that the ELI vaccine should mediate enhanced multivalent CD8 T cell responses compared with unmodified gag-pol Ags. In this study, we have performed a direct comparison between the non-CO ELI vaccine and plasmids expressing either non-CO gag-pol or CO gag-pol. We find that all of the vaccines initially generate varied multivalent CD8 T cell responses, with the ELI vaccine generating responses 2- to 10-fold higher than those of the gag-pol vaccines. Although all primed multivalent CD8 responses, boosting with each vaccine demonstrated striking differences in each vaccine’s ability to maintain these multivalent responses. Boosting with both of the gag-pol plasmid vaccines increased CD8 responses only against immunodominant epitopes and decreased the existing responses against subdominant epitopes. In contrast, the ELI vaccines boosted all of the primed multivalent responses with no apparent immunofocusing. These data suggest that genetic vaccines expressing full-length, structurally intact gag and pol proteins may bias T cell responses toward immunodominant epitopes. In contrast, genetically re-engineered Ags that break down the structure of HIV Ags and immunologically target them appear to avoid this immunofocusing and drive resilient multivalent T cell responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Mice transgenic for HLA-A*0201/Kb (line 6) were generously provided by Dr. L. Sherman from The Scripps Research Institute (La Jolla, CA) (12). These mice were crossed with C3H mice, and the F1 progeny were used for these experiments as in Ref.11 .

Plasmids

The HIV-1 ELI vaccine was constructed as described in Ref.9 . Briefly, the entire HIV-1 (HTLV-IIIb) genome was broken in to 32 overlapping fragments by PCR using 32 sets of primers, and each fragment was cloned in pCMVi plasmid downstream of the CMV enhancer/promoter and the pCI synthetic intron. Each fragment was fused to the C terminus of ubiquitin for cytoplasmic expression and proteasome targeting. The relative position of each library member expressing a fragmented HIV-1 Ag is shown in a cartoon format in Fig. 1. wt and CO gag-pol plasmids in the pCI plasmid (with CMV promoter and pCI synthetic intron) were generously provided by R. Sutton (Baylor College of Medicine) and Oxford BioMedica (Oxford, U.K.) (13), respectively. All plasmids were purified on Qiagen (Valencia, CA) endotoxin-free columns.

Genetic immunization

Four- to 6-wk-old mice were genetically immunized by gene gun transfection of the epidermis using the Helios biolistic device (Bio-Rad, Hercules, CA) using 400 psi of helium. Plasmid DNA (3.2 µg/shot of 1.6-µm gold particles) was delivered according to the instructions provided by manufacturer of the device. In most cases, four shots (a total of 12.8 µg of plasmid DNA) were delivered into the backs of the ears on each mouse at each immunization except where noted in the text, and the immune responses were measured 1 mo later. For boosting experiments, mice were primed for a month and were then boosted for another month with same plasmid(s). For all experiments, five animals per group were vaccinated.

Target cell lines expressing HLA-A*0201 and ubiquitin-HIV-1 ELI gag-pol library members

A panel of HLA-A*0201-expressing 10T/2 cell lines (derived from a C3H/J mouse (H-2K) were generated as described in Ref.11 . Each cell line stably expresses both HLA-A*0201 and 1 of the 32 plasmids from the HIV-1 ELI vaccine (see Fig. 1).

Splenocytes stimulation and intracellular IFN-{gamma} staining

Splenocytes were recovered from nonimmunized and immunized mice and processed as described in Ref.11 . Fresh lymphocytes were stimulated in vitro (1 x 106 cells/sample) for 6 h at 37°C in a CO2 incubator with the indicated 10T1/2 cells or 10T1/2-HLA-A*0201 stable cells as shown in the figures and described in Ref.11 . The cells were then assayed by flow cytometry for intracellular IFN-{gamma} production of CD3/CD8 double-positive cells as described by Singh et al. (11).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Comparison of HLA-restricted CD8/IFN-{gamma} T cell responses against immunodominant gag and pol epitopes

Of 32 different plasmids that together constitute the HIV-1-ELI vaccine and represent the whole HIV-1 genome (Fig. 1), plasmids 1–13 express the gag-pol region as overlapping protein fragments fused to ubiquitin. Plasmids 14–16 express ubiquitin-fused regions of vif and vpr and are mismatched for gag and pol. To compare HLA-restricted CD8 responses, HLA-A*0201 x C3H mice were immunized by gene gun with plasmids 1–16 (ELI gag-pol vaccine) of the library or were immunized with single plasmid expressing wt or CO gag-pol. Each group of mice (n = 5) received a total of 12.8 µg of DNA divided in four gene gun shots (for ELI gag-pol: 0.8 µg of each member x 16 members = 12.8 µg of DNA). Another group of mice (n = 5) were immunized with above plasmid(s) and then were immunized a second time with the same plasmid(s) a month later to compare the effect of boosting on the CD8 T cell responses. Splenocytes from the mice were then tested for intracellular IFN-{gamma} production of CD3/CD8 cells by flow cytometry (Fig. 2).



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  		FIGURE 2. Effect of homologous boosting on IFN-{gamma}+CD3+CD8+ T cell responses induced by ELI gag-pol, CO gag-pol, and wt gag-pol vaccines. Two sets of 6-wk-old HLA-A*0201 transgenic mice (n = 5/vaccine) were genetically immunized with 12.8 µg of plasmid DNA encoding either ELI gag-pol vaccine, CO gag-pol vaccine, or wt gag-pol vaccine by gene gun as described in Materials and Methods. One set of mice was assayed for immune responses after 1 mo of immunization, whereas the second set of mice was boosted for another month (ELI gag-pol boosted with ELI gag-pol, CO gag-pol boosted with CO gag-pol, and wt gag-pol boosted with wt gag-pol). Fresh lymphocytes (1 x 106/sample) were stimulated for 6 h at 37°C in the presence of 5 µg/ml brefeldin A with irradiated (6000 rad) 10T/2-HLA-A*0201 stimulator cell lines loaded with indicated peptide at responder:stimulator ratio of 10:1. Intracellular IFN-{gamma} staining was performed using Cytofix/Cytoperm kit (BD Pharmingen, San Diego, CA) as described in Materials and Methods. CD3/CD8 double-positive cells (5–10 x 104/sample) were analyzed for intracellular IFN-{gamma} production on a FACScan using CellQuest software. A, Bar graph showing IFN-{gamma} production of CD3/CD8 cells in variously immunized mice. Data shown are mean ± SD (n = 5). B, A representative staining of a single mouse per group of five is shown.

 
Mice immunized with ELI gag-pol, wt gag-pol, or the CO gag-pol vaccine, all generated CD8 T cells specific for the immunodominant gag76–84 and pol468–476 epitopes as compared with the background responses against control APCs 10T/2 and 10T/2-HLA, and APCs labeled with mismatched epitopes env (P18), and nef190–198 (Fig. 1). ELI gag-pol vaccine induced highest number of gag- and pol-specific CD8 T cell responses followed by CO gag-pol and wt gag-pol (Fig. 2), indicating the immunostimulatory potential of all three HIV-1 genetic vaccines.

Comparison of HLA-restricted CD8/IFN-{gamma} T cell responses mediated by single immunization

To identify new or subdominant epitope-directed CD8 T cell responses, splenocytes from the immunized mice were tested against a panel of 16 Ag presenting 10T/2 cell lines each expressing HLA-A*0201/Kb and one ELI vaccine plasmid (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) (Fig. 1 and Ref.11). These cells express 150- to 200-aa fragments of gag-pol bearing a number of HLA-A2-restricted epitopes allowing a low-level epitope screen for CD8 T cell responses against different regions of gag and pol. Ag H1 expresses the HLA-restricted immunodominant gag epitope gag76–84. H8 and H9 express the immunodominant pol epitope pol468–476.

When the HLA mice were immunized with non-CO wt gag-pol, CD8 T cell responses were provoked predominantly against Ags H1–H3 and H7–H9 with highest response against cell line H8 expressing pol468–476 (Fig. 3E). The CO gag-pol vaccine elicited CD8 T cell responses that were in general increased 2-fold when compared with the non-CO gag-pol vaccine (Fig. 3C). Both generated responses against all of the cell lines, with the exception of cell lines H14–H16, which express vif and vpr and are mismatched for gag-pol Ags.



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  		FIGURE 3. Repertoire of Ag-specific CD8/IFN-{gamma} responses induced by ELI gag-pol, CO gag-pol, and wt gag-pol vaccines, and effect of boosting on the repertoire. Transgenic mice (n = 5/vaccine) were genetically immunized with 12.8 µg of plasmid DNA encoding either ELI gag-pol, CO gag-pol, or wt gag-pol vaccine for a month. Mice were also vaccinated with 0.8 µg of plasmid DNA encoding either CO gag-pol or wt gag-pol vaccine for a month in combination with 12 µg of pCMVi plasmid DNA; therefore, all mice received a total of 12.8 µg of DNA. Vaccinated mice were divided into two groups. Group 1 was assayed for immune responses after a month, whereas the second group was boosted with the same plasmid used for priming for another month as explained in Fig. 2. Splenocytes (1 x 106/sample) were stimulated for 6 h with irradiated (6000 rad) 10T/2 stimulator cell lines stably transfected with HLA-A*0201 molecule and individual member of ELI gag-pol vaccine (H1–H16 for 16 library members) at responder:stimulator ratio of 10:1. CD3/CD8 double-positive cells (5–10 x 104/sample) were analyzed for intracellular IFN-{gamma} production on a FACScan using CellQuest software. Data shown are mean ± SD (n = 5).

 
Comparison of the two non-CO vaccines demonstrated that the ELI vaccine generated CD8 responses 2- to 10-fold higher than wt gag-pol. When compared with CO gag-pol, the non-CO ELI vaccine generated responses slightly higher than those mediated by the CO vaccine (Fig. 3, compare B with C and E). This suggests that there is some benefit to the re-engineering in the ELI vaccine, because this non-CO vaccine outperforms the CO one.

Molar comparison of the ELI and gag-pol vaccines

In these studies, we delivered 16 plasmids of the ELI vaccine. Because of this, when the same amount of DNA is delivered, each epitope is delivered in one-sixteenth to one-eighth the molar amount than the single gag-pol (i.e., gag76 is present once in the library; pol468 is present twice on two fragments due to overlap; Fig. 1). To compare the vaccines on an approximately equimolar basis, 0.8 µg (one-sixteenth) of the CO and wt gag-pol plasmids was delivered to the HLA transgenic mice diluted with 12 µg of vector plasmid pCMVi to equalize the total DNA amount (Fig. 3, D and F). Delivery of 0.8 µg of the wt gag-pol plasmid mediated barely detectable immune responses focused only on H1 and H8 cell line. Similarly, delivery of 0.8 µg of CO gag-pol vaccine induced detectable, but substantially reduced CD8 T cell responses against all of the cell lines, with the exception of H1 and H8, which display the immunodominant gag76–84 and pol468–476 epitopes (Fig. 3D). Therefore, in this comparison, delivering similar molar amounts of each epitope by each vaccine, the responses by even the CO gag-pol construct were markedly less than that mediated by the ELI vaccine. Furthermore, these responses by CO gag-pol were predominantly directed against only a subset of Ag domains from gag and pol. In contrast, the ELI vaccine generated responses against all of the gag-pol Ags (Fig. 3B).

Vaccine boosting and immunofocusing

As shown above, each vaccine generated variable levels of multivalent CD8 T cell responses. Although these priming responses were robust, work with simian-human immunodeficiency virus vaccines suggests that multiple immunizations generate immunofocused responses onto dominant epitopes (4, 5). To test this, mice were primed and boosted with the vaccines, and CD8 responses were assessed. Boosting with wt gag-pol increased CD8 responses only against cell lines H1, H7, and H8 (Fig. 3, E and F). Although these responses were boosted, second immunization concomitantly reduced or ablated the responses against all of the other Ags. Similarly, second immunization with CO gag-pol boosted CD8 responses against H1, H7, and H8, but also decreased the responses against all other Ags (Fig. 3, C and D). In marked contrast to the immunofocusing observed by the two gag-pol plasmids, second immunization with the ELI vaccine boosted not only the immunodominant responses, but also the responses against all of the other Ags. These data suggest that vaccination with intact HIV gag and pol Ags generates immunofocusing with vaccine boosting. In contrast, it appears the ELI vaccine evades this immunofocusing to produce a wider repertoire of CD8 T cell responses at priming and with boosting.

Cross-clade reactivity of CD8 T cells generated by gag-pol and ELI vaccines

HIV-1 vaccines built from one clade of the virus may have only limited abilities to cross-control evolutionarily divergent viruses from different clades, because epitopes targeted by a vaccine may have point mutations. To test the ability of these clade B genetic vaccines to cross-react against other clades, mice were immunized a single time with 0.8 or 12.8 µg of plasmid DNA of each vaccine with or without boosting, and CD8 T cell responses were tested against a panel of HLA-A*0201-restricted pol132–140 peptides from clades A, B, C, and D that bear point mutations when compared with clade B (Table I). Splenocytes were tested against these mutant epitopes and also against the clade B positive-control immunodominant pol468–478 peptide and the negative-control nef190–198 peptide (Fig. 4). Priming with the wt gag-pol plasmid generated low-level CD8 responses that cross-reacted well with the mutant peptides from different clades (Fig. 4, E and F). CO gag-pol generated higher CD8 T cells than wt gag-pol that also cross-reacted against the mutant epitopes (Fig. 4, C and D).


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  		Table I. Amino acid sequences of peptides used to stimulate splenocytesa

 


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  		FIGURE 4. Effect of boosting on cross-clade-reactive immune responses induced by ELI gag-pol, CO gag-pol, and wt gag-pol vaccines. Transgenic mice (n = 5/vaccine) were genetically immunized with 12.8 µg of plasmid DNA encoding either ELI gag-pol, CO gag-pol, or wt gag-pol vaccine, or with 0.8 µg of plasmid DNA encoding CO gag-pol or wt gag-pol vaccine plus 12 µg of pCMVi plasmid DNA without or with boosting as detailed in Fig. 3. Primed, and primed and boosted splenocytes (1 x 106/sample) were stimulated for 6 h with irradiated (6000 rad) 10T/2 stimulator cell lines stably transfected with HLA-A*0201 molecule at responder:stimulator ratio of 10:1 in the presence of indicated peptide (5 µg/ml). CD3/CD8 double-positive cells (5–10 x 104/sample) were analyzed for intracellular IFN-{gamma} production on a FACScan using CellQuest software. Data shown are mean ± SD (n = 5).

 
Although both gag-pol plasmids did generate cross-reactive CD8s after one immunization, boosting with the plasmids drastically attenuated the level of responses against the cognate and mutant forms of the subdominant pol132–140 epitope (Fig. 4, C and E). In contrast, the ELI gag-pol vaccine induced equal or higher levels of cross-clade-reactive CD3/CD8 T cells on priming than either CO or wt gag-pol plasmids. More importantly, second immunization with the ELI vaccine boosted the responses against both the cognate and mutant epitopes.

Comparison of responses against mutated HLA-A*0201 epitopes from protease drug-resistant HIV-1 viruses

To further test the ability of the genetic vaccines to cross-react to HIV-1 variants, responses were measured against two HLA-A*0201-restricted epitopes in protease (Table I) that are mutated during antiretroviral therapy in drug-resistant viral variants (14, 15, 16, 17). HLA transgenic mice were immunized once with each of the vaccines with or without boosting, and splenocytes were tested for their ability to recognize wt (45-B and 75-B) or mutant peptides (45mt-B and 75mt-B) for pol45–54 and pol75–84 (Table I). Similar to the responses against clade mutants in Fig. 4, we observed that boosting with both gag-pol plasmids drastically attenuated the repertoire of cross-reactive CD8 T cell responses against the subdominant cognate and mutant epitopes from the drug-resistant virions (Fig. 5). Again, the HIV-1 ELI vaccine generated stronger cross-reactive CD8 responses after priming, and all of these responses were boosted after second immunization. These data indicate that gag-pol genetic vaccines expressing intact HIV proteins drive immunofocused CD8 T cell responses against immunodominant epitopes. In contrast, the ELI vaccine avoids immunofocusing, drives stronger responses against dominant and subdominant epitopes, and also maintains cross-reactive responses against mutated epitopes.



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  		FIGURE 5. Effect of boosting on immune responses against protease inhibitor-induced mutant epitopes provoked by ELI gag-pol, CO gag-pol, and wt gag-pol vaccines. Transgenic mice (n = 5/vaccine) were genetically immunized with 12.8 µg of plasmid DNA encoding either ELI gag-pol, CO gag-pol, or wt gag-pol vaccine, or with 0.8 µg of plasmid DNA encoding CO gag-pol or wt gag-pol vaccine plus 12 µg of pCMVi plasmid DNA without or with boosting as detailed in Fig. 3. Primed, and primed and boosted splenocytes (1 x 106/sample) were stimulated for 6 h with irradiated (6000 rad) 10T/2 stimulator cell lines stably transfected with HLA-A*0201 molecule at responder:stimulator ratio of 10:1 in the presence of indicated peptide (5 µg/ml). CD3/CD8 double-positive cells (5–10 x 104/sample) were analyzed for intracellular IFN-{gamma} production on a FACScan using CellQuest software. Data shown are mean ± SD (n = 5).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, humanized mice transgenic for HLA-A*0201/Kb chimeric MHC class I molecule were immunized with non-CO ELI gag-pol vaccine, CO gag-pol vaccine, and wt gag-pol vaccine to assess and compare the following: 1) the breadth of CD8 T cell responses induced by each vaccine, 2) the effect of boosting on the above immune responses, 3) the ability of each vaccine to induce cross-clade-reactive CD8 T cells, and 4) the potential of each vaccine to induce CD8 T cell responses that can recognize mutated epitopes generated in drug-resistant virions.

Single immunization by gene gun with 12.8 µg of plasmid DNA of each vaccine resulted in robust CD8/IFN-{gamma} responses against the immunodominant epitopes gag76–86 and pol468–476. Using this amount of DNA, direct comparison between the vaccines demonstrated that ELI gag-pol vaccine and CO gag-pol are similarly efficient at inducing CD8 responses against these two immunodominant epitopes. The ability of the ELI vaccine to generate equal or better responses than the CO gag-pol vaccine was striking, because the ELI vaccine is not CO. The enhanced activity of the non-CO ELI vaccine was further demonstrated by its head-to-head comparison to the non-CO gag-pol vaccine. In this case, the ELI vaccine generated CD8 responses that were 2- to 10-fold higher than the conventional genetic vaccine. The potency of the ELI vaccine was also supported by the molar comparison of it vs the two gag-pol plasmid vaccines. In this case, approximately equimolar delivery of epitopes by the vaccine demonstrated that the ELI vaccine generated responses that were higher and that were substantially more multivalent than both gag-pol vaccines.

To assess the repertoire of CD8 responses, we performed a low-resolution Ag epitope screen of gag and pol using ELI library members expressed in target cells. Each library member expresses an Ag of 150–200 aa, including from one to four known HLA-A2-restricted epitopes from the HIV Molecular Immunology Database (Fig. 1). Of these 17 known HLA epitopes, we also directly tested five of these as synthetic peptides. By these screens, we demonstrated that all of the vaccines generated varied levels of multivalent CD8 responses after single immunization. However, there was a striking dichotomy in the maintenance of these CD8 responses after boosting by the ELI vaccine and the gag-pol vaccines. When tested against the library member Ags, boosting with either gag-pol vaccine attenuated the existing CD8 responses against 10 of the 13 Ag targets. Similarly, when tested with the seven synthetic peptides, gag-pol boosting increased responses only against the dominant gag and pol epitopes, but attenuated the pre-existing CD8 responses against the five subdominant epitopes. In contrast, boosting with the ELI vaccine maintained CD8 responses against all of the Ags and epitopes, both dominant and subdominant. These data suggest that the use of structurally intact gag and pol proteins as immunogens may drive immunoevasive, immunofocused T cell responses that can restrict the repertoire of epitopes that are targeted by these vaccines. This observation is consistent with work in nonhuman primates, where immunofocused responses were generated primarily against a single epitope in SIV gag (4, 5). Although this immunofocused CD8 response transiently controlled viremia, it was also readily escaped by virions that mutated the single targeted epitope (6). Work is underway to determine at the peptide level what fraction of the known 17 HLA-A2-restricted peptides are actually targeted. Work is also underway to determine the relative affinity of the CD8 cells that target the range of epitopes, because these initial in vitro tests likely overestimate the functionality of these T cells for targeting HIV-1-infected cells.

The robust CD8 T cell responses by the ELI vaccine could be due to several factors. The ELI library for HIV-1 (9) was based on earlier work fusing Ags to ubiquitin to target them to the proteasome to enhance CD8 responses (10). Ubiquitin fusion increases CD8 responses against dominant and subdominant epitopes (9, 18). We speculate that the apparent ability of the ELI vaccine to avoid immunofocusing may be related to the fact that all of the HIV-1 Ags are expressed as fragmented proteins in which native HIV protein secondary structure is disrupted. Previous work demonstrated that fusion of ubiquitin to intact HIV-1 gag protein fails to generate detectable CTL responses in mice against three H-2-restricted gag epitopes in mice (9). In contrast, when ubiquitin was fused to the fragmented ~150-aa fragments of gag in the ELI library, these library members generated CTLs against all three epitopes. These data suggest that ubiquitin targeting by itself may not be sufficient for Ags like gag that may be difficult to degrade at the proteasome. In contrast, breaking down the secondary structure of gag in the ELI vaccine combined with ubiquitin targeting may allow these gag proteins to be effectively degraded by the proteasome after ubiquitin targeting. Therefore, we speculate that the fragmented Ags in the HIV-1 ELI vaccine may be more efficiently degraded by the proteasome to yield a better repertoire of epitopes to avoid immunofocusing.

Our data suggests that the use of unmodified HIV Ags can drive problematic immunofocused responses against a limited set of epitopes. We anticipate that the same effects will be observed during prime-boost strategies in which DNA is used for priming and viral vectors like adenovirus or vaccinia virus are used for boosting. Whether the more robust Ag expression mediated by these viral vectors overcome this immunofocusing remains to be determined. Our data also suggests that genetic re-engineering of HIV proteins to break down their secondary structure and target them to the proteasome may avoid this immunofocusing and drive better multivalent responses against dominant and subdominant epitopes. These observations suggest that genetically re-engineering HIV Ags to convert immunoevasive proteins into immunostimulatory Ags has promise in producing multivalent CD8 T cell responses to control HIV-1 from different clades or to control viruses that have become drug resistant during highly active antiretroviral therapy.


   Acknowledgments
 
We thank Mary E. Barry and Jared Abrahim for their excellent technical assistance.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant AI042588 (to M.A.B.) and in part by the Baylor Center for AIDS Research Core Support Grant AI36211. This work was also supported by the use of reagents supplied by the National Institutes of Health AIDS Research and Reference Reagent Program. Back

2 Address correspondence and reprint requests to Dr. Michael A. Barry, Center for Cell and Gene Therapy, Baylor College of Medicine, One Baylor Plaza, BCM505 Houston, TX 77030. E-mail address: mab{at}bcm.tmc.edu Back

3 Abbreviations used in this paper: ELI, expression library immunization; CO, codon-optimized; wt, wild type. Back

Received for publication February 6, 2004. Accepted for publication July 12, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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