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* Epimmune, San Diego, CA 92121; and
Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The use of epitope-based vaccines represents one approach to induce HTL responses of this nature. These type of vaccines have the added advantage of potentially focusing the immune responses on conserved epitopes, a feature that is likely to be important for rapidly mutating pathogens such as HIV, hepatitis C virus, and malaria. In addition, epitope-based vaccines may overcome T cell tolerance (7, 8), thus facilitating the development of cancer vaccines. Finally, epitope-based vaccines might overcome immunodominance and generate broad responses simultaneously targeting multiple Ags (9).
Recently, the identification of broadly cross-reactive HTL epitopes with high population coverage enables the development of epitope-based vaccines. However, no study to date has addressed the use of multiple HLA-DR-restricted epitopes to elicit multiple HTL responses. In certain cases, vaccines containing multiple human HTL epitopes have been constructed, but their evaluation has been hampered by the genetic restriction of MHC responses. Although HLA-DR transgenic mice have been developed as a means of addressing this limitation (10), they have not been used to evaluate vaccines destined for human use. An alternative strategy relies on cross-reactive binding between human and murine class II molecules. Anecdotal evidence of such cross-reactivity has been reported (11); similarly, our group has observed that the binding motifs for I-Ab and HLA-DR molecules share common anchor residues (12).
With regard to designing immunogens for the inductions of HTL responses, various strategies such as multiple Ag peptide (MAP) conjugates (13) and sequential arrangement of epitopes into a single polypeptide (14) have been investigated. MAP constructs have been shown to be potent (15), but have been challenging to produce in large quantities. Linear polypeptides can be produced by recombinant techniques, thus overcoming these manufacturing concerns.
Creation of junctional epitopes is a serious concern in the design of linear polypeptides. A junctional epitope is defined as a neoepitope, created by the juxtaposition of two authentic epitopes. The new epitope is composed of a C-terminal section from the first epitope and an N-terminal section derived from a second epitope. The presence of such a junctional epitope could create undesired immunodominance effects, redirecting the immune response to irrelevant epitopes and in some cases suppressing the induction of responses to the desired epitopes. Gefter and colleagues (16) were the first to demonstrate the recognition of a junctional epitope in a murine model that used a linear arrangement of two class II epitopes. The effect was so marked that recognition of the component epitopes could be completely silenced by the presence of the junctional epitope (17).
The present study defines a rational strategy to design and test HLA-DR-restricted multiepitope vaccine constructs. Epitopes cross-reactive between murine I-Ab and human HLA-DR class II molecules were used to evaluate different delivery systems. These studies revealed significant heterogenicity and suggested that the presence of junctional epitopes can be overcome by the introduction of spacer amino acids designed to retard the binding of the junctional epitope. These findings have broad practical applications in the design of epitope-based vaccines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peptides and MAP constructs were synthesized using FMOC chemistry, as described previously (18). After the synthesis was completed, the peptide was cleaved from the resin, the protecting groups were removed, and the peptides were then purified by reversed-phase HPLC. The purity of the peptides was substantiated by mass spectrometry and/or composition analysis and found to be routinely greater than 95%.
DNA expression constructs were constructed using standard PCR techniques. The multiepitope minigenes were cloned into the CMV-driven expression vector pcDNA3.1 (Invitrogen, San Diego, CA); DNA for immunization was purified using endotoxin-free Qiagen kits (Valencia, CA).
MHC purification and peptide-binding assays
Human and murine class II molecules were purified by affinity chromatography from EBV-transformed homozygous cell lines, as previously described (19). In short, cell lysates were filtered twice through two precolumns of inactivated Sepharose CL4-B and protein A-Sepharose, and then passed over a column of Sepharose CL-4B beads coupled with the appropriate anti-class II mAbs. Class II molecules were eluted with 50 mM diethylamine in 0.15 M NaCl containing 0.4% N-octylglucoside, pH 11.5. The eluate was then concentrated by centrifugation in Centriprep 30 concentrators at 2000 rpm (Amicon, Beverly, MA).
Peptide-binding affinity was measured by incubating a dose range of the unlabeled test peptides and 1–10 nM 125I-radiolabeled probe peptides with purified class II molecules (5–500 nM) for 48 h. In preliminary experiments, the class II preparation was titered in the presence of fixed amounts of radiolabeled peptides to determine the concentration necessary to bind 10–20% of the total radioactivity. All subsequent inhibition and direct binding assays were then performed using these concentrations. The percentage of HLA-MHC-bound radioactivity was determined by capturing MHC/peptide complexes on LB3.1 Ab (anti-HLA-DRA; American Type Culture Collection, Manassas, VA)-coated Optiplates (Packard Instrument, Meriden, CT) and measuring bound cpm using the TopCount (Packard Instrument) microscintillation counter. In appropriate stoichiometric conditions, the IC50 of an unlabeled test peptide to the purified class II molecule is a reasonable approximation of the affinity of interaction (Kd).
Immunizations
Ag, peptides, and polypeptides were suspended in PBS and emulsified in 50% CFA (Sigma-Aldrich, St. Louis, MO). H-2b mice were immunized with 100 µl emulsion s.c. at the base of the tail. For DNA immunizations, mice were pretreated by injecting 50 µl 10 µM cardiotoxin bilaterally into the tibialis anterior muscle; 4 days later, the same muscle received 100 µg plasmid DNA diluted in PBS. Eleven to fourteen days after immunization, epitope-specific HTL responses were measured using either proliferation or ELISPOT assays. In the case of peptide immunization, responses were measured from CD4 cells purified from the para-aortic and inguinal lymph nodes. Responses induced by DNA immunization were measured from CD4 cells purified from the spleen.
Proliferation assay
CD4+ T cells were purified from single cell suspension of para-aortic and inguinal lymph nodes using Dynal beads (Great Neck, NY). CD4+ T cells were detached from the beads and washed in RPMI, 2% FCS. Cells were plated in 96-well flat-bottom plates at a density of 2 x 105/well. Irradiated syngenic splenocytes (5 x 105 cells/well) were added as a source of APCs. Cells were stimulated with various concentrations of peptide ranging from 0.05 to 20 µg/ml. Background proliferation was measured in the absence of peptide. Cultures were incubated 3 days at 37°C and pulsed with 1 µCi [3H]thymidine. Cultures were incubated an additional 16–18 h. Cellular DNA was harvested on glass fiber mats and analyzed for 3H incorporation. Stimulation indexes were calculated using T cell responses observed from cells stimulated with 1 µg/ml peptide.
ELISPOT assay
Membrane-backed 96-well ELISA plates (Millipore, Bedford, MA)
were coated with anti-IFN-
mAb (BD PharMingen, San Diego, CA)
overnight at 4°C and then blocked with medium containing 10% FCS.
Purified CD4+ cells (4 x
105/well) were added to the microplate wells and
cultured with 1 µg/ml peptide and irradiated splenocyte cells
(105 cells/well) for 20 h at 37°C. The
number of IFN--secreting cells was detected by incubation with
biotinylated anti-mouse IFN- Ab (BD PharMingen), followed by
incubation with avidin-peroxidase complex (Vectastain). Finally, the
plates are developed using AEC (3-amino-9-ethylcarbazole;
Sigma-Aldrich), washed, and dried. Spots are counted using the Zeiss KS
ELISPOT reader (Oberkochen, Germany).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously reported a set of highly conserved
HIV-1-derived HLA-DR-restricted HTL epitopes suitable for development
of a multiepitope vaccine (20). In the present study, we
used four of these epitopes (Pol 711, Gag 171, Pol 303, and Pol 335) to
address basic issues related to the design of such multiepitope
vaccines. These epitopes were selected because in addition to binding
several different HLA-DR molecules, they also bound the murine class II
molecule-bound I-Ab with affinities in the
100–600 nM range (Table I
). This range
of binding affinities is comparable with the affinity of a known
I-Ab-restricted epitope, the universal HTL
epitope PADRE (21).
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), was
included as a negative control. Specifically,
H-2b mice were immunized s.c. with 140 µg of
the various epitopes emulsified in CFA. Eleven to fourteen days
postimmunization, CD4+ T cells were purified from
the draining lymph nodes, and epitope-specific recall proliferative
responses were quantitated based on
[3H]thymidine incorporation. As expected, all
four I-Ab-binding HIV-derived epitopes induced
significant proliferative responses. The Gag 171, Pol 303, and Pol 335
epitopes induced responses similar in magnitude to the response induced
by the PADRE epitope (Fig. 1). The
epitope Pol 711 was the most immunogenic and induced an 
10-fold
stronger response. As anticipated, Pol 758, which binds
I-Ab weakly, also failed to induce an HTL
response. Collectively, these data identified four HIV-derived
HLA-DR-restricted epitopes that are also immunogenic in
H-2b mice. Accordingly, these data defined a
murine model system suitable to evaluate strategies for effective
delivery of multiple HTL epitopes in vivo.
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Although peptide pools are a straightforward means of delivering
multiple epitopes, clinical development of peptide pools may be
associated with significant manufacturing and formulation concerns,
particularly if delivery of a large number of epitopes is required. For
these reasons, the development of a single vaccine construct capable of
delivering multiple epitopes would be desirable. Accordingly, we
examined the immunogenicity of two different single component
multiepitope Ags. Specifically, a MAP containing the four selected HIV
HTL epitopes (Fig. 2A) was
synthesized (18). The MAP is a branched structure in which
the epitopes are linked together through a C-terminal lysine residue.
In addition, we also characterized the immunogenicity of a polypeptide
consisting of a sequential arrangement of the Pol 711, Gag 171, Pol
335, and Pol 303 epitopes all colinearly synthesized without
intervening flanking residues.
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, the peptide pool primed
significant HTL responses to all four epitopes. Stimulation indexes
were typically in the 4–10 range, and the response magnitudes were
relatively comparable for the various epitopes. Thus, at least under
the conditions tested, little or no interepitope competition was
occurring, and no particular epitope appeared to be immunodominant over
the other epitopes.
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). Although a strong
response was measured against the Pol 303 epitope, the response to the
Pol 711 epitope was relatively weak (stimulation index (SI) =
3.3), and virtually no recall response was detectable against Gag 171
and Pol 335. By comparison, the sequential polypeptide induced stronger
HTL responses to three of the four component epitopes. Responses to the
Pol 711 and Gag 171 epitopes were similar in magnitude to the responses
induced by the peptide pool. Significant recall responses to Pol 303
were also induced, although of lower magnitude than those induced by
the peptide pool. Finally, the linear polypeptide failed to prime for
responses directed against the Pol 335 epitope. In conclusion, priming for balanced HTL responses against all four epitopes was not achieved with either the MAP or linear polypeptide. Because a pool of individual peptides could prime for HTL responses to all four epitopes, these data suggest that factors inherent in the design of the MAP and the linear polypeptide hindered their capacity to induce responses to all epitopes.
Juxtaposition of defined epitopes created a junctional epitope overlapping Pol 335
Because of the higher intrinsic activity demonstrated by the multiepitope polypeptide, we concentrated on optimizing this construct. We hypothesized that a junctional epitope might have been created by the juxtaposition of the component HIV-derived epitopes and that this might interfere with effective priming of responses to Pol 335. To test this hypothesis, six different peptides spanning the epitope junctions were synthesized. Each of the junctional peptides encompasses 10 aa of one HIV epitope and 5 aa of the adjacent HIV epitope.
These junctional peptides were first characterized by measuring their
binding affinity to I-Ab molecules. The
junctional peptides, 3 and 4, which correspond to the junction between
Gag 171 and Pol 335, bound I-Ab with affinities
of 14 and 75 nM, respectively. These affinities are on average 25-fold
higher than the binding affinity of the Pol 335 epitope. (Table II). By contrast, the junctional peptides
1, 2 and 5, 6 bound purified I-Ab poorly (1474 nM
for peptide 6) or not at all (peptides 1, 2, and 5).
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). The magnitude of this response
(SI = 5.7) was comparable with those observed for Pol 711, Gag
171, and Pol 303, which stimulated mean responses of SI = 5.1 (see
Fig. 2 for comparison). In conclusion, the binding and immunogenicity
data demonstrate the presence of a junctional epitope created by the
juxtaposition of the Gag 171 and Pol 335. The presence of this
junctional epitope may be associated with the inability of the linear
polypeptide to effectively prime Pol 335-specific responses.
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Peptide binding to class II molecules is predicated on specific sequence motifs based on the occurrence of certain amino acid residues with defined spacing. Disrupting these specific binding motifs, through either the alteration of primary anchor residues or their relative spacing, is predicted to deleteriously affect peptide binding. Both these approaches could be used to disrupt the junctional epitope in the polypeptide; however, changing the anchor residues responsible for junctional peptide binding would require changing the sequence of the epitopes themselves, and thereby may be associated with altering the specificity of the HTL response. Consequently, we sought to disrupt the junctional epitope through the introduction of spacer amino acids between the HIV epitopes.
Although MHC class II molecules typically bind peptides of 12–20 residues in length, a nine-residue core region is usually the main determinant of the binding energy. In the case of the binding motifs associated with most human class II molecules, positions 1 and 6 (referred to as P1 and P6, respectively) are generally the most important anchor residues (19). The I-Ab class II molecule also appears to share a similar motif and anchor spacing (12). Accordingly, we reasoned that a spacer with a minimum of five residues would be required to disrupt potential peptide-binding motifs occurring as the result of sequentially linking two different epitopes. We also reasoned that to be effective, the spacer should be made of residues not commonly allowed at either P1 or P6.
A survey of the side chain specificities for main anchor residues of
prevalent MHC class II molecules (Table III) highlighted that in most
instances, Y, F, I, L, V, or W is preferred at P1. Small or hydrophobic
residues such as S, A, T, or C at P6 are also important for most
peptide-DR interactions. As G and P are residues not typically found at
main anchor positions, we predicted that introduction of a GPGPG spacer
between epitopes would have a high likelihood of preventing the
formation of most junctional epitopes.
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To test this approach directly, a peptide was synthesized in which
a GPGPG spacer was inserted into the peptide spanning the Gag 171-Pol
335 junction. The binding of the spacer peptide relative to that of the
junctional peptide was measured for several common HLA-DR molecules
(Table IV). Binding to HLA-DR molecules
was measured because our goal is to develop a strategy applicable to
multiepitope vaccines for human use.
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The ability of the GPGPG spacer to disrupt I-Ab binding was also evaluated. As presented in previous sections, the peptide spanning the Gag 171-Pol 335 junction binds I-Ab with high affinity of 14 nM. Insertion of a GPGPG spacer decreased the binding affinity to 313.3 nM, a greater than 20-fold decrease in binding affinity compared with the native peptide without spacers. By comparison with the effects seen on DR binding, the impact on I-Ab binding was relatively modest. We reasoned that the use of the murine model system would represent a stringent functional test of the GPGPG spacer.
Immunogenicity of a GPGPG spacer containing HTL polypeptide
Based on these results, the sequential polypeptide was redesigned
to include GPGPG spacers between all the epitopes. To determine the
impact this modification would have on immunogenicity,
H-2b mice were immunized with equimolar doses of
either the sequential polypeptide (112 µg/mouse) or the GPGPG spacer
polypeptide (140 µg/mouse) emulsified in CFA. The presence of the
GPGPG spacer restored the immunogenicity of the Pol 335 epitope that
was lost in the context of the sequential polypeptide (Fig. 5). In addition, the GPGPG spacer did not
appreciably effect responses to the Pol 711, Gag 171, and Pol 303
epitopes. In each of those cases, the magnitude of the responses was
indistinguishable from those obtained from mice immunized with the
sequential polypeptide.
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ELISPOT assay. CD4 T cells were
purified from animals immunized with the GPGPG spacer polypeptide, and
epitope-specific responses were directly measured in the absence of
restimulation to expand the T cell population. As shown in Fig. 6A, responses against all four
epitopes were detected in these experiments. The frequency of
IFN--secreting CD4 cells was in the range of 90–500 spot-forming
cells/106 cells. These data illustrate
that the spacer polypeptide effectively induces multiple HTL responses
characterized by both the ability to secrete IFN- and proliferate in
responses to specific epitopes. More importantly, these data
demonstrate how junctional epitopes can be disrupted and how a
spacer-optimized polypeptide could simultaneously prime responses
against all the HTL epitopes incorporated into the construct.
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Although DNA immunization has been an effective means to induce CTL responses, the approach has been less effective in stimulating HTL responses. To further examine the applicability of the GPGPG spacer design, we constructed a minigene encoding the GPGPG spacer polypeptide.
Mice were immunized i.m. with 100 µg plasmid expressing the GPGPG
polypeptide. The induction of epitope-specific CD4 T cells was measured
using a primary ELISPOT. IFN--secreting T cells were detected for
all epitopes (Fig. 6B). The HTL responses were in general
comparable with those induced by polypeptide immunization, thus
underlying the flexibility of this approach in the induction of
multispecific HTL responses.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The definition of peptides that bind and are immunogenic in the context of different HLA-DR molecules enables the design of epitope-based vaccines capable of being recognized by the vast majority of the human population. In general, human and murine class II molecules are thought to recognize different ligands. This different specificity restricts the experimental testing of these constructs in laboratory animals. To overcome this difficulty, transgenic mice expressing human class II molecules have been derived (10). We describe an alternative strategy based on the observation that peptide-binding motif for the murine class II molecule I-Ab (12) overlaps with the motif recognized by several common DR molecules (19). Accordingly, we predicted that it should be possible to identify DR-restricted epitopes capable of binding I-Ab molecules and inducing epitope-specific HTL responses in nontransgenic mice. Our results validate this concept and enable the use of H-2b mice as an experimental system to model HLA-DR-restricted responses.
One fundamental question concerning the development of multiepitope vaccines is whether or not it is possible to simultaneously induce responses to a number of epitopes. In general, immune responses are only directed at a small fraction of the possible peptides derived from a given pathogen are recognized as epitopes. This phenomena of immunodominance can be attributed to a number of factors, including binding affinity, holes in the T cell repertoire, or epitope processing (9). Immunodominance primarily focused on one or a few epitopes is more commonly observed in murine systems, as a number of studies have shown human responses are often directed against multiple epitopes (20, 22). We find in the present study that immunization with a pool of peptides, each of which individually induces potent HTL responses, was nearly as effective at inducing responses as the individual peptides. Some caution should be exercised in interpreting these results, as dose-response experiments were not performed. Nevertheless, these data were encouraging, in that they alleviated possible concerns over immunodominance and demonstrated that it is possible to prime for a balanced multispecific HTL response at least up to a tetravalent epitope construct. Current experiments are examining whether there is a limit to the number of epitopes that can be recognized.
Although pools of peptides have been used clinically to elicit multispecific immune responses (23), practical issues relating to the manufacture and formulation limit the usefulness of this approach to the development of a vaccine designed to incorporate multiple HTL epitopes. An alternative approach is the use of MAP conjugates in which distinct epitopes are chemically linked into a single molecule. Such constructs have been shown to be immunogenic, capable of inducing both humoral and cellular immune responses to Plasmodium falciparum epitopes (15). An alternative approach is to produce an Ag representing a string of select epitopes. Using a baculovirus expression system, Shi et al. (14) produced an immunogenic polypeptide composed of 21 P. falciparum epitopes. Although both Ags induced immune responses that conferred in vitro protective efficacy, there was no direct comparison of immunogenicity of the Ags, and means of optimizing immunogenicity were not addressed. In fact, the polypeptide was found to contain two junctional epitopes that may contribute to reduced potency (24). This study represents the first direct comparison of these delivery strategies and examines means to improve vaccine design through the use of specialized spacer sequences.
The presence of junctional epitopes could redirect the immune response to irrelevant and possibly even immunodominant epitopes. In the present study, we observed similar effects in a sequential tetravalent polypeptide. This problem was overcome by the introduction of a GPGPG spacer. The use of spacers has been used in constructs consisting of helper and Ab epitopes to preserve conformational dependent immunogenicity. Spacers have also been used between two neighboring CTL epitopes to facilitate epitope processing (25, 26). In this study, the spacers are applied to eliminate junctional epitopes and tailor the specificity of the immune response. The introduction of GPGPG spacers does not preclude the possibility that such linear arrangements of epitopes might contain other cryptic epitopes. Nonetheless, the use of GPGPG spacers should be broadly applicable at minimizing purely junctional epitopes because the approach is not context dependent.
The receptor-binding pockets of MHC class II molecules are best suited to bind amino acids with specific biochemical properties and spacing. The characterization of these parameters has led to the definition of detailed peptide-binding motifs. Although detailed knowledge of these binding motifs has been used for epitope identification, it can also be used to minimize the creation of artificial epitopes, such as junctional epitopes, in the design of specialized epitope-based vaccines. The peptide-binding motifs of common MHC class II molecules are typically based around two primary anchor residues separated by 4 aa. As such, the introduction of a 5-aa spacer between epitopes would preclude the creation of a junctional epitope that uses amino acids from both epitopes as anchor residues. To prevent the formation of junctional epitopes, it becomes a matter of using a spacer based on residues that are infrequently used as primary anchors.
Among various possible spacers, GPGPG was selected for two reasons.
First, GPGP extensions around the core binding region greatly decrease
binding affinity, thus suggesting that GPGPG-containing epitopes would
not bind efficiently unless the spacer was removed by Ag processing.
Consequently, we felt that this spacer would maximize the likelihood
that the relevant epitope would be regenerated with little or no
extraneous sequences attached to its N and C terminus. Second, the
GPGPG spacer was selected because regions rich in G and P are known to
be associated with 
turns (27). The presence of this
spacer at 15–20 residue intervals might help create some secondary
and possibly tertiary structure, thereby facilitating Ag expression and
potential purification.
Polypeptides are frequently used as a means of delivering HTL epitopes, as such Ags are likely to be endocytosed by APC, where they could directly access the endosomes in which class II molecules are loaded. Recent data suggest that HTL epitopes may also be effectively delivered by DNA immunization (28). Our data demonstrate that the GPGPG spacer can also be applied to DNA-based vaccines, underlying the broad applicability of spacers in optimizing the potency of multiepitope HTL vaccine constructs.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Brian Livingston, Epimmune, 5820 Nancy Ridge Drive, Suite 100, San Diego, CA 92121. E-mail address: blivingston{at}epimmune.com
3 Abbreviations used in this paper: HTL, Th lymphocyte; MAP, multiple Ag peptide; SI, stimulation index.
Received for publication January 18, 2002. Accepted for publication March 22, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TH2 switch is a critical step in the etiology of HIV infection. Immunol. Today 14:107.[Medline]
- and -turns in proteins revisited: a new set of amino acid turn-type dependent positional preferences and potentials. J. Biosci. 25:143.[Medline]
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