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1 Molecular Virology and Microbiology Graduate Program, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
2 Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
3 Department of Veterinary Science, Gluck Equine Research Center, University of Kentucky, Lexington, KY 40516, USA
Correspondence
Ronald C. Montelaro
rmont{at}pitt.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Whilst human and other animal lentiviruses cause progressive degenerative or immune-suppressive disease, EIAV infection of horses is characterized by dynamic recurring disease cycles in the first year of infection. Following this chronic stage of disease, most infected horses progress to a long-term, inapparent carrier state in which disease and virus replication are strictly controlled for the lifespan of the horse. This inapparent state is not attributable to attenuation of the infecting virus, as whole-blood transfers from inapparent carriers can induce disease in naïve recipient horses and immune suppression of inapparent horses can induce a recurrence of disease, indicating the potential virulence of the infecting EIAV (Issel & Coggins, 1979; McGuire et al., 1971; Montelaro et al., 1993). In addition, inapparent carriers of EIAV appear to be highly resistant to additional exposure to diverse and highly virulent strains of EIAV, suggesting that the immune response in inapparent carriers not only controls the existing infection, but also provides a high level of prophylactic immunity (Montelaro et al., 1993). Thus, the EIAV system is useful in examining the natural immunological control of a pathogenic lentivirus infection, despite the array of persistence mechanisms employed by these viruses.
During the past 20 years, we have evaluated a number of experimental EIAV vaccines, with the results revealing that EIAV-specific immune responses are a double-edged sword that can protect or enhance viral disease and infection upon virus challenge of experimentally vaccinated horses (Craigo et al., 2005; Hammond et al., 1999; Issel et al., 1992; Li et al., 2003; Lichtenstein et al., 1995; Raabe et al., 1998; Wang et al., 1994). As observed in similar studies of experimental vaccines in the simian immunodeficiency virus (SIV) and feline immunodeficiency virus systems, live-attenuated EIAV vaccines have to date produced the highest level of vaccine immunity against virulent EIAV challenge (Bogers et al., 2000). Interestingly, however, these attenuated lentiviral vaccines do not achieve optimum protective immunity until 6 months post-inoculation, indicating a progressive maturation of virus-specific immunity. In addition to the protective immunity observed in EIAV inapparent carriers, the mature immunity elicited by attenuated EIAV vaccines provides a complementary model for defining critical immune correlates of enduring broadly protective vaccine immunity to a lentiviral infection.
Based on a comparison of the quantitative and qualitative properties of lentiviral envelope protein (Env)-specific antibodies, we have defined an association between immature/non-protective or enhancing and mature/protective virus-specific immunity in the EIAV/horse (Hammond et al., 1999), SIV/monkey (Clements et al., 1995; Cole et al., 1997) and simian–human immunodeficiency virus/monkey (Cole et al., 1998) vaccine systems. Thus, we propose that lentiviral vaccines must drive this immune maturation sufficiently to achieve protective efficacy. Additionally, we hypothesize that the EIAV Env is a major determinant of vaccine efficacy and that the maturation to protective immunity is associated with a redirection of host immune responses from immunodominant, variable Env domains to relatively immunorecessive, conserved Env domains.
There has been, to date, only limited characterization of cellular and humoral immune responses associated with enduring protective EIAV immunity. Clearance of the acute viraemia typically correlates with the appearance of cytotoxic T lymphocytes (CTLs) (McGuire et al., 1994) and non-neutralizing antibodies (Perryman et al., 1988; Rwambo et al., 1990). Whilst levels of CTLs fluctuate throughout the disease course, neutralizing antibodies generally do not appear until 2 months post-infection and increase in titre and breadth of specificity during the first year post-infection (Ball et al., 1992; Hammond et al., 1997; Howe et al., 2002; O'Rourke et al., 1988). Recently, however, Mealey et al. (2005) have shown the presence of neutralizing antibodies immediately after the resolution of acute disease, perhaps suggesting an earlier role for neutralizing antibodies in controlling EIAV replication in experimentally infected horses. During the chronic phase of EIAV infection, there is a high propensity for the selection of antigenic variants of EIAV that are able to temporarily escape the existing neutralizing antibodies directed to Env proteins or CTL responses directed to Gag protein epitopes (Chung et al., 2005; Howe et al., 2002; Hussain et al., 1987; Mealey et al., 2003). The prevalence of antibody and CTL antigenic variants is consistent with a role for these specific immune responses in controlling EIAV infection. McGuire and colleagues have thoroughly examined the specificity of CTL responses to EIAV Gag and Pol proteins to identify broadly reactive T-helper (Th) and CTL epitopes in these proteins (Chung et al., 2005; Howe et al., 2002; Hussain et al., 1987; Mealey et al., 2003). However, vaccines based on these broadly reactive T-cell epitopes have failed to elicit protective immunity against EIAV challenge (Fraser et al., 2005), indicating that immune responses to other viral proteins, such as Env, may be an important component of the establishment of protective immunity to EIAV.
To explain the observed requirement of maturation for protective immunity, we have proposed that the progression towards protection is associated with recognition of conserved immunorecessive epitopes of the Env proteins. A necessary foundation for testing this hypothesis is a detailed characterization of humoral and cellular epitopes in the Env proteins that are recognized during protective immunity. We have previously examined in detail the neutralizing epitopes of EIAV Env proteins and the effects of sequence variation on viral neutralization properties (Ball et al., 1992; Craigo et al., 2002; Howe et al., 2002). In contrast to the detailed studies of Env neutralization determinants, there have been limited studies of the Th and CTL epitopes of EIAV Env proteins. Thus, in the current study, we have used 12 horses immunized with a highly protective live-attenuated EIAV vaccine and two experimentally infected asymptomatic carrier horses, with the infecting virus strain in both groups of horses expressing identical Env proteins, to elucidate regions of EIAV Env that are broadly recognized by Th and CTL. The results of these studies for the first time provide a comprehensive mapping of Env-specific Th and CTL peptides associated with enduring protective immunity to EIAV and a foundation for determining the effects of Env variation on immune recognition.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Serological typing of the major histocompatibility complex (MHC) I locus was performed by the University of Kentucky Equine Blood Typing Laboratory using established techniques for the 11 A-locus antigenic specificities of the equine lymphocyte alloantigen (ELA-A) (Table 1) (Bailey, 1980, 1983). Direct sequencing of the DRA and DQA MHC II loci was performed by using previously published protocols (Albright-Fraser et al., 1996; Fraser & Bailey, 1998). Horses C23, C66 and B81 were not MHC II-typed due to lack of cells. After allowing the horses 7 months to establish a mature immune response to the vaccine, blood was drawn from each horse and peripheral blood mononuclear cells (PBMCs) were isolated over a Histopaque gradient. On the following day, the vaccinated horses were challenged intravenously with a reference virulent EIAVPV strain, using a low-dose, multiple-exposure challenge to mimic the predominant natural route of EIAV infection by horsefly bites (Li et al., 2003).
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Production of synthetic peptides.
Eighty-five 20-mer peptides overlapping by 10 residues, representing the entire EIAVUK (GenBank accession no. AF016316
[GenBank]
) Env, were synthesized in the Biomedical Research Support Facilities Peptide Synthesis Core of the University of Pittsburgh, using an Advanced Chemtech model 396 Omega synthesizer. All peptides were HPLC-purified and confirmed by mass spectrometry. The peptides were dissolved at 2 mg ml–1 in 100 % DMSO and stored at –80 °C until further use. Pools of nine to ten peptides were constructed in a standard matrix such that each peptide was represented in two pools. The pools were used to stimulate PBMCs from the vaccinated and control horses in lymphoproliferation assays and to pulse target cells for chromium-release assays. The design of the matrix was such that reactive peptides were indicated by a proliferative or a cytolytic response to the two distinct pools containing the peptide. Reactive peptides were then assayed individually to determine their reactivity in the separate assays (Roederer & Koup, 2003).
Lymphoproliferation assays.
PBMCs from the 12 vaccinated, two infected and five uninfected horses were tested for recognition of EIAV Env peptides in a standard 7 day thymidine-incorporation assay, as described previously (Fraser et al., 2002) with minor modifications. Cryopreserved PBMCs from the vaccinated, challenged and naïve horses were >80 % viable upon thawing and maintained viability throughout the assays, as measured by trypan blue exclusion. PBMCs, at 2x105 cells per well, were assayed in six replicates in complete RPMI 1640 medium (10 % fetal equine serum, 1 % penicillin/streptomycin, 1 % L-glutamine, 55 µM 
-mercaptoethanol). In initial experiments, we observed relatively high lymphoproliferation backgrounds in PBMCs in the absence of peptide stimulation, apparently resulting from in vitro amplification of vaccine or challenge virus infection in the cultured cells. The addition of AZT (5 µM; Sigma) to the PBMCs upon plating, and its replenishment every 2 days, abrogated virus production and eliminated the observed background lymphoproliferation (data not shown). Cell-viability assays with trypan blue exclusion and MTT (Roche) staining indicated there were no deleterious effects of AZT on PBMC viability. Pokeweed mitogen (PWM) and acetone-extracted EIAV (AE-EIAV) were used as positive proliferative controls throughout the assays at 2.5 and 10 µg ml–1, respectively. The peptide pools were used at 20 µg ml–1 and individual peptides were used at 10 µg ml–1. DMSO was added to the medium-control wells to match the DMSO concentration in the peptide wells. The PBMCs were incubated with PWM for 48 h, AE-EIAV for 4 days and the peptides for 6 days prior to labelling with 0.75 µCi (27.75 kBq) [3H]thymidine (Amersham Biosciences). The cells were incubated for an additional 16–18 h with [3H]thymidine before being harvested and quantified for 3H incorporation by liquid scintillation counting. Stimulation indices (SI) were calculated by dividing the mean c.p.m. of stimulated cells by the mean c.p.m. of non-stimulated cells. A positive response to the peptide pools was an SI >1. A positive response to the individual peptides was set at at least 2x the naïve PBMC stimulation, SI >2.5. Reactive peptides were defined as having >2.5 SI in >50 % of the horses and a P value <0.05 in the Wilcoxon ranked sums test.
MHC-blocking assay.
To determine the MHC specificity of the observed peptide-specific lymphoproliferation, a blocking assay was performed by using anti-equine MHC I (H58A) and MHC II (EqT2) antibodies (VMRD). Preliminary blocking assays showed the optimal antibody concentration for MHC I and MHC II to be 10 µg ml–1. The optimal concentration for the IgG control was 5 µg ml–1. The antibodies were incubated with PBMCs (2x106 cells ml–1) for 1 h at 37 °C, 5 % CO2, before stimulation with peptides at 20 µg ml–1. After 1 h peptide stimulation in the presence of antibody, the cells were washed three times with 1x PBS/1 % horse serum (HS) and resuspended in 200 µl RPMI complete medium. A standard 7 day thymidine-incorporation assay was then performed as described above. Proliferation was considered to be MHC II-restricted when the SI of the MHC II antibody-treated PBMCs was decreased by >50 % compared with the SI of PBMCs treated with IgG or MHC I antibodies. As MHC II molecules present peptides to CD4 Th cells, it can be inferred that MHC II-restricted peptides cause Th proliferation.
CTL assays.
Standard chromium-release assays previously employed for the identification of EIAV CTL epitopes in Gag and Pol were used for these studies, with minor modifications (Hammond et al., 1998; McGuire et al., 1994, 1997; Ridgely et al., 2003; Zhang et al., 1998). PBMCs from the vaccinated or infected horses were expanded in vivo for 7–10 days with either 2.5 µg PWM ml–1 or 10 µg gradient-purified EIAV ml–1 in complete RPMI medium supplemented with 20 U recombinant human interleukin-2 (Hoffmann–La Roche Inc.). After expansion, the PWM-stimulated cells were labelled with 100 µCi (3.7 MBq) 51Cr (Na51CrO) (MP) for 1 h at 37 °C, 5 % CO2. Cells were washed four times with 1x PBS/1 % HS and 30 000 cells per well were plated prior to peptide pulsing. Peptide pools were used at a final concentration of 20 µg ml–1 and individual peptides were used at a final concentration of 10 µg ml–1. The target cells were pulsed for 1 h at 37 °C, 5 % CO2. EIAV-stimulated cells were used as effector cells at a 20 : 1 effector : target cell ratio. After the effector cell addition, the cells were incubated for 12–16 h prior to being harvested. Following this incubation, 25 µl cell supernatant was added to 175 µl OptiPhase SuperMix scintillation fluid (Perkin Elmer) and analysed for 51Cr release with a MicroBeta reader (Perkin Elmer). Maximum 51Cr release was determined by plating 51Cr-labelled target cells with the non-ionic detergent Nonidet P-40 to lyse cells. Background spontaneous lysis was determined by plating 51Cr-labelled target cells with 0.1 ml medium alone. Percentage specific lysis was calculated as follows: (51Cr release in peptide wells– spontaneous 51Cr release)x100/(51Cr release by NP-40–spontaneous 51Cr release)=specific lysis (%). Reactive peptides were defined as causing >10 % specific lysis in >50 % of the reactive horses and to have a P value of <0.05 in the Mann–Whitney test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Before the horses were challenged with the reference virulent EIAVPV strain, whole blood was drawn and PBMCs were isolated for use in cellular immune assays. All 12 horses were protected from virulent virus challenge, as evidenced by lack of equine infectious anaemia signs and detectable challenge virus in plasma. Two naïve horses were infected experimentally with EIAVPV to serve as infection and virulence controls for the EIAVPV challenge. PBMCs from these EIAVPV-infected horses were isolated after at least 6 months post-infection, when mature EIAV Env-specific immune responses were documented. PBMCs from five naïve horses were included in the lymphoproliferation assays to establish appropriate background levels.
Proliferation in response to the synthetic peptide pools
Preliminary proliferation assays were performed by using PWM or AE-EIAV as stimulants in a standard thymidine-incorporation lymphoproliferation assay. All of the horses responded well to PWM, with SI values of >50 (Table 1
). In response to AE-EIAV, all horses displayed a positive proliferation with SI values ranging from 3 to 140, presumably reflecting the variation in virus-specific immune responses among this outbred population of horses (Table 1). Proliferation in response to PWM and AE-EIAV demonstrated that the PBMCs maintained proliferative capabilities following cryopreservation and thawing. Based on these preliminary observations, PWM and AE-EIAV were used as positive controls for lymphoproliferation in parallel assays to the peptide stimulations.
An inclusive scan of all potential Th peptides was conducted by using a peptide-matrix strategy. Eighty-five peptides (20-mers overlapping by 10 aa), encompassing the EIAVUK Env proteins gp90 (SU) and gp45 (TM), were synthesized and used in pools of nine to ten peptides (Fig. 1a) to stimulate PBMCs from the vaccinated and infected horses in a standard thymidine-incorporation assay and also to label target cells for the chromium-release assay. Fig. 1(b) is a representative graph of the matrix results for the lymphoproliferation assay. An SI of >1.0 was used as the cutoff for determining positive proliferation. Two independent assays were conducted on PBMCs from each horse for a total of 28 assays. If a peptide was identified as positive (SI >1) in eight of the 28 assays (approx. 30 %), the peptide was subsequently tested individually to determine peptide-specific proliferative properties (Table 2). Based on these criteria, 21 peptides were identified and tested individually (Table 2). An additional five peptides were also chosen (23, 60, 73, 74 and 77) because the corresponding peptide pools produced high SI values in PBMCs from several horses, but not in the minimum of eight horses.
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0.9. We defined a reactive peptide as (i) having an SI of >2.5 (greater than two times the background) in seven of the 14 horses and (ii) having a P value of <0.05 in the Wilcoxon ranked sums test.
Overall, the PBMCs from the vaccinated and infected horses proliferated in response to a variety of peptides (Fig. 2). Of the 26 tested peptides, 23 peptides were positive for proliferation in seven of the 14 horses (Fig. 3a). Only peptides 17, 48 and 58 did not cause proliferation in 50 % of the horses and were therefore not considered reactive. For the 23 peptides that were positive for proliferation in 
50 % of the horses, the Wilcoxon ranked sums test was used to establish the statistical significance of the SI values. This test takes into consideration the number of positive events and also the magnitude of stimulation. Ranks 1, 2 and 3 were defined as SI values of 2.5–5, 5–7.5 and >7.5, respectively (Fig. 2). All 23 peptides that caused stimulation in seven of the 14 horses also had P values of <0.05 and were thus considered to be reactive (Fig. 3a).
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). Five of the peptides demonstrated a pattern that indicated that both CD4 and CD8 cells were proliferating in response to those peptides. Peptides 7, 60, 66, 68 and 77 all decreased in SI when MHC I or MHC II antibodies were used. Further studies are needed to verify the results. Peptides 11, 19, 76 and 83 failed to produce significant stimulation in this assay; therefore, we were unable to determine the CD4/CD8 restriction of these peptides (Fig. 3b).
Cytolytic response to the synthetic peptide pools
Preliminary CTL assays were conducted to determine the cytolytic capabilities of the PBMCs from the panel of vaccinated and infected horses against a pool of sequential Env 20-mer peptides representing the entire gp90 protein sequence. PBMCs from the 12 vaccinated horses displayed cytolytic activity to the Env peptide pool, with specific lysis values ranging from 0 to 30 % (Table 1). The PBMC samples from the two infected horses failed to display significant CTL activity against the Env peptide pool at the 6 month post-infection time point.
To identify reactive CTL peptides, the Env peptide-pool matrix was again utilized to screen CTL activity of PBMCs from four of the vaccinated horses. Fig. 1(c) is a representative graph of the matrix results for the CTL assay. The cutoff for a positive cytolytic response was set at 10 % specific lysis and each peptide had to cause lysis in all four assays to be tested individually. With these criteria, 20 peptides were chosen for further analysis as individual peptides (Table 2). Additionally, peptides 50, 52 and 54 were chosen because they caused high specific lysis in three of the four CTL assays.
Peptide-specific CTL responses
To determine which peptides were recognized by CTL cells, the 23 peptides identified with the peptide matrix were tested individually in chromium-release assays (Fig. 4). Of the 12 vaccinated-horse PBMC samples, eight had clear CTL activity against individual envelope peptides. PBMCs from horses C15, 266 and C9 did not have detectable CTLs to the individual peptides at the day of challenge time point and were therefore not used to determine which peptides were reactive. Due to insufficient B61 PBMCs, this horse was also excluded from determining reactive peptides. Specific lysis of PBMCs from untreated and non-reactive horses was typically <2.0 %, so a 10 % specific lysis cutoff was set for determining which peptides were positive.
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). The remaining 16 peptides had <5 % specific lysis in this cohort of horses. The Mann–Whitney test was used to determine the significance of the specific lysis in the responder horses compared with non-responder horses. Peptides 5, 9, 17, 23, 27, 50 and 52 had P values of <0.05 (Fig. 5) and were subsequently considered to be reactive. Although peptide 28 did have a P value of <0.05, it was not considered reactive because only three horses responded to that peptide.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Fraser et al., 2002, 2005; Mealey et al., 2003, 2005).
For a peptide to be considered broadly reactive, the peptide must be presented by the majority of expressed MHC alleles. Two MHC II loci, which present peptides to Th cells, were typed in this study, and nine of the 11 typed horses had distinct DRA/DQA combinations. C9 and C16 both shared DRA*101/DRA*301 and DQA*301/DQA*1301, but still differed in their proliferative response to the individual peptides. Therefore, it can be reasoned that they differ at another MHC II locus. Allelic restriction at the less polymorphic DRA allele is not evident with the 23 reactive peptides. At the more polymorphic DQA allele, seven different haplotypes were observed. The dominant allele of the horses typed was DQA*301, with six of the 11 horses expressing that allele. None of the 23 reactive peptides appear to be DQA*301-restricted, as horses with and without DQA*301 reacted to the same peptides. Peptides 29, 68 and 74 appear not to be presented by DQA*301, but seem to be widely presented by the remaining alleles. The next dominant allele was DQA*1301, which was expressed in four of the 11 horses typed. Peptides 76 and 83 are the only two peptides excluded from Th recognition in animals expressing DQA*1301. On closer examination, it is found that only horses with DQA*301, 401 and 501 can recognize peptide 76 and horses expressing DQA*101, 401 and 701 can recognize peptide 83. With fewer than half the expressed DQA alleles recognizing these two peptides, they are not broadly reactive. Whilst peptides 79 and 81 are not presented by the dominant DQA*301 and DQA*1301 alleles, they are considered to be broadly reactive because they seem to be presented by the remaining five alleles. Peptides 7, 9, 11, 19, 23, 25, 27, 35, 60, 66, 70 and 77 appear to be presented by four or more (>50 %) of the DQA alleles and are thereby broadly reactive. Of the 23 identified reactive peptides, it appears that 17 are broadly reactive across a majority of DQA haplotypes (Fig. 6).
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). Additionally, there are problems with serological typing, such as allele mismatching. It was reported that target cells from one horse with an ELA-A5 haplotype were not lysed by memory CTLs from another horse with an ELA-A5 haplotype, indicating that the serological reagents used for haplotyping can recognize more alleles non-specifically than thought originally (Zhang et al., 1998). Until a more discriminating MHC I typing procedure is developed, we cannot definitively assign ELA-A allelic restriction to the defined peptides. It does appear that peptide 50 may not be broadly reactive, in that only one horse without the A2 allele recognizes that peptide. The remaining six peptides are recognized by a range of diverse ELA-A types and can therefore be considered broadly reactive (Fig. 6).
Of the 17 broadly reactive Th peptides, nine were located in the gp90 protein and eight in the gp45 protein. gp90, which is the more variable portion of Env, has eight defined variable domains that are assumed to occupy the outer exposed loops on a three-dimensional structure of the protein, like the HIV-1 gp120 protein (Leroux et al., 1997). Of the nine broadly reactive Th peptides from the gp90 sequence, four are located in these variable domains (Fig. 6a). Peptide 19 is in V3, peptide 23 is in V4 and peptides 27 and 29 are both in V5. Only two of the five CTL peptides in this protein are contained in variable domains; peptide 23 is located in the V4 domain and peptide 27 in the V5 domain. The principal neutralization domain (PND), along with other major neutralization-antibody determinants (V4 and V5), are located in the variable loops of gp90. These antibody determinants have considerable overlap with Th and/or CTL peptides. The remaining Th and CTL peptides are in more conserved regions of gp90 that are shielded by the variable loops and are perceived to be immunorecessive.
There are no predetermined variable domains in the gp45 protein, but it is accepted that the cytoplasmic tail is more conserved than the ectodomain. Only one Th peptide (peptide 60) is found in the ectodomain, along with the only CTL peptide of this protein (peptide 52). The remaining seven Th peptides are found in the putative intracytoplasmic tail (Fig. 6b).
Lentiviral Env proteins are some of the most heavily glycosylated proteins characterized to date. The role of N-linked glycosylation in antigen presentation has yet to be established. The EIAV gp90 protein has 18 predicted N-linked glycosylation sites, yet the majority of the identified broadly reactive Th and CTL peptides lack potential glycosylation sites. Additionally, gp45 contains four potential N-linked glycosylation sites and none of them are included in the identified cellular peptides. These data are consistent with the concept that lentiviruses sequester glycosylated Env sequences from cellular recognition, as observed for the SIV Env protein (Sarkar et al., 2002).
With the current characterization of Th and CTL peptides of EIAV gp90 and gp45 associated with protective immunity, we now have the necessary foundation to examine the evolution of these cellular immune responses during maturation to protective immunity and to evaluate these peptides as correlates of protective vaccine immunity.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 17 July 2006;
accepted 16 December 2006.
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P value was determined by using the Wilcoxon ranked sums test. 
Reactive peptides were defined by two criteria: (i) a peptide causing proliferation in seven of the 14 horses tested and (ii) the peptide having a P value of <0.05 in the Wilcoxon ranked sums test. (b) MHC-blocking assay. PBMCs were blocked with MHC I, MHC II or IgG antibody prior to stimulation with the broadly reactive peptides. A decrease in stimulation of >50 % compared with the SI of PBMCs treated with IgG or MHC I antibody is considered significant. CD4-restricted peptides are underlined.
