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The Journal of Immunology, 2002, 168: 4001-4011.
Copyright © 2002 by The American Association of Immunologists

Detailed Analysis of CD4+ Th Responses to Envelope and Gag Proteins of Simian Immunodeficiency Virus Reveals an Exclusion of Broadly Reactive Th Epitopes from the Glycosylated Regions of Envelope1

Surojit Sarkar*, Vandana Kalia{dagger}, Michael Murphey-Corb*,{dagger} and Ronald C. Montelaro2,*,{dagger}

* Department of Infectious Diseases and Microbiology, University of Pittsburgh Graduate School of Public Health, and {dagger} Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ag-specific CD4+ Th cells play a key role in the development, maturation, and maintenance of pathogen-specific humoral and cellular immune responses. To define the fine specificity of broadly reactive Th responses associated with mature immunity in a lentiviral system, we analyzed peptide-specific Th responses in eight macaques chronically infected with a reference live attenuated SIV at 12–14 mo postinoculation. All macaques had stable immunocompetent Th cells at the time of analysis, and a unique array of Th responses to 20-mer overlapping peptides from envelope (Env) and Gag was identified for each macaque, which were then used to define a set of 31 broadly reactive peptide epitopes. Only 5 of the 31 broadly reactive Th epitope peptides mapped to the surface (SU) domain of Env. Interestingly, these were all confined to two conserved nonglycosylated regions toward the carboxyl terminus of SU, suggesting a structural influence of glycosylation on development of Th responses. Gag and the Env transmembrane proteins contained the majority of broadly reactive peptide epitopes (12 and 14 peptides, respectively), which were uniformly distributed throughout their sequence. This study defines for the first time broadly reactive Th epitope peptides of SIV Env and Gag proteins that are associated with enduring broadly protective vaccine immunity to attenuated SIV, which may be used for the design and evaluation of experimental vaccines. Moreover, the data suggest that extensive glycosylation of SU may provide yet another immune escape mechanism developed by lentiviruses to restrict the breadth of Th repertoire to SU, a major immunologically exposed protein of the virus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Correlates of protective immunity against HIV-1 remain uncertain, making the development of a vaccine against HIV-1 a formidable task. The importance of CTLs (1, 2) in effective control of primate lentiviral infections and the role of neutralizing Ab responses (3, 4) in protection against i.v. and mucosal challenge with SIV or chimeric SIV/HIV viruses is well established. Thus, there is growing consensus that both humoral and cellular immunity may be critical to the success of a vaccine against HIV-1 (5).

Pivotal to both humoral and CTL responses is the role of CD4+ Th responses. While CD4+ Th responses are known to be essential for the maintenance of effective immunity in several chronic viral infections (6, 7), their importance in SIV and HIV-1 infections is becoming increasingly evident (8, 9, 10). Until recently, few studies detected HIV-1-specific CD4+ T cell proliferative responses, as initial attempts to detect virus-specific proliferative responses were made in patients with progressive disease when the cellular responses are significantly impaired (11, 12, 13). However, recent studies with long-term nonprogressors have shown that vigorous virus-specific CD4+ T cell responses correlate with long-term control of HIV-1 replication (14). A clear inverse correlation between virus-specific Th responses and plasma HIV-1 viremia has been established in untreated patients with chronic HIV-1 infection (15). Structured treatment interruption studies in HIV-1-infected patients (15, 16, 17) also support the functional significance of persistent, strong HIV-1-specific Th responses in HIV-1 infection. HIV-1 variation in the CD4+ T cell epitopes has also been shown to result in diminished CD4+ T cell recognition (18), implicating an important functional role of virus-specific CD4+ T cells. In the SIV/macaque model, induction of strong type 1 Th responses is observed upon immunization with live attenuated SIV (19), and virus-specific CD4+ T cell responses are also observed in DNA-vaccinated macaques protected from clinical AIDS (9, 10). The functional relevance of virus-specific CD4+ T cells in control of viral replication after structured treatment interruption has also been shown recently in the SIV/macaque model (20, 21, 22).

Functionally, CD4+ T cells are central to the development, coordination, and regulation of both Ab and CD8+ T cell responses to viral infections (23, 24). For example, CD4+ T cell loss is associated with waning anti-HIV-1 Ab responses (25), and the role of Th cells in priming of HIV-1-specific CTL responses is also well documented (26). Furthermore, in HIV-1 infection, a direct association between HIV-1 specific memory CTL precursor frequencies and Gag-specific proliferative responses is observed (27). Thus, CD4+ T cells occupy a critical position in the generation and maintenance of antiviral immunity and have been clearly shown to play a crucial role in control of HIV-1 and SIV replication. However, our knowledge of the precise epitopes of the different SIV proteins targeted by CD4+ T cells is limited. Furthermore, the significance of these responses in SIV infection is not clearlyunderstood. Thus, functional characterization, fine specificity analysis, and quantification of virus-specific CD4+ T cells are critical to defining their role in antivirus immune mechanisms.

Using whole viral proteins as immunogens in lymphoproliferation assays, it has been shown that Th responses to SIV (28) and HIV-1 (29) surface (SU) are considerably lower than to Gag and other nonglycosylated viral proteins. From the perspective of immunogenicity, glycosylation on SU has been shown to restrict both Ab (30) and CTL responses (31). Furthermore, studies with recombinant point-deglycosylated HIV gp120 immunogens (32) suggest that N-linked carbohydrates within an epitope can limit Ag recognition by T cells. Using HIV subunit gp120 prime-boost immunization in mouse models, structural influences have been suggested to affect localization of CD4 Th epitope hot spots in the envelope (33). Although this system provides for studying an exhaustive gp120-specific Th repertoire in a specific MHC class II (MHC-II)3 background, the breadth and actual specificity of Th responses associated with protective immunity in a heterogeneous population cannot be addressed in this mouse model. The current study was designed to better understand the epitope-specific CD4+ Th cell repertoire associated with mature immunity in the SIV/macaque model to elucidate structural influences of SU on Th responses in comparison with other viral proteins.

Attenuated SIV vaccines have, to date, been uniquely successful in generating potent, enduring, broadly protective immunity against challenge with pathogenic strains of SIV (34, 35). Immunization with live attenuated SIV generates neutralizing Abs (35), CTL responses (36), type-1 Th responses, and {beta}-chemokine production (19). Moreover, the attenuated SIV model system is suitable for detailed analyses of Th responses as it presents an intact CD4+ Th repertoire with no significant T cell depletion or unresponsiveness being induced. However, the fine specificity of Th repertoire associated with broad and enduring protection, which is important for the design and evaluation of vaccine strategies, is not known. This study is the first comprehensive report of both envelope (Env) and Gag peptide-specific Th proliferative responses at a single CD4+ T cell level in monkeys with an intact Th cell repertoire associated with mature vaccine immunity elicited by a reference attenuated SIV vaccine (37). This fine specificity analysis reveals a lack of broadly reactive CD4+ Th responses in glycosylated regions of Env and is suggestive of a potential Th cell immune evasion mechanism developed by lentiviruses. Furthermore, immunization with conserved Th epitopes derived from nonglycosylated regions of Env, which are associated with protective immunity, may be central to inducing appropriate humoral and cellular immune responses for broad and enduring protection against lentiviruses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Immunization of animals

Eight adult rhesus macaques were inoculated with attenuated SIV 17E-Fred, a macrophage-tropic infectious molecular clone previously described (37) and characterized (38) in the rhesus macaque system. All procedures for inoculation and monitoring of immunologic and virologic parameters have been described previously (34). We have previously reported that inoculation of macaques with this attenuated SIV strain produces enduring broadly protective immunity by ~8 mo postinoculation (34). In agreement with these previous observations, experimental challenge with pathogenic SIVB67O (34) of the current eight macaques inoculated with attenuated SIV-17E-Fred strain demonstrated protection from immunodeficiency disease and apparent prevention of detectable challenge virus in plasma by highly sensitive real-time RT-PCR (M. Murphey-Corb, unpublished data). Thus, the eight monkeys used in these studies have achieved vaccine-induced mature immune responses associated with enduring, broadly protective immunity to pathogenic SIV exposure. For comparison, three naive SIV-negative rhesus macaques were used as a source of control PBMC samples.

Peptides for proliferation assays

Panels of 20-mer peptides with 10-aa overlap, representing the entire protein sequence of SIVmac239 (GenBank accession no. AAA47637), Env (SU and transmembrane (TM); 87 overlapping peptides: 50 from SU and 37 from TM), and SIVsmmH4 (GenBank accession no. CAA32483) Gag (50 peptides), were provided by National Institutes of Health AIDS Research and Reference Reagent Program. Peptides were dissolved at a concentration of 10 mg/ml as per the manufacturer (Anaspec, San Jose, CA). Single peptides diluted from this stock were used in the proliferation assays.

Proliferation assays using CFSE and Ki-67

Isolation of PBMCs. PBMCs were isolated from fresh heparinized blood by density gradient centrifugation (Ficoll 1077; Sigma-Aldrich, St. Louis, MO) and resuspended in AIM V medium (Life Technologies, Carlsbad, CA) supplemented with 10% human AB serum (Gemini Biologicals, Woodland, CA). About 25% of the total PBMCs were frozen in 90% FCS plus 10% DMSO for later use as APCs.

Resting PBMCs in culture. The remaining PBMCs were cultured for 24–48 h to minimize nonspecific proliferation. The activation status of CD4+ T cells was determined by comparing CD69 and Ki-67 expression immediately after isolation and following 24–48 h rest in culture.

CFSE staining. The frozen APCs were thawed and mixed with the rested PBMCs and resuspended at 2 x 106 cells/ml. CFSE staining was performed as described previously (39).

Stimulation with individual peptides. Approximately 200,000–300,000 CFSE-stained cells were stimulated with a single 20-mer peptide (20 µg/ml). Each peptide was tested for inducing background proliferation in PBMCs from three naive animals. A 20-mer peptide derived from the env of equine infectious anemia virus was used as negative control in triplicate. Acetone-extracted SIVB7 viral protein lysate (40) was used as positive whole virus Ag control and staphylococcal enterotoxin B (SEB) was used as positive mitogen control. Stimulated PBMCs were cultured in a CO2 incubator at 37°C for 5 days.

Immunofluorescent staining and analysis of proliferation by flow cytometry. Cells were stained with CD4 allophycocyanin (BD Biosciences, San Jose, CA), CD8 PerCP (BD Biosciences), and Ki-67 PE (DAKO, Carpinteria, CA) for monitoring CD4+ Th cell proliferation by flow cytometry as described previously (41). A minimum of 50,000 live, CD4-positive, and CD8-negative gated events were acquired on a FACSCalibur (BD Biosciences) flow cytometer and analyzed using FlowJo batch analysis software (Treestar, San Carlos, CA) for CFSE and Ki-67. Gates for proliferation were determined using isotype control IgG PE in case of Ki-67 and using cells cultured in medium alone for CFSE. Proliferation was expressed as the percentage of total CD4+ T cells staining positive for proliferation markers. The proliferation induced by medium control was subtracted from test samples to determine proliferation induced by each peptide. To account for variations in basal T cell activation states of each animal, individual cutoff values (background) for each animal were established as two times their respective negative controls (run in triplicate). Peptides were classified as responders when they stimulated CD4+ T cell proliferation to levels greater than the defined background proliferation value for each monkey.

MHC-II blocking assay

A preliminary MHC-II blocking assay was done with 10 responder peptides using three dilutions (1/50, 1/100, and 1/200) of anti-MHC-II, -MHC class I (MHC-I), and isotype IgG Abs to determine the effective blocking Ab concentration for the assay (representative data for one responder peptide is shown in Fig. 8). A dilution of 1/100 for all the Abs was subsequently chosen to test MHC-II restriction of all the responder peptides. Briefly, anti-MHC-II Ab (1/100) was added to cell culture and incubated at 37°C for 2 h before stimulation of the cells with defined responder peptides, followed by proliferation assays, which were conducted as described above. Anti-MHC-I and isotype IgG control Abs were used as negative controls. Proliferation induced by a peptide was considered MHC-II restricted when anti-MHC-II Ab inhibited proliferation by >50% compared with control Abs.

MHC-II background of the experimental rhesus macaques

Highly polymorphic exon 2 of MHC-II DRB subregion was PCR amplified from genomic DNA of eight immunized and three unimmunized macaques as previously described (42). The PCR product was denatured at 94°C for 3 min and immediately cooled on ice. The resulting conformationally polymorphic single strand products were resolved using a 20% polyacrylamide gel. Bands were then visualized by standard silver staining.

Selection of broadly reactive Th epitopes

Peptides were classified as nonresponders, low responders, or high responders based on fold proliferation induced above cutoff and a numerical score of 0 (<1-fold proliferation above cutoff), 1 (1- to 2-fold above cutoff), or 2 (>2-fold above cutoff) was assigned to each peptide. Each peptide was then compared with the negative control group across all eight immunized macaques using a paired two-tailed Student’s t test, and responder peptides with p < 0.05 were classified as broadly reactive Th epitope peptides.

Amphipathicity calculations

Windows NT version of Antheprot 2000 v 5.0 release 1.0.5 was downloaded from ftp://ftp.ibcp.fr/pub/ANTHEPROT/WINDOWS and used to perform amphipathicity calculations for env and gag sequences in an {alpha}-helical structural context with a window size setting of seven amino acids. Helical hydrophobic moment values (43) at each amino acid residue were plotted, with the hydrophobic moment being normalized for local hydrophobicity.

Amino acid conservation determination

Fourteen SIV Env and Gag protein sequences were downloaded from the Los Alamos Immunology Database and were aligned with SIVmac239 sequence using Macintosh (Apple Computer, Cupertino, CA) version of Clustal (MacVector 6.0.1, Oxford Molecular, Oxford, U.K.). The GenBank accession numbers of the reference SIV sequences that were used for alignment and percent conservation calculations are as follows: M33262, D01065, L22809, M19499, M16403, M32741, X14307, U04982, L03295, L20008, M31325, L09212, M90048, M76764, and M83293. Alignment parameters included gap-opening penalty of 10 and a gap extension penalty of 0.10, and protein weight matrix Gonnet 250 was used.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Evaluation of CD4+ Th cell proliferation assays using CFSE and Ki-67

Mitogen-induced proliferation, as measured by CFSE and Ki-67 markers, has been shown to be comparable in PBMCs from both sooty mangabeys and rhesus macaques (41). However, measurement of peptide- or whole Ag-induced proliferation with these markers in rhesus PBMCs has not been demonstrated. To validate a flow cytometry-based assay system for the measurement of peptide Ag-induced CD4+ T cell proliferation in macaque PBMCs, we first compared CFSE and Ki-67 as markers of proliferation. Fig. 1Go demonstrates proliferative responses of PBMCs from monkey M0798 to responder peptide 5 from the SU panel, as measured by CFSE and Ki-67. Background proliferation stimulated by the negative control peptide was only minimally higher than the medium control for all experimental macaques. However, under similar conditions, PBMCs that were not rested before CFSE staining and stimulation showed considerably higher (~7–10%) backgrounds in medium and negative controls (data not shown). These observations may be explained by a higher percentage of background proliferation in these animals. Chakrabarti et al. (41) previously reported that ~12% of circulating CD4+ T cells are in the proliferative cycle in SIV-infected macaques as compared with 1–2% in uninfected animals. Thus, resting of the PBMCs in culture, in the absence of any antigenic stimulation, was used to reduce the activation of CD4+ T cells to basal levels and thereby minimize backgrounds in subsequent proliferation assays. As shown in Fig. 1Go, the percentage of live CD4+ Th cells undergoing proliferation, as measured by CFSE and Ki-67, were very similar in both mitogen- (~50%) and peptide (~15%) Ag-stimulated PBMCs. Thus, these data validated the use of CFSE or Ki-67 as equally sensitive markers in macaque PBMCs for the detection of peptide-induced CD4+ Th cell proliferation. These markers were then used in the next set of experiments for the detailed fine specificity analyses of CD4+ Th responses in PBMCs derived from attenuated SIV-inoculated rhesus macaques.



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  		FIGURE 1. Comparison of CFSE and Ki-67 as markers of peptide Ag-induced CD4+ T cell proliferation. PBMCs derived from monkey M0798 were rested in vitro for 2 days and stimulated for 5 days with 20 µg/ml of a representative responder peptide (peptide 5) from the SU peptide panel. A 20-mer peptide derived from the Env glycoprotein of equine infectious anemia virus served as a negative control for background proliferation along with medium control. Mitogen SEB was used as a positive control. Cells were stained with CD4 allophycocyanin, CD8 PerCP and CFSE, or Ki-67 PE for monitoring CD4+ Th cell proliferation by flow cytometry. CD4+ cells were gated from the live lymphocyte subset for proliferation analysis. The gates for proliferation were drawn using the isotype control IgG PE in case of Ki-67 PE. The proliferation gate for CFSE was drawn on cells cultured in medium alone and was further adjusted with mitogen-stimulated cells. Proliferation was expressed as a percentage of gated CD4+ T cells staining positive for proliferation markers.

 
Immunocompetence of CD4+ T cells from macaques immunized with live attenuated SIV to respond to mitogenic and viral antigenic stimuli

An important prerequisite for performing a fine specificity analysis is the presence of an intact and appropriate Ag-specific CD4+ Th cell repertoire. Thus, an attenuated SIV/macaque model system was used for this purpose. We have previously demonstrated that the development of mature protective immunity in monkeys inoculated with SIV 17E-Fred requires a complex evolution of immune responses over 8–10 mo postinoculation (44). Thus, the fine specificity analysis of CD4+ Th cells was performed at 12–14 mo postinoculation with attenuated SIV. At the time of sampling, all inoculated monkeys had <100 copies of viral RNA per milliliter of plasma and stable CD4 T lymphocyte counts representing 40–60% of the total lymphocytes of each animal (data not shown). Because all the animals were healthy with stable CD4+ T cell levels, the SIV Ag-specific Th cell repertoire in these animals was considered intact for fine specificity analysis studies. The appropriateness of the Th cell immune repertoire in terms of functionality was further studied. We thus evaluated the general immunocompetence of CD4+ T cells from inoculated animals and compared their ability to respond to mitogenic and antigenic stimuli induced by SEB and SIVB7 whole viral protein lysate (AE-B7) (Table IGo) with that of CD4+ T cells from unimmunized macaques. Upon stimulation with SEB for 48 h, 37–56% CD4+ T cells from both inoculated and uninfected macaques entered the proliferative cycle as indicated by Ki-67 expression. This demonstrated that the CD4+ T cells from these animals were immunocompetent and were capable of proliferating in response to stimuli induced through MHC-TCR interaction. AE-B7 induced 2–23% proliferation in CD4+ T cells from inoculated animals, thereby demonstrating the generation of Ag-specific CD4+ Th responses in the inoculated animals. The wide range in the Ag-specific in vitro proliferative responses observed is indicative of the diverse genetic backgrounds of the macaques used in this study. The PBMCs from three naive animals showed Ag-induced background proliferative responses of <0.5%. Thus, these proliferative data demonstrated a general immunocompetence of CD4+ cells in the panel of monkeys inoculated with the attenuated SIV, which were then used for the Th peptide epitope mapping analyses.


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  		Table I. Proliferative capacity of CD4+ T cells isolated from experimental macaques1

 
SU, TM, and Gag peptide epitope-specific CD4+ T cell proliferative responses associated with mature immunity in macaques immunized with live attenuated SIV

A detailed Th fine specificity analysis was performed using a comprehensive panel of Env and Gag peptides to identify responder peptides for each of the eight inoculated macaques, as described in Materials and Methods. Detailed peptide-specific lymphoproliferation data for all the peptides from individual monkeys are shown in Figs. 2Go, 3Go, and 4Go. None of the peptides from Env and Gag panels stimulated a proliferative response above background in PBMCs from three naive control animals (data not shown). As expected with an outbred population, there were marked variations between monkeys in terms of proliferative responses to individual SIV peptides. The observed differences in response were both qualitative (specificity) and quantitative (intensity). Apart from differences in peptide specificity, qualitative differences also reflected differences in the breadth of the fine specific CD4+ Th responses generated by the individual animals. It is noteworthy that the differences in the quality and quantity of Th responses were not reflective of the circulating levels of viral Ag in these monkeys, as all the animals had similar viral loads during this study. These variations reflect the highly heterogeneous genetic backgrounds of these monkeys. The MHC-II heterogeneity of the eight experimentally immunized and three naive animals was confirmed by PCR-single strand conformational polymorphism analysis (42) of the macaque MHC-II DRB gene (data not shown). These data defined a set of Th epitope responder peptides from the Env and Gag proteins of the virus capable of stimulating proliferative responses in CD4+ cells derived from our experimental monkeys.



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  		FIGURE 2. Th epitope mapping of the SU domain of Env. CD4+ T cell proliferative responses were evaluated individually in response to stimulation with 20 µg/ml of 50 overlapping 20-mer peptide Ags spanning the length of SU. Each panel represents proliferative responses of individual immunized macaques. The percentage of CD4+ T cells proliferating in response to negative control peptide is shown as the first filled bar in each panel. The horizontal dashed line represents CD4+ T cell proliferation value that is 2-fold greater than that observed with negative control peptide for each experimental macaque. MHC-II-restricted CD4+ T cell responder peptides are shown as filled bars. Peptides that were not able to induce any proliferation above the cutoff value were classified as nonresponders (shown by the open bars) for that experimental macaque. Gray bars indicate peptides that stimulated MHC-II-unrestricted proliferative responses.

 


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  		FIGURE 3. Th epitope mapping of the TM domain of Env. A panel of 37 overlapping 20-mer peptides representing the full-length of TM domain of SIVmac239 Env was used to stimulate PBMCs from eight macaques immunized with attenuated SIV. Peptides were used at a concentration of 20 µg/ml and CD4+ T cell proliferation was assessed following stimulation for 5 days. As described in Fig. 2Go, MHC-II-restricted responder peptides are indicated by filled bars, nonresponder peptides by open bars, and MHC-II-unrestricted responder peptides by gray bars.

 


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  		FIGURE 4. Th epitope mapping of SIVmac239 Gag protein. Th epitope responder peptides in Gag were determined for eight immunized macaques following stimulation of PBMCs individually with 20 µg/ml of 50 overlapping 20-mer peptides from Gag panel. The shading scheme of the bars is same as used in Figs. 2Go and 3Go.

 
Identification of MHC-II-restricted CD4+ Th epitope responder peptides from SU, TM, and Gag proteins

The proliferative responses shown in Figs. 2Go, 3Go, and 4Go were gated on CD4+ populations and the possibility of bystander, MHC-II unrestricted proliferation of CD4+ cells by cytokines secreted from other proliferating cells in culture could not be excluded. To differentiate between MHC-II-restricted and unrestricted (bystander) CD4+ Th cell proliferation and to identify a set of MHC-II-restricted responder peptides from Env and Gag, we performed MHC-II blocking assays for each of the responder peptides identified in the previous section. MHC-II Ab-blocking assay for 10 responder peptides in monkey M0798 was first performed at three different Ab dilutions (1/50, 1/100, and 1/200) to determine the optimum concentration of anti-MHC-II Abs to be used. Anti-MHC-I and IgG isotype control Abs at same dilutions and a no-Ab control were used to determine nonspecific and background blocking of proliferation, respectively. Representative data for one peptide from monkey M0798 is shown in Fig. 5Go. As exemplified by the data in Fig. 5Go, specific blocking of peptide-specific proliferation by anti-MHC-II Abs was observed in a dose-dependent manner when using anti-MHC-II Abs at various dilutions (1/50, 1/100, and 1/200). Blocking of proliferation due to specific binding of MHC-II Abs decreased with the increasing dilution of blocking Ab. In contrast, <5% blocking was observed with MHC-I and isotype IgG Abs, and this level of blocking did not change with increasing dilutions of the Abs, thereby demonstrating their nonspecific nature. Subsequently, all the assays for individual responder peptides for each macaque were conducted at 1/100 dilution of blocking and control Abs. A peptide was considered MHC-II restricted when the reduction in proliferation was >50% when compared with control without Ab. From this analysis, ~1–5 MHC-II-unrestricted Th epitope peptides were identified in each peptide panel for each macaque (see Figs. 2Go, 3Go, and 4Go, gray bars), which were further excluded from subsequent analyses. Thus, individual macaque-specific sets of MHC-II-restricted CD4+ Th epitope peptides associated with mature immunity were identified for the Env and Gag proteins of SIV, which are summarized in Fig. 6Go.



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  		FIGURE 5. MHC-II restriction of proliferation induced by CD4+ Th responder peptides. MHC-II Ab-blocking assays were performed for 10 responder peptides in monkey M0798 at three different Ab dilutions to determine optimum anti-MHC-II blocking Ab concentrations. Data for one such responder peptide, peptide 5 from SU panel, is shown. Anti-MHC-II Ab (1/50, 1/100, and 1/200) was added to the cell culture and incubated at 37°C for 2 h before peptide stimulation. Cells were stimulated for 5 days with 20 µg/ml of the responder peptide, following which they were assessed for proliferation using CFSE and Ki-67. Percentages of proliferating CD4+ T cells are shown. Anti-MHC-I, IgG isotype control Abs at same dilutions, and no-Ab control were used to determine nonspecific and background blocking of proliferation, respectively.

 


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  		FIGURE 6. Summary of MHC-II-restricted SU, TM, and Gag Th epitope peptide-specific reactivity. CD4+ T cell proliferative responses in attenuated SIV-inoculated macaques were evaluated following stimulation with single 20-mer overlapping peptides spanning the entire length of SU, TM, and Gag proteins. This chart summarizes MHC-II-restricted proliferative responses induced by individual peptides from SU, TM, and Gag panels of peptides for all eight macaques ( Figs. 2–4GoGoGo). As indicated, the level of proliferation induced by each peptide was graded into four classes based on the intensity of response. A filled bar above the peptide number column indicates a broadly reactive peptide epitope with a value of p < 0.05. This data for responder peptides is representative of at least three independent assays.

 
The strength of proliferative response to each peptide was graded into four classes and shaded with increasing intensity based on the strength of response. As summarized in Fig. 6Go, ~90% (126 of 137 total) of the test peptides induced a MHC-II-restricted proliferative response in at least one of the eight immune animals. Taken together, these MHC-II-restricted CD4 T cell proliferation data clearly indicate a unique combination of breadth and strength of CD4+ Th responses to SU, TM, and Gag proteins for each monkey. For example, animals M0798 and M1998 showed a robust and broad CD4+ Th cell repertoire to a range of peptides among the SU, TM, and Gag proteins. This showed the generation of a robust and broad SIV Ag-specific CD4+ Th cell repertoire in these two animals. At the other end of the response spectrum was monkey M6598, with weak CD4+ Th proliferative responses to only a limited number of peptides from Env and Gag, which also correlated with its weak proliferative response to whole Ag, AE-B7. The remaining monkeys showed a mixed response in terms of strength and breadth of Ag-specific Th responses. Two monkeys, M6798 and M7098, generally mounted a strong epitope-specific Th proliferative response to SU, TM, and Gag, although they differed in the breadth of Th responses to the individual proteins. For example, the breadth of epitope-specific response to SU was quite limited in animal M7098, whereas a limited range of peptide specificity was observed to both SU and Gag proteins in case of M6798. Conversely, M7198 and M7298 demonstrated broad epitope specificity but differed in the intensity of fine specific Th responses to the individual SIV Ags. Finally, M6998 showed a unique mixture of Th responses in terms of qualitative and quantitative properties of SIV Ag-specific responses. In terms of the localization of MHC-II-restricted Th responder peptides, in general, they were uniformly distributed over the entire sequence of TM and Gag proteins. In contrast, the SU protein evidently contained a relatively limited number of Th epitopes that were predominantly located toward the C terminus of the protein. In fact, 11 peptides (nos. 3, 4, 6, 9, 11, 14, 15, 20, 22, 28, and 29) of SU did not induce MHC-II-restricted proliferative responses in any experimental animal and were classified as absolute nonresponders, while there was no such nonresponder peptide in TM or Gag (Fig. 6Go). Next we sought criteria to define a set of broadly reactive MHC-II-restricted Th responder epitope peptides.

Definition and identification of broadly reactive Th epitope peptides in the Env and Gag proteins

More than 75% (98 of 126) of the overlapping responder peptides from Env and Gag stimulated proliferative responses in at least two animals, suggesting the presence of broadly reactive Th epitopes. Based on the ability of each responder peptide to induce a statistically significant (p < 0.05) proliferation across all the eight monkeys, a set of 31 broadly reactive MHC-II-restricted Th epitope peptides from Env and Gag was defined (indicated by solid horizontal bars above the peptide number in Fig. 6Go). It was observed that all the peptides that satisfied the criteria for being broadly reactive induced a proliferative response in four or more inoculated macaques. A close examination of the location of the 31 broadly reactive peptides revealed an interesting and informative distribution of Th epitopes among the Gag, SU, and TM protein sequences (Fig. 7Go). Gag contained 12 Th epitope peptides spanning six distinct regions of the protein. Similarly, TM contained 14 broadly reactive Th epitope peptides localizing to five distinct regions. However, in contrast to Gag and TM, the SU domain of the Env contained only five broadly reactive Th epitope peptides localizing specifically to two distinct regions (amino acids 341–360 and 421–470) in the C terminus of the protein; no broadly reactive Th peptides were detected in the N-terminal 340 residues. These observations suggested a strong structural influence on localization of Th determinants among the different SIV proteins, which was further investigated.



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  		FIGURE 7. Properties of MHC-II-restricted broadly reactive CD4+ Th epitope peptides. Primary sequence of Env and Gag proteins of SIVmac239 was assessed for amphipathic potential using Antheprot, and percentage of sequence conservation was determined using Clustal at each amino acid residue. Amphipathicity (thick black line) and percentage of conservation (thin gray line) of SIVmac239 SU domain of Env and TM domain of Env and Gag proteins are plotted across the length of the proteins. Filled bars above each panel represent the location of broadly reactive Th epitope peptide regions. Gray bars labeled V1–V5 represent the variable loops classically defined in SU. Oval CHO symbols (carbohydrates) indicate the potential N-linked glycosylation sites in SU.

 
Properties of MHC-II-restricted broadly reactive CD4+ Th epitope peptides

A careful comparative analysis of the location of 31 broadly reactive peptides with structural properties of SU, TM, and Gag protein sequences revealed several relevant characteristics.

Broadly reactive Th epitopes from the Env SU and TM were located in the nonglycosylated regions. First, the greater concentration of broadly reactive Th epitopes to the TM and Gag proteins relative to SU suggested an influence of glycosylation on the breadth of Th responses. Therefore, the location of the broadly reactive Th epitopes of the heavily glycosylated SU protein was compared with that of Gag and TM proteins (Fig. 7Go), which are predominantly nonglycosylated. First, we observed that the broadly reactive Th epitopes from SU were concentrated in two clusters toward the carboxyl terminus of the protein. In contrast, the broadly reactive Th epitopes of Gag and TM were located evenly along their protein sequence. This showed a selective bias of the broadly reactive Th epitopes for being located at the carboxyl terminus of the SU protein. Second, when we compared the glycosylations of the SU with the location of Th epitopes, we noticed that these broadly reactive Th epitopes were located predominantly outside the heavily glycosylated regions of SU. It is noteworthy that 9 of the 11 absolute nonresponder peptides identified in SU contained one or more N-linked glycosylation sites within their 20-mer sequences (Table IIGo). The remaining two SU peptides (nos. 6 and 9) were flanked by heavily glycosylated regions. Taken together these data suggest that the heavy glycosylation of the SU protein may be important in limiting the generation of broadly reactive Th responses to this largest immunologically exposed protein of the virus.


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  		Table II. Potential glycosylation properties of absolute nonresponder peptides1

 
Broadly reactive Th epitope peptide regions were located in highly conserved sequences of SU, TM, and Gag proteins. Previous studies of SIV genomic variation have defined conserved and variable regions of the viral proteins, particularly the SU protein. An evaluation of the location of the broadly reactive Th epitopes with respect to the conservation of amino acid sequences of SU across different SIV isolates indicated that the broadly reactive Th peptides mapped predominantly to highly conserved domains of SU (Fig. 7Go) and outside of the variable regions of this protein. Interestingly, while there is much less variation in the SIV TM and Gag proteins, the broadly reactive Th peptide epitopes identified in these two proteins also localized predominantly to their most conserved segments. These observations suggest that the identified broadly reactive Th epitopes should be conserved among variant strains of SIV and are therefore relevant to immune responses to diverse SIV isolates.

The broadly reactive Th epitope peptides were located predominantly in highly amphipathic regions of Env and Gag proteins. Finally, numerous studies in the past have documented a correlation of Th function with protein sequences possessing a high potential for amphipathic helical structure with specific amino acid sequences matching MHC binding site specificities (45). Because the detailed structures and binding clefts of various MHC-II molecules of rhesus macaques are not fully elucidated, the amphipathicity analysis provides an initial indication of potential broadly reactive Th epitopes in lieu of the availability of detailed MHC-II binding motifs. In this study, the identification of broadly reactive Th epitopes was done using sequential overlapping peptides. These overlapping broadly reactive Th epitope peptide regions were plotted across the respective amphipathicity of the protein sequences (Fig. 7Go). All the defined broadly reactive Th epitope peptides of SU mapped to two segments of the protein sequence that were predicted by computer algorithms to have the highest amphipathic helical potential within SU (Fig. 7Go). Furthermore, it was observed that broadly reactive Th epitopes regions in Gag and TM corresponded to regions of medium to high amphipathic potential, indicating differences between predicted and experimental Th epitope regions. This observation is concordant with previous studies (45) and lends a high degree of confidence to our criteria for defining broadly reactive Th epitopes.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study for the first time provides a high resolution analysis of both Env (SU plus TM) and Gag peptide-specific Th responses in a relatively large number of monkeys with established mature vaccine immunity from parallel inoculation with the same attenuated SIV strain. Because all the animals were healthy with stable immunocompetent CD4+ T cell levels, the SIV Ag-specific Th repertoire in these animals was considered appropriate for fine specificity analysis. The results of these comprehensive Th epitope-mapping studies using primary CD4+ T cells reveal that there is a relative lack of Th responder epitope peptides in the SU domain of SIV Env. Furthermore, the few broadly reactive Th epitope peptides identified in SU are concentrated in its C-terminal end, evidently excluded from potentially glycosylated regions. Thus, these data reveal for the first time a previously unrecognized limitation on Th responses to the SIV SU protein, even during a persistent infection for over a year, where both the virus and the immune system are allowed to mature to a steady state of enduring broadly protective immunity. This comparative fine mapping of SIV glycosylated and nonglycosylated proteins provides a potential structural explanation for the previously observed low levels of lymphoproliferation to SU proteins from HIV-1 and SIV compared with Gag and has important implications in vaccine design.

Among our set of eight inoculated monkeys, we observed a wide variation in Th responses in terms of both peptide specificity and relative intensity ( Figs. 2–4GoGoGo and 6). In light of the outbred nature of monkeys used in these studies (data not shown), we believe that these qualitative and quantitative variations reflect genetic differences related to Ag processing and presentation on host MHC-II. Despite the observed individual variations in Th responses, a generalized lack of Th reactivity to SU panel of peptides was observed for all the monkeys. Furthermore, despite the diversity of response, we found that 31 of the 126 MHC-II-restricted responder peptides were broadly reactive (p < 0.05) and induced peptide epitope-specific Th responses in four or more monkeys. The broadly reactive Th peptides identified in our study also fulfilled the dual requirements of mapping to amphipathic helical and conserved segments of the Env and Gag proteins (Fig. 7Go), thereby lending confidence to our criteria for definition of broadly reactive CD4+ T cell epitopes. In absence of detailed information on macaque MHC-II binding motifs, finer peptide mapping of the broadly reactive Th epitopes is required to identify the sequences of the minimal Th epitopes.

Previous studies of Th responses to attenuated SIV in experimentally inoculated monkeys have focused on bulk lymphoproliferation assays to complete Gag or Env proteins (28), thus not providing information on the nature of Th epitopes that may be associated with the evolution of protective immunity. Dittmer et al. (46) described a comprehensive analysis of Env SU-specific Th responses by using cultured short-term T cell lines from four monkeys inoculated with an attenuated SIV, while excluding comparative analysis of fine Th responses elicited to other SIV proteins. Thus, the current comparative analysis of the broadly reactive Th epitope peptides from SU and Gag showed that, despite similarity in protein lengths, SU contained only ~15% of the mapped broadly reactive Th epitope peptides, while Gag contained 40% of the epitopes. In fact, the SU protein contained all of the 11 absolute nonresponder peptides, which were unable to induce lymphoproliferation in even a single monkey (Table IIGo). The observed relative lack of Th responses to SU is in agreement with previous studies that indicate stronger Th responses to Gag compared with SU proteins (28, 29). Moreover, Th responses were generated to only some variable regions of SU in certain monkeys, and these responses were often very weak (Fig. 6Go), while no broadly reactive Th responses localized to the variable regions SU (Fig. 7Go). Due to the highly variable nature of these regions, one can speculate that specific sequences from variable regions are not presented consistently to the CD4+ T cells, thereby hindering the generation of a stably large memory CD4+ T cell repertoire for detection. Likewise, the presence of Th epitopes in conserved regions of Env and Gag suggests that the identified broadly reactive Th epitopes should be conserved among variant strains of SIV and therefore would be relevant to the generation of immune responses to diverse SIV isolates.

The observed lack of broadly reactive Th epitope peptide regions in SU was suggestive of a structural influence on the generation of CD4+ Th responses. A further analysis revealed that there was in general an exclusion of the broadly reactive Th epitopes from regions of SU containing potential N-linked glycosylation sites and presumably modified with complex oligosaccharides (Fig. 7Go). No Th responses were observed to the V2 region of SU, which notably also happens to contain multiple glycosylation sites. Absence of broadly reactive Th epitopes in certain highly amphipathic regions of SU containing glycosylation sites, coupled with the observation that multiple glycosylation sites were present on majority of the absolute nonresponder peptides in SU (9 of 11), or in flanking regions (peptides 6 and 9 of SU panel) (Table IIGo), suggests a role of glycosylation in hindering Ag processing, presentation, and/or recognition of glycopeptides by CD4+ T cells. Recent studies indicating that protein glycosylation can reduce the efficiency of Ag processing or presentation for both MHC-I (31, 47)- and MHC-II (32, 48)-mediated immune responses further support this hypothesis. In this context, it has been demonstrated that glycosylation of HIV-1 SU protein limits CTL responses (31) and Ab responses (30) as well as Ag recognition by CD4+ T lymphocytes (32).

Based on these observations, we speculate that the restricted Th functions intrinsic to the SIV SU protein may represent another mechanism that lentiviruses have evolved to confound antiviral humoral and cellular immune responses. With the current detailed analyses of Env Th responses associated with mature and enduring vaccine immunity, it will be of interest to perform a longitudinal analysis of the fine specificity of SIV Ag-specific Th responses during the first 8–10 mo postinoculation with attenuated SIV, during which the maturation of protective immune responses occurs (44). On a related issue, the current important new fundamental information about the specificity of Th responses associated with protective immunity may be used to improve Env immunogenicity design by engineering additional Th epitopes to enhance protective host Ab and CTL responses.


   Acknowledgments
 
We thank Drs. Charles Rinaldo, Albert Donnenberg, and Simon Barratt-Boyes for many helpful discussions and Dawn McClemens-McBride for excellent animal technical assistance. We also thank Opendra Sharma at the National Institutes of Health AIDS Research and Reference Reagent Program for providing the Env and Gag overlapping peptide panels.


   Footnotes
 
1 This study was supported by grants 5PO1 AI28243 and 1RO1 AI47758 from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Ronald C. Montelaro, Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, W1144 Biomedical Science Tower, Pittsburgh, PA 15261. E-mail address: rmont{at}pitt.edu Back

3 Abbreviations used in this paper: MHC-II, MHC class II; MHC-I, MHC class I; Env, envelope; SU, surface; TM, transmembrane; SEB, staphylococcal enterotoxin B. Back

Received for publication December 5, 2001. Accepted for publication February 12, 2002.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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