Ratto-Kim, Silvia*; Loomis-Price, Lawrence D.*; Aronson, Naomi†‡; Grimes, Janelle*; Hill, Cristin*; Williams, Chevelle*; El Habib, Raphaelle§; Birx, Deborah L.∥; Kim, Jerome H.‡∥
It has been shown that T helper cells are an essential component of immune response to viruses (1–4). In HIV-1 infection, CD4+ T helper cells are also the targets of the virus, and studies have shown that CD4+ T–helper cell function is affected in many ways by HIV-1 infection (5–9). Reduction of CD4 level and the impairment of CD4 function complicate the generation and maintenance of an effective immune response against HIV-1. Evidence that certain T-helper responses may play a role in long-term nonprogression and that more effective early control of viral replication may alter the T-helper response to infection critically highlights the need to understand and target the CD4+ T-helper response to HIV-1 in vaccine development (10–13).
To date, many experimental HIV-1 vaccines have been tested; among the most promising are those utilizing a “prime-boost” strategy (Canarypox vector containing various HIV-1 genes “prime” the immune system for a “boost” using subunit proteins, typically envelope gp120 or gp160). These vaccines have been tested in thousands of individuals and have been shown to be safe and immunogenic, eliciting CD8+ CTL responses and neutralizing antibodies and T-helper responses (14–18). Studies of the CTL response induced by these vaccines have defined epitope recognition and cross-clade reactivity of vaccine-induced CTLs (19,20). Also, it is known that the vCP205 prime and Env boost regiments induce functional antibodies that neutralize T-cell line–adapted (laboratory) strains of HIV-1 but not primary (peripheral blood mononuclear cell [PBMC]–derived) strains (21,22). On the other hand, the CD4+ T-helper response has not been studied as extensively.
In this study, we sought to investigate the epitope reactivity of the CD4+ T-helper response induced by HIV-1 vaccine. Gp160MN/LAI-2–specific CD4+ T-cell lines were established from participants in an ALVAC-HIV(vCP205) prime with gp160MN/LAI-2 boost vaccine study. The Env-specific lines were established from volunteers in three different arms of the study: gp160MN/LAI-2 subunit alone, ALVAC-HIV (vCP205) alone, and ALVAC-HIV(vCP205) + gp160MN/LAI-2 prime-boost combination. GP160MN/LAI-2–specific T-cell lines from these HIV-1–noninfected individuals were compared with gp160MN/LAI-2–specific T-cell lines from HIV-1–infected individuals.
MATERIALS AND METHODS
Nine HIV-1–seronegative volunteers enrolled in an HIV-1 vaccine protocol, and 6 HIV-1–infected volunteers were studied. The 9 HIV-1–seronegative volunteers participated in a phase I/II trial of various combinations of ALVAC-HIV(vCP205) (Aventis Pasteur, Lyon, France) and gp160MN/LAI-2 (recombinant chimeric Env-protein; Aventis Pasteur). The individuals chosen for this study were 3 volunteers who received four doses of gp160MN/LA-2 (100 μg) formulated in adjuvant, 3 volunteers who received four doses of vCP205, and 3 volunteers who received a combination of the above (four doses of vCP205 and two doses of gp160MN/LAI-2 [100 μg] given in conjunction with the third and fourth doses of vCP205). The vaccine was administered at months 0, 1, 3, and 6. The 6 HIV-1 chronically infected volunteers had nadir CD4+ counts >400/mm3; all but 1 (Patient 3) HIV-1–infected volunteers were on antiretroviral therapy. Five of 6 HIV-1–positive volunteers had viral loads <400 copies/mL, and the remaining HIV-positive volunteer (Patient 3) had limited viral replication (viral load ∼5000 copies/mL, data not shown) throughout the study.
The research was approved by the Walter Reed Army Institute of Research Human Use Review Committee and the Human Subjects Research Review Board under the office of the US Army Surgeon General. All subjects voluntarily agreed to participate and gave written informed consent.
Peripheral blood mononuclear cells were obtained by Ficoll separation of heparinized venous blood. For the vaccinees, the first blood draw to establish CD4 T-cell lines was obtained at day 168, the day of the fourth vaccination.
Medium and Antigens
Lymphocytes were cultured in complete medium composed of X-vivo 15 (Biowhittaker, Walkersville, MD, U.S.A.) supplemented with 4 mM of L-glutamine, 5 × 10−5 M of 2-ME, 100 U/mL penicillin, 100 μg/mL streptomycin, and 5% heat-inactivated normal human AB serum (Gemini Bio-products, Woodland, CA, U.S.A.). Recombinant gp160MN/LAI-2 was obtained from Aventis Pasteur.
Synthetic HIV-1 Peptides (T-Scan Peptides)
The entire sequence of the protein immunogen rgp160MN/LAI-2 was modeled with 15-mer peptides overlapping by 10 amino acids (135 peptides) (Fig. 1). Peptides are denoted by N-terminal amino acid, starting with peptide 31TEKLWVTVYYGVPVW45, then peptide 36VTVYYGVPVWKEATT50 (amino acids 1–30 constitute the signal region, which was not included in the immunogen), and ending with peptide 701RHLPTRGPDRPEGI715 just before the transmembrane crossing region of gp41 (14). Peptides were synthesized on the heads of synthetic pins with cleavable diketopiperazine linkages (Chiron Mimotopes, San Diego, CA, U.S.A.) using Fmoc chemistry as previously described (23,24). Peptides were cleaved from the pins in 0.2 M of sodium bicarbonate, pH 8.0, at a 60:40 water/acetonitrile ratio and stored at −80°C in 96-well plates until used. The synthesis quality was checked by amino acid analysis on control peptides and specific peptides of interest. Previous results using this method have typically yielded short peptides of greater than 80% purity (25); we observed comparable results. Peptides were used without further purification.
Human Leukocyte Antigen Typing
Human leukocyte antigen (HLA) typing was performed on all subjects using DNA extracted from autologous B-lymphoblastoid cell lines (BLCLs) by the American Red Cross National Histocompatibility Laboratory at the University of Maryland Medical Center, employing standard molecular techniques (26). Ambiguities in the dot blot analysis were resolved by DNA sequencing, plasmid DNA was prepared from transformed and expanded Escherichia coli colonies, and the insert was sequenced by the dideoxy chain termination method on an ABI 373 DNA sequencer (Applied Biosystems, Foster City, CA, U.S.A.).
Generation of Specific T-Cell Lines
The lines were established from PBMCs as previously described (27). Briefly, PBMCs at 1 × 107 cells/mL were pulsed with gp160MN/LAI-2 (30 μg/mL) for 4 hours at 37°C. Cells were diluted in complete medium, plated at 2 × 106 cells/mL in 24-well plates (Costar, Cambridge, MA, U.S.A.) and incubated at 37°C in 5% CO2. After 4 days, the cells were fed with 10 U/mL recombinant interleukin-2 (rIL-2) (Boehringer-Mannheim, Germany); after 1 week of culture and every 2 to 3 days thereafter, half of the medium was replaced with fresh medium and rIL-2.
After 15 days, the T cells were harvested, washed, and resuspended at a final concentration of 5 × 105 cells/mL. A total of 10 x 106 autologous PBMCs were pulsed with gp160MN/LAI-2 as described above and irradiated with 30 Gy from a 137Cs source. After 4 hours of incubation at 37°C, antigen-pulsed PBMCs were diluted to 1 × 106 cells/mL; 1 × 106 antigen-presenting cells were added to 5 × 105 of the harvested T cells in a 24-well plate. After 2 days, rIL-2 was added, and the cultures were further expanded and split after 5 days. The third stimulation, after 15 days of culture, was performed as described above.
The proliferative response of the T-cell lines was measured by incubating 3 × 104 T cells in a 96-well flat-bottom plate (Costar) with either 1 × 105 autologous antigen-pulsed or nonpulsed irradiated PBMCs; the assays were done in duplicate. After 2 days of incubation, the cells were pulsed with 1.67 μCi per well of 3H thymidine for 18 hours, harvested using the Tomtec Mach3M (EG&G Wallac, Gaithersburg, MD, U.S.A.) and counted in a 1450 microbeta trilux (EG&E Wallac). The data are expressed as a lymphocyte stimulation index (LSI) ([T cell + antigen-pulsed PBMCs cpm]/[T cell + unpulsed PBMCs cpm]) to define antigen specificity. Lines were arbitrarily designated as antigen specific if their LSI was greater than or equal to 3 (27). All the specific T-cell lines were challenged in a proliferation assay with gp160MN/LAI-2 envelope peptides (see Fig. 1) (T scan). T cells (3 × 104) were incubated with irradiated autologous PBMCs (1 × 105) for the HIV-1–infected individuals or with BLCLs (5 × 104) or PBMCs (1 × 105) for the vaccinees and 5 μg/mL of each peptide or medium alone. As controls, responses to the whole gp160MN/LAI-2 and to the cleavage cocktail were assessed along with the peptides. Cells were pulsed with 3H-thymidine after 2 days and processed as described above. All assays were done in duplicate.
T-scan data are shown as z scores (i.e., as reactivity in SDs [σ]) from the median reactivity to all the peptides. SDs were determined in each experiment using this equation: σ = (median − first quartile)/0.675. Values above z = 3.29 σ were considered significant in this assay.
Establishment of the T-Cell Lines from Seronegative Vaccinees and HIV-1–Infected Individuals
The T-cell lines from uninfected individuals were established from three groups of the prime-boost protocol. PBMCs were started in culture on the day of the fourth vaccination; hence, specificity of the T-cell lines reflects immunity acquired after three vaccinations.
After three stimulation cycles, a total of six of nine T-cell lines were specific for gp160MN/LAI-2 (Table 1). All six lines were tested with the T-scan peptides using autologous BLCLs and/or PBMCs as antigen-presenting cells. From the 6 HIV-1–infected individuals, a total of four of six T-cell lines were specific for gp160MN/LAI-2 (see Table 1). All individuals were HLA typed (Table 2), and flow cytometric measurements showed that all specific T-cell lines were >90% CD4+, consistent with previous studies (data not shown) (27).
Peptide Mapping Using T-Cell Lines
The specific T-cell lines were challenged with a series of overlapping peptides spanning the entire gp160MN/LAI-2 (see Fig. 1) (T scan). The peptides are 15 amino acids long overlapping by 10. The T-scan technique allows simultaneous testing of each peptide and control. Statistics described in the Materials and Methods section define positivity by clearly differentiating positive responses from background responses.
Table 3 summarizes epitope recognition by the specific T-cell lines. Repetition of a T scan using the same T-cell line yielded consistent results. Although the reactivity to some peptides was observed only once, this was usually in close proximity to repeatedly recognized peptides (e.g., Patient 5 cluster in gp41; see Table 3). This phenomenon was probably due to the recognition of an epitope or part of an epitope that was shared by contiguous peptides. The peptides recognized in this study overlapped in whole or in part with peptides already described in the literature and available from the HIV databases (28).
There were no striking similarities in the pattern of epitope recognition among the T-cell lines tested, probably due to HLA diversity among the volunteers (Table 2). T-cell lines established from HIV-1–infected volunteers had poor recognition in C1 and C5. Two of four T-cell lines had some reactivity in the V3 region. In contrast, the T-cell lines established from vaccinees had no reactivity in the V3 region but had scattered reactivity along the entire gp160MN/LAI-2. Of note, the T-cell lines from Patients 176 and 184, who received only the vCP205 vaccine, had poor T-scan peptide recognition (Table 3). The prime-boost group yielded the greatest number of specific T-cell lines (three of three lines) (Table 3).
Reactogenicity of the Specific T-Cell Lines
There was a great diversity in reactogenicity toward T-scan peptides between T-cell lines established from HIV-1–infected individuals and vaccinees. Figures 2 and 3 show the combined reactogenicity of specific T-cell lines of vaccine recipients and HIV-1–infected individuals, respectively. HIV-1–infected individuals had poor reactogenicity to the T-scan peptides with σ ranging from −2.3 to 21.1, whereas the vaccinees had σ ranging from −6.4 to 116.4. Of note, three peptides (131,161, and 191) were so nonreactive with the CD4 T-cell line established from Volunteer 18 (σ < 3.29) as to suggest that they might be inhibitory. The alternative explanation (that these peptides were toxic) seemed unlikely because they did not inhibit growth in other T-cell lines. Of note, we have observed apparent inhibition of proliferative responses in spleen cells from mice immunized with gp160LAI using peptides having sequences overlapping with peptides 131 and 161 (data not shown).
The aim of this study was to identify CD4+ T-helper epitopes in a cohort of individuals vaccinated with an experimental HIV-1 vaccine and to compare the responses with those generated by chronic infection in HIV-1–infected individuals.
The vaccine is composed of ALVAC-HIV-1(vCP205) with or without booster doses of gp160MN/LAI-2. For this study, individuals enrolled in the gp160MN/LAI-2 only, vCP205 only, and prime-boost arms were studied for CD4+ T-helper epitope recognition. CD4+ epitope mapping was studied using T-cell lines obtained from the volunteers tested against a panel of 15 overlapping amino acid peptides spanning the entire sequence of the immunogen. This method allowed mapping to a single epitope in one experiment. T-cell lines were chosen as a method to test the peptides because they had been used successfully to map epitopic responses in HIV-1–infected individuals, were shown to reflect in vivo immunogenicity, and require minimal amounts of PBMCs for input initially (27–30). This was of paramount importance due to the paucity of PBMCs available from the ongoing trial. This was also the reason for establishing the lines on the day of the fourth vaccination, because the protocol demanded all the following blood draws for immunogenicity testing.
Nonetheless, we were able to establish CD4+ T-cell lines from 6 of 9 of the vaccinees and from 4 of 6 HIV-1–infected individuals. The protein-only group yielded only one specific line of three, although the numbers are too small to draw any conclusions. The breadth of the response was heterogeneous among the vaccinees (Table 3) compared with HIV–infected persons. Often, positive responses were found for contiguous peptides, probably due to the epitopic localization in overlapping regions of the peptides. All the epitopes defined in this study have been described, and they are reported in the Los Alamos database (28). Of note, the T-cell lines established from vaccinees had no reactivity in the V3 region, whereas T-cell lines established from HIV-1–infected individuals, albeit weak, demonstrated reactivity to peptides from the V3 region. Among the vaccinees, it was immediately clear that the T-cell lines established from the vCP205-only group, although specific to the whole protein, were poorly reactive in the T-scan assay. The T-cell line from Patient 176 was tested twice with the T-scan peptides, and although the median LSI reactivity to the whole protein was 21, only one T scan yielded one weakly positive peptide (691, 5σ). Studies have shown that in standard LPA assays, the CD4+ proliferative responses from recipients of vCP205 alone are less robust than those from recipients of vCP205 prime and Env-boost (H.J. Kim, manuscript in preparation) (15,16). It seems that the vaccination with vCP205 alone had expanded a pool of CD4+ T-cell clones that recognized epitope(s) different from those presented in the T-scan assay. These T-cell lines appear to recognize the subunit gp160 antigen but are nonreactive to the T-scan peptides. This effect could be due to a differential processing of the vCP205 vector compared with the subunit gp160. These two antigens may undergo different pathways for processing and presentation, because the Env protein is produced intracellularly by the vCP205 vector. In addition, it has been shown that mammalian cells infected in vitro with vCP205 can produce pseudovirions containing gag-env proteins (31). These pseudovirions could yield Env proteins conformationally different from the recombinant subunit gp160MN/LAI-2, and processing and presentation of these products could yield different epitopes not represented in the set of peptides used in this study.
The responses in the T scans of the T-cell lines generated from HIV-1–infected individuals were different from those of the vaccinees; notably, the reactogenicity was much lower (Fig. 2 and 3). One possible explanation is that the acquired natural immunity generated by viruses is different from the immune response generated by the vaccine. This has been shown for HIV-1–specific CD8 CTL responses in vaccinated individuals compared with HIV-1–infected persons (20). Further, the peptide sequences were identical to the strain of HIV-1 used in the vaccine; in HIV-1–infected individuals, the immune response was targeted toward wild-type viruses whose sequences may diverge from the immunogen used to generate the T-cell lines and may not cross-react with the strain used in the vaccine. This was probably the case for Patient 2, in whom a non-clade B infection was identified by sequencing of the autologous virus (segment of pol aligned with subtype A virus). As mentioned previously, class II responses may be induced by the processing of HIV-1 viral proteins that may generate reactivity to peptide sequences not represented in the T-scan peptides used in this study.
In conclusion, we demonstrated that it is feasible to epitope map CD4+-helper responses using T-cell lines and the T-scan peptide mapping technique. These methods could prove useful in the evaluation of candidate HIV vaccines due to the limited number of cells needed for testing. The vCP205 vaccine in combination with gp160MN/LAI-2 generated strong and broad responses to autologous sequence T-scan peptides. Individuals vaccinated with vCP205 only, despite sequence identity of the envelope gene, manifested poorer responses when tested with the T-scan peptide panel. Finally, although T-cell lines were easily established from HIV-1–infected individuals, these lines had limited reactogenicity in both breadth and amplitude of T-helper response.
These results highlight the complexity of major histocompatibility complex (MHC) class II presentation and the CD4+ antigen-specific reactivity, emphasizing the need to better understand these crucial T–helper cell responses if prolonged T-helper memory or specific T-helper responses are critical to the success of an HIV vaccine.
The authors thank all the volunteers who made this study possible, Josephine Cox for donating the autologous B-lymphoblastoid cell lines and for her critical review of the manuscript, Kathleen Duffy for patient coordination at the HIV clinic, and David Larson for technical support.
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