A novel HLA (HLA-A*0201) transgenic rabbit model for preclinical evaluation of human CD8+ T cell epitope-based vaccines against ocular herpes

Abstract

We introduced a novel humanized HLA-A*0201 transgenic (HLA Tg) rabbit model to assess the protective efficacy of a human CD8(+) T cell epitope-based vaccine against primary ocular herpes infection and disease. Each of the three immunodominant human CD8(+) T cell peptide epitopes from HSV-1 glycoprotein D (gD(53-61), gD(70-78), and gD(278-286)) were joined with a promiscuous human CD4(+) T cell peptide epitope (gD(49-82)) to construct three separate pairs of CD4-CD8 peptides. Each CD4-CD8 peptide pair was then covalently linked to an N(epsilon)-palmitoyl-lysine residue via a functional base lysine amino group to construct CD4-CD8 lipopeptides. HLA Tg rabbits were immunized s.c. with a mixture of the three CD4-CD8 HSV-1 gD lipopeptides. The HSV-gD-specific T cell responses induced by the mixture of CD4-CD8 lipopeptide vaccine and the protective efficacy against acute virus replication and ocular disease were determined. Immunization induced HSV-gD(49-82)-specific CD4(+) T cells in draining lymph node (DLN); induced HLA-restricted HSV-gD(53-61), gD(70-78), and gD(278-286)-specific CD8(+) T cells in DLN, conjunctiva, and trigeminal ganglia and reduced HSV-1 replication in tears and corneal eye disease after ocular HSV-1 challenge. In addition, the HSV-1 epitope-specific CD8(+) T cells induced in DLNs, conjunctiva, and the trigeminal ganglia were inversely proportional with corneal disease. The humanized HLA Tg rabbits appeared to be a useful preclinical animal model for investigating the immunogenicity and protective efficacy of human CD8(+) T cell epitope-based prophylactic vaccines against ocular herpes. The relevance of HLA Tg rabbits for future investigation of human CD4-CD8 epitope-based therapeutic vaccines against recurrent HSV-1 is discussed.

FIGURE 1

Schematic representation of prototypes human CD4–CD8 lipopeptide vaccines. The C-terminal end of a promiscuous CD4⁺ T cell peptide epitope (gD_49–82) was joined in line with the N-terminal end of one of three different HSV-1 gD CD8 T cell epitopes: gD_53–61 (A), gD_70–78 (B), or gD_278–286 (C). The N-terminal end of each resulting CD4–CD8 peptide was extended by a lysine covalently linked to one molecule of palmitic acid (a lipid). This results in three separate pairs of CD4–CD8 lipopeptides.

FIGURE 2

Detection of HLA-A*0201 molecules in cornea, TG, and PBMC of HLA transgenic rabbits. A, Cornea and TG sections from either HLA Tg rabbit or from wild type nontransgenic rabbits (control) were immunostained with FITC (green) conjugated BB7.2 mAb (anti-human HLA-A*0201) and analyzed by fluorescence microscopy (see Materials and Methods). Cell nuclei are shown in red. Dashed circles delineate neuron bodies in the TG (original magnification 320). B, Coexpression of HLA-A*0201 (green) and rabbit MHC class I (red) on the surface of a PBMC derived from an HLATg rabbit and detected by fluorescence microscopy. PBMCs from HLA Tg rabbit were double stained with two different primary Abs HLA-A2.1 mAb (BB7.2) and a rabbit MHCI followed by staining with secondary Abs conjugated with two different fluorescence probes, either Alexa Fluor 594 (green) or Alexa Fluor 488 (red), respectively. Merged yellow signals indicate the colocalization of these two molecules on the surface of PBMCs. C, PBMCs from either HLA Tg rabbit or from wild type nontransgenic rabbits were first stained with BB7.2 mAb, then with PE conjugated anti-mouse secondary IgG Ab, and analyzed by flow cytometry (original magnification ×20).

FIGURE 3

HLA-A*0201 Tg rabbit recognizes human HLA-A*0201–restricted CD8⁺ T cell epitopes from gD. Five HLA Tg rabbits and five wild type rabbits were infected ocularly with 2 × 10⁵ PFU per eye of HSV-1. Twelve days p.i., spleens were harvested and the percentage HLA-A*0201/tetramer (+) CD8⁺ T cells specific to each of 10 HSV-1 gD epitopes was determined by FACS. In the numbering system used for gD peptides in this study, the first amino acid in the mature gD is indicated as AA1. Thus, amino acids in the 25 aa leader sequence are indicated as negative numbers relative to AA1. Similar tetramer analysis was performed in PBMCs derived from five HSV-seropositive HLA-A*0201⁽⁺⁾ human blood samples. The results are representative of two independent experiments. Asterisk indicates a significant T cell response (p < 0.0005) compared with an irrelevant tetramer. The backgrounds were determined for all ten tetramers using PBMCs from each of the five different HLA-A2⁽⁺⁾ HSV-uninfected individuals, five uninfected HLA Tg rabbits, and five uninfected wild type rabbits (controls). The dashed vertical lines in each panel designate maximum background cutoff levels of epitope-specific CD8⁺ T cells in uninfected HLA-Tg rabbits, uninfected WT rabbits, and seronegative HLA-A2⁺ individuals.

FIGURE 4

Induction of gD_49–82-specific CD4⁺ T cell responses in HLA Tg rabbits immunized with human CD4–CD8 lipopeptides. HLATg rabbits (four in experiment 1, four in experiment 2) were immunized s.c. three times with the mixture of human CD4–CD8 lipopeptides as described in Materials and Methods. Ten days after the third immunization, all rabbits were ocularly challenged with 2 × 10⁵ PFU per eyeof HSV-1 (McKrae). Mock-immunized HLA Tg rabbits (two in experiment 1, two in experiment 2) were ocularly challenged as above. Mock-immunized, uninfected HLATg rabbits (two in experiment 1, two in experiment 2) were used as negative controls. Two weeks after HSV-1 challenge, DLNs were collected from each rabbit, and cells suspension were prepared, labeled with CFSE (2 mM), and incubated with the promiscuous gD_49–82 CD4⁺ T cell epitope peptide (10 mg/ml) for 5 d. The cells were washed and stained for CD4 molecule expression. Dividing CD4⁺ T cells were gated and analyzed by flow cytometry. A, Dot plot representation of CFSE intensity in dividing CD4⁺ T cells, from lipopeptide-immunized or mock-immunized rabbits, stimulated with gD_49–82 peptide. CFSE staining is shown on the x-axis and CD4 staining on the y-axis. The percentage in the quadrant represents the fraction of dividing CD4⁺CFSE^low T cells among total lymphocytes. B, Absolute number of dividing CD4⁺ T cells following in vitro stimulation with the promiscuous gD_49–82 CD4⁺ T cell epitope (left panel). PHA stimulation (right panel, positive control). The percentage of dividing lymphocytes under stimulated conditions was calculated as: N^br of dividing lymphocytes = N^br of gated CD4⁽⁺⁾ CFSE^(low) lymphocytes × N^br of total CD4⁺ lymphocytes—(CD4⁽⁺⁾ CFSE^(low) + CD4⁽⁺⁾ CFSE^(high) lymphocytes)/N^br of total gated lymphocytes) × 100. The ratio ([CD4⁽⁺⁾ CFSE ^(low) + CD4 ⁽⁺⁾ CFSE^(high) lymphocytes]/total lymphocytes) serves as normalization factor between one sample to another and one experiment to another.

FIGURE 5

Frequency and absolute numbers of dividing HSV-gD epitope-specific CD8⁺ T cells in HLA Tg rabbits immunized with human CD4–CD8 lipopeptides. CD8 T cells were isolated from DLNs, conjunctiva, and TG from the rabbits described in Fig. 4. The cell suspensions were im-munostained with an FITC-labeled mAb specific to rabbit CD8 and with a PE-labeled human HLA-A*0201/tetramer specific to each of the three human CD8⁺ T cell epitopes. A, Representative FACS analysis from individual rabbits. The numbers show the average number of tetramer-positive/CD8⁺ T cells from all the rabbits in the indicated group. B, Average absolute numbers for each epitope-specific HLA-A*0201/tetramer/CD8⁺ T cells. The dashed horizontal lines designate the maximum frequency levels of epitope-specific CD8⁺ T cells in mock-immunized/uninfected HLA-Tg rabbits. C, Average absolute number of dividing CD8⁺ T cells in each group following in vitro stimulation with gD_53–61, gD_70–78, or gD_278–286 epitope peptide. The number of dividing CD8⁺ lymphocytes under stimulated conditions was calculated as follows: N^br of dividing lymphocytes = N^br of gated CD8⁽⁺⁾ CFSE^(low) lymphocytes × N^br of total CD8⁺ lymphocytes—(CD8⁽⁺⁾ CFSE^(low) + CD8⁽⁺⁾ CFSE^(high) lymphocytes)/N^br of total gated lymphocytes. The dashed horizontal lines designate the maximum response levels of T cells in mock-immunized/uninfected HLA-Tg rabbits. The results are representative of two independent experiments.

FIGURE 6

Immunization with human CD4–CD8 lipopeptides protects against HSV-1 replication in the eyes of HLA Tg rabbits. HLA Tg rabbits were immunized with the three human CD4–CD8 lipopeptides (four in experiment 1, four in experiment 2) or mock-immunized (two in experiment 1, two in experiment 2). Two weeks after the third immunization, all rabbits were ocularly challenged with 2 × 10⁵ PFU per eye of HSV-1. Tears were collected daily from all eyes (20 eyes per group) on the days indicated, and the amount of infectious virus was determined by standard plaque assays. The p value is calculated using data from days 4 and 5 analyzed by ANOVA followed by Dunnett’s post test to identify differences between groups.

FIGURE 7

Immunization of HLA Tg rabbits with human CD4–CD8 lipopeptides protects the severity of ocular herpes disease. A, Representative slit lamp images of lipopeptide immunized/HSV-1 infected (four in experiment 1, four in experiment 2) and mock-immunized/HSV-1 (two in experiment 1, two in experiment 2). Infected HLA Tg rabbit eyes 12 d after ocular infection. One drop of 1% fluorescein dye was placed in each eye to identify the corneal damage under cobalt blue light. The amount of disease was estimated by determining the surface area stained, in square millimeters. B, The amount of corneal involvement in disease is plotted for each eye. C, Correlation between virus titers on day 5 and eye disease on day 12.

FIGURE 8

Immunized HLA Tg rabbits with more epitope-specific CD8⁺ T cells have less corneal disease. A, The results obtained from the HLA Tg rabbits that were immunized and ocularly challenged with HSV-1 (from Figs. 6 and 7) were divided into two groups, based on the amount of corneal disease on day 12 p.i. (low disease, < 10 mm²; high disease, > 30 mm²). The average absolute number of tetramer positive/CD8⁺ T cells specific to each of the three human CD8⁺ T cell epitopes are shown. The results are representative of two independent experiments. B, The relative amount of IFN-γ mRNA produced by conjunctiva- and cornea-derived CD8⁺ T cells of low and high corneal disease eyes was determined by quantitative RT-PCR.