Characterization of the early cellular immune response induced by HPV vaccines

Abstract

Introduction: Current human papillomavirus (HPV) vaccines consist of virus-like particles (VLPs) which are based on the L1 protein, but they are produced by different expression systems and use different adjuvants. We performed in-depth immunophenotyping of multiple innate and adaptive immune cells after vaccination with bivalent versus nonavalent HPV vaccines.

Method: Twenty pre-menopausal HPV-seronegative women were enrolled and randomized to receive three-doses of either the bivalent or the nonavalent HPV vaccine. Blood samples were collected at multiple time points from baseline up to 7 months after first vaccination. Four extensive EuroFlow flow cytometry antibody panels were used to monitor various immune cell subsets. Additionally, HPV-specific memory B- and T cells were determined by ELISPOT and HPV-specific antibody levels were measured by a VLP-based multiplex immunoassay.

Results: In both cohorts, the numbers of plasma cells expanded in the first week after both primary and tertiary vaccination. HPV16 and HPV18-specific antibody levels and memory B and T-cell responses were higher in the bivalent than in the nonavalent vaccinees one month post third vaccination. For HPV31 and HPV45-specific antibody levels this pattern was reversed. Monocytes showed an expansion one day after vaccination in both cohorts but were significantly higher in the bivalent vaccine cohort. Large heterogeneity in responses of the other cell subsets was observed between donors.

Conclusion: This pilot study showed a consistent response of monocytes and plasma cells after vaccination and a considerable variation in other circulating immune cells in both types of HPV vaccines between donors.

Keywords: B cells; T cells; antibodies; human papillomavirus (HPV); innate cells; vaccines.

Figure 1

Study design. Adult healthy pre-menopausal women (n=20) seronegative for HPV16/18/31/45 received either the bivalent or the nonavalent HPV vaccine according to a three-dose schedule, the second vaccination at 1 month and the third vaccination at 6 months. (A) In the preliminary experiments a blood sample was taken just before vaccination (day 0), and after the first vaccination at day 1, 2, 3, 6, 7, 10 and 14. Numbers of subsets of innate cells (blue), T cells (green), and B cells (red) were studied in 3 bivalent and 2 nonvalent recipients. The arrows indicate the specific time points used per cell type. (B) In the follow up (*) blood samples were drawn of the other 7 bivalent and 8 nonavalent vaccine recipients just before vaccination (day 0), and after the first vaccination at day 1, 3, and 7. At two months a second vaccination was given to all 20 study participants (N=10 per cohort). After 6 months (day 180) the third vaccination was given and blood samples were drawn at day 181 (1 day), 183 (3 days), 187 (7 days) and 208 (28 days post third vaccination. At day 0, 7, 180, 187 and 208 HPV IgG serum antibodies specific for HPV16/18/31 and 45 were determined. At 28 days post third vaccination (day 208) PBMCs were isolated for studying HPV-type specific memory B cells and T-cell responses.

Figure 2

Monocyte responses. (A) Kinetics of circulating monocytes upon bivalent (purple-blue) or nonavalent (orange-red) vaccination at day 0, 1, 3, 180, 181 and 183 post vaccination, presented as ratio compared to baseline cell numbers (cells/μL blood) depicted per individual participant. Participant identification by color is indicated on the right. The dotted vertical line split the results post 1^st and 3th vaccination. (B) Kinetics of monocyte subsets upon bivalent (left) or nonavalent (right) vaccinations at day 0, 1, 3, 180, 181 and 183, presented as the ratio in numbers compared to baseline numbers (cells/μL blood) indicated per vaccine cohort. Solid lines indicate the mean ratio of each cohort and the dotted lines represent the upper and lower confidence intervals. cMo= classical monocytes (blue), iMo= intermediate monocytes (red) and nMo= non-classical monocytes (green). At day 180, participants received the third HPV vaccination, therefore, this day served as a new ‘baseline’ moment to normalize the cell numbers from day 181 and 183, 1 and 3 days post third vaccination, respectively.

Figure 3

T-cell responses. (A) Kinetics of circulating CD4+ T-cells upon bivalent (purple-blue) and nonavalent (orange-red) vaccination at day 0, 3, 7, 180, 183, 187 and 208 post vaccinations presented as ratio in cell numbers compared to baseline cell numbers (cells/μL blood) depicted per participant. Participant identification by color is indicated on the right. At day 180, participants received the third HPV vaccination. therefore, this day served as a new ‘baseline’ moment to normalize the cell numbers from day 181 and 183, 1 and 3 days post third vaccination respectively. The dotted vertical line split the results post 1^st and 3th vaccination. (B) Numbers of HPV16/18/31/45-type specific memory CD4+ T cells producing IFN-γ at day 208, 28 days post third vaccination. PBMCs have been stimulated with HPV-type specific virus like particles (VLPs) for 4 days after which IFN-y producing cells have been enumerated by ELISPOT assay per 100.000 PBMCs. *indicates significant difference between the cohorts p<0.05.

Figure 4

B-cell responses. (A) Increases in numbers of plasma cells upon bivalent (purple-blue) and nonavalent (orange-red) vaccination at day 0, 7, 180, 187 and 208 post vaccinations presented as ratio in numbers of cells compared to baseline numbers (cells/μL blood) per participant, presented per vaccine cohort indicated. Participant identification by color is indicated on the right. At day 180, participants received the third HPV vaccination, therefore, this day served as a new ‘baseline’ moment to normalize the cell numbers from day 181 and 183, 1 and 3 days post third vaccination respectively. The dotted vertical line split the results post 1^st and 3th vaccination. (B) Numbers of HPV16/18/31/45 specific memory B cells at day 208, 28 days post third vaccination. Purified B-cells have been stimulated polyclonally for 5 days and subsequently numbers of HPV-type specific numbers of memory B-cells have been measured by ELISPOT assay in which plates have been coated with HPV-type specific virus like particles (VLPs).

Figure 5

IgG antibody responses. IgG antibodies concentrations (Luminex units/mL blood) specific for (A) HPV16, (B) HPV-18, (C) HPV-31 and (D) HPV-45 upon bivalent (blue) and nonavalent (red) vaccinations at day 0, 7, 14, (post first vaccination) and day 180, 187 and 208, respectively 0, 7 and 28 days post third vaccination. The lines represent the geometric mean concentrations (GMCs) over time per vaccine cohort. The dotted lines represent the upper and lower limit of the 95% confidence interval of the geomean. At day 180, participants received the third HPV vaccination. The high upper limit in the nonavalent group for HPV45 is due to the measurement of antibodies at day 14 instead of day 7 in just two donors, which gave a high variation.