Divergent memory responses driven by adenoviral vectors are impacted by epitope competition

Abstract

Adenoviral vectors induce robust epitope-specific CD8⁺ T cell responses. Within the repertoire of responses generated both conventional memory evolution and the phenomenon of memory inflation are seen. The rules governing which epitopes inflate are not fully known, but may include a role for both antigen processing and competition. To investigate this, we looked at memory generated from vectors targeting the Gp33-41 (KAVYNFATC/K9C) epitope from the gp of lymphocytic choriomeningitis virus (LCMV) in mice. This well-described epitope has both the Gp33-41 and Gp34-41 epitopes embedded within it. Vaccination with a full-length gp or a minigene Ad-Gp33/K9C vector-induced conventional memory responses against the immunodominant Gp33/K9C epitope but a strong inflationary response against the Gp34/A8C epitope. These responses showed sustained in vivo function, with complete protection against LCMV infectious challenge. Given the unexpected competition between epitopes seen in the minigene model, we further tested epitope competition using the full-length Ad-LacZ (β-galactosidase) model. Generation of an Ad-LacZ vector with a single amino acid disruption of the inflationary β-gal_96-103 /D8V epitope transformed the β-gal_497-504 /I8V epitope from conventional to inflationary memory. This work collectively demonstrates the importance of epitope competition within adenoviral vector inserts and is of relevance to future studies using adenoviral vectored immunogens.

Keywords: LCMV; adenoviral vectors; immunoproteasome; memory inflation; minigenes.

Figure 1

(A) Gp33/K9C and Gp34/A8C tetramer‐specific responses by flow cytometry in blood at time‐points 21, 50, 75, 100, and 150 post‐immunization with Ad‐Gp33 compared to naïve mice. Data pooled from three separate experiments up to day 50: n = 13, Day 75 onwards is one experiment (n = 4). Mean ± SEM shown. (B) A8C and K9C tetramer‐specific CD8⁺ T cells showing CD44, CD62L, and CD127 positivity by flow cytometry at day 21 and 50‐post immunization with Ad‐Gp33. Data shown from one experiment (n = 6), and representative of two independent experiments (n = 12). Mean ± SEM shown. (C) A8C and K9C tetramer‐specific responses by flow cytometry in organs at day 75‐post immunization with Ad‐Gp33, compared to naïve mice. Data shown from one experiment (n = 4), and representative of two independent experiments (n = 8). Mean ± SEM shown. (D) Demonstrates LCMV plaque assays performed on spleens (i) and lymph nodes (inguinal) (ii) at day 4‐post LCMV_WE challenge of C57BL/6 mice pre‐immunized (day 50) with Ad‐Gp33 and Ad‐Gp compared to naïve mice. Data are pooled from two separate experiments (n = 6). Mean ± SEM shown.

Figure 2

(A) Gp34/A8C (i) and Gp33/K9C (ii) tetramer‐specific responses by flow cytometry in blood at time‐points 14, 21, and 50 post‐immunization with Ad‐Gp34 compared to Ad‐Gp33 and naïve mice. Data pooled from two separate experiments: n = 12. Mean ± SEM shown. Unpaired t‐test with Welch's correction: *p ≤ 0.01. (B) A8C and K9C tetramer‐specific CD8⁺ T cells showing CD44, CD62L, and CD127 positivity by flow cytometry at day 21 and 50‐post immunization with Ad‐Gp34. Data shown from one experiment (n = 6), and representative of two independent experiments (n = 12). Mean ± SEM shown. (C) Gp34/A8C (i) and Gp33/K9C (ii) tetramer‐specific responses by flow cytometry in organs at day 75‐post immunization with Ad‐Gp34, compared to Ad‐Gp33 immunized and naïve mice. Data taken from two pooled experiments (n = 12). Mean ± SEM shown.

Figure 3

(A) β‐gal_96‐103/D8V (i) and β‐gal_497‐504/I8V (ii) tetramer‐specific responses by flow cytometry in blood at time‐points 14, 21, 50, and 91 post‐immunization with either Ad‐Disrupted D8V (LacZ) or Ad‐LacZ compared to naives. Data pooled from two separate experiments up to day 50: n = 12 and a single experiment at day 91: n = 6. Mean ± SEM shown. Unpaired t‐test with Welch's correction: ***p ≤ 0.0006. (B) I8V tetramer‐specific CD8⁺ T cells following Ad‐disrupted D8V (LacZ) and D8V tetramer‐specific CD8⁺ T cells following Ad‐LacZ immunization showing CD44, CD62L, and CD127 positivity by flow cytometry at day 21 and 50‐post immunization. Data shown from one experiment (n = 6), and representative of two independent experiments (n = 12). Mean ± SEM shown. (C) D8V (i) and I8V (ii) tetramer‐specific responses by flow cytometry in organs at day 75‐post immunization with Ad‐disrupted D8V (LacZ), compared to Ad‐LacZ immunized and naïve mice. Data from two pooled experiments (n = 12). Mean ± SEM shown.