Adjuvant-enhanced CD4 T Cell Responses are Critical to Durable Vaccine Immunity

Abstract

Protein-based vaccines offer a safer alternative to live-attenuated or inactivated vaccines but have limited immunogenicity. The identification of adjuvants that augment immunogenicity, specifically in a manner that is durable and antigen-specific, is therefore critical for advanced development. In this study, we use the filovirus virus-like particle (VLP) as a model protein-based vaccine in order to evaluate the impact of four candidate vaccine adjuvants on enhancing long term protection from Ebola virus challenge. Adjuvants tested include poly-ICLC (Hiltonol), MPLA, CpG 2395, and alhydrogel. We compared and contrasted antibody responses, neutralizing antibody responses, effector T cell responses, and T follicular helper (Tfh) cell frequencies with each adjuvant's impact on durable protection. We demonstrate that in this system, the most effective adjuvant elicits a Th1-skewed antibody response and strong CD4 T cell responses, including an increase in Tfh frequency. Using immune-deficient animals and adoptive transfer of serum and cells from vaccinated animals into naïve animals, we further demonstrate that serum and CD4 T cells play a critical role in conferring protection within effective vaccination regimens. These studies inform on the requirements of long term immune protection, which can potentially be used to guide screening of clinical-grade adjuvants for vaccine clinical development.

Keywords: Adjuvant; BME, beta mercaptoethanol; CD, cluster of differentiation; DSCF, Dwass, Steel, Critchlow-Fligner; Durable protection; ELISA, Enzyme linked immunosorbent assay; ELISPOT, enzyme-linked immunospot assay; Ebola virus; FACS, fluorescence activated cell sorting; FBS, fetal bovine serum; GP, glycoprotein; IACUC, Institutional Animal Care and Use Committee; IM, intramuscular; IP, intraperitoneal; IQR, interquartile range; Immune correlates; LN, lymph node; MPLA, monophosphoryl lipid A; NAb, neutralizing antibody; Ns, not significant; PBS, phosphate buffered saline; PRR, pattern recognition receptor; Pfu, plaque forming unit; PsVNA, pseudovirion neutralization assay; TLR, Toll-like receptor; USAMRIID, United States Army Medical Research Institute of Infectious Diseases; VLP, virus-like particle; Vaccine; ma-EBOV, mouse-adapted Ebola virus.

Supplemental Fig. 1 Effective adjuvants increase the frequency of whole glycoprotein-specific T cells. IFNγ ELISPOT analysis of splenocytes from C57BL/6 mice vaccinated two times with indicated vaccine and adjuvant combination. Splenocytes were collected on day 4 or day 147 after the second vaccination, which occurred on day 21. Data is pooled from two separate experiments each containing 3–4 mice per group, as described in Fig. 3a and d. Recombinant Ebola GP was used as antigen. Mean and IQR shown. Comparisons between animals receiving VLP with or with adjuvant are shown, where “*” indicates 0.01 < p < 0.05 (Kruskal–Wallis test). Supplemental Fig. 2 CD4-deficient mice are not protected from challenge after vaccination with VLP. (A) Mice were treated IM twice with saline or VLP. Four weeks after the second treatment, mice were challenged IP with 1000 pfu of ma-EBOV. Closed symbols represent wild type C57BL/6 mice and open symbols indicated CD4-deficient mice. (B) Two weeks after the second vaccination and one week prior to challenge, blood was collected from vaccinated animals and evaluated for anti-GP IgG antibody titers. N = 5–10/group with survival data shown pooled from two separate studies.

Fig. 1

Adjuvants impact the durability of protection conferred by eVLP vaccination. (a) C57BL/6 mice were vaccinated IM two times with 10 μg of eVLP and challenged four (short-term) weeks or twenty-two (long-term) weeks after the vaccine boost. Data in A are pooled from 8 individual studies with 6–10 animals/group. Fisher's exact test: survival in the short-term group was significantly higher than in the long-term group (p < 0.0001). (b) C57BL/6 mice were vaccinated IM two times with 10 μg of eVLP and challenged at the indicated days after the second vaccination. n = 9 or 10/group. Cochran-Armitage test: percentage surviving declined as time to challenge increased (p = 0.0046). There was a significant difference (p = 0.03) between survival on Day 77 and Day 175. (c) Serum samples collected from animals in (B) one week prior to challenge were subjected to an ELISA for the evaluation of anti-GP IgG, IgG1, and IgG2c antibody titers. Red symbols indicate titers of animals that succumbed to challenge while black indicate titers of survivors; red symbols with black outlines indicate that one of these two animals succumbed to challenge, but animal tags were indeterminate after challenge. Median and IQR shown. (d) C57BL/6 mice were vaccinated two times (IM) with VLP, with or without the indicated adjuvants. Animals were challenged twenty-two weeks after the vaccine boost. Data in D are pooled from at least 4 separate studies with a total of at least 35 animals per group. P-values comparing VLP alone to vaccination with VLP and adjuvant are shown, calculated using Fisher's exact tests with stepdown Bonferroni correction. V = VLP, VP = VLP + PolyICLC, VC = VLP + CpG, VM = VLP + MPLA, and VA = VLP + alhydrogel.

Fig. 2

Adjuvants have variable impact on IgG subclasses and antibody neutralization. (a) Serum was collected 14 days and 147 days after the vaccine boost (days 35 and 168, respectively) and evaluated for anti-GP IgG, IgG1, IgG2c, and IgG3 levels using an ELISA. Data shown are pooled from at least two separate experiments per group. (b) Pairwise comparison using DSCF multiple pairwise comparison was used and p values greater than 0.05 are shown as “ns”. (c) Summary of results shown in A, B and D. (d) Neutralizing antibody titers were evaluated using the PsVNA, with titers giving 80% neutralization shown; median and IQR shown. Samples within each group were selected randomly from three separate studies for evaluation in the assay. Pairwise comparison using post-hoc Tukey's studentized range test procedure was used to evaluate differences between groups at both days 35 and day 168, where “*” indicates 0.01 < p < 0.05, “**” indicates 0.001 < p < 0.01, “***” indicates 0.0001 < p < 0.001, and “****” indicates p < 0.0001.

Fig. 3

Effective adjuvants increase the frequency of antigen-specific T cells. (a) IFNγ ELISPOT analysis of splenocytes from C57BL/6 mice vaccinated two times with indicated vaccine and adjuvant combination. Splenocytes were collected on day 4 after the second vaccination. Data is pooled from four separate experiments each containing 2–3 mice per group; median with IQR shown. (B&C) Frequency of IFNγ +, IL2 +, or TNFα + cells after vaccination with VLP and polyICLC or VLP and CpG; median shown. Gating is on viable T cells and then CD4 + CD44high T cells (b) or CD8 + CD44high T cells (c). (d) IFNγ ELISPOT analysis of splenocytes from C57BL/6 mice vaccinated with indicated vaccine and adjuvant combination. Animals received the standard two vaccinations and then received a third vaccine boost 22 weeks after the second vaccination. Splenocytes were collected 4 days later. Data is pooled from two separate experiments each containing 3–4 mice per group; median with IQR shown. (e) Four weeks after the second vaccination, splenocytes were collected from animals and T cells were isolated using negative bead selection. T cells were then sorted to collect central memory, effector memory, and CD44int/low cell populations. Gating strategy is shown. (f) Sorted T cells were cultured with peptide-exposed, T cell-depleted, naïve splenocytes and the frequency of cytokine-positive CD4 and CD8 T cells in each sorted population was quantified after 5 days of culture. Gating is on CD4 or CD8 T cells and data shown are the frequency of cells expressing IFNγ, TNFα, or IL2; median is shown. “*” indicates 0.01 < p < 0.05.

Fig. 4

CD8 T cell deficiency does not impact short term or long term survival of vaccinated C57BL/6J mice. (a) Mice were treated IM twice with saline, VLP, VLP and polyICLC, or VLP and CpG. Four weeks after the second vaccination, mice were challenged. Closed symbols represent wild type C57BL/6J mice and open symbols indicate CD8-deficient mice. (b) Mice were vaccinated on the same schedule as in A, but challenge occurred 22 weeks after the second vaccination. (c) Two weeks after the second vaccination and 1 week prior to challenge, blood was collected from vaccinated animals and evaluated for anti-GP IgG antibody titers. (D) A subset of vaccinated animals was euthanized 4 days after the vaccine boost to evaluate CD4 + T cell responses. Median response of C57BL/6J mice and CD8-deficient mice is shown. N = 8–10 per treated group for A–C and D presents data pooled from two separate evaluations of 4–8 mice per group each. “*” indicates 0.005 < p < 0.05.

Fig. 5

Tfh frequencies are increased by adjuvants associated with protection from challenge. C57BL/6 mice were vaccinated a single time IM with VLP, with or without adjuvant. Seven days after vaccination, the draining popliteal LN was isolated. (a) Untouched T cells were isolated from the LN (n = 3 or 4/group). RNA was isolated from the purified T cells and pooled for each vaccination group. Samples were then evaluated using the SABiosciences T and B cell activation PCR array in triplicate. For the volcano plot, the fold difference is the average of triplicate, and the p value was calculated comparing animals vaccinated with VLP to those vaccinated with VLP and polyICLC. (b) Seven days after vaccination, cells from the draining LN were evaluated for Tfh populations. Cells were gated on viable lymphocytes after doublet exclusion, and then on CD4 + T cells expressing CXCR5 and PD1. Median and IQR shown. (c) CD3 + CD4 + CXCR5 + PD1 + cell population in mice vaccinated with VLP and polyICLC. ICOS and Bcl6 expression of this population is shown. Data shown are pooled from three separate vaccination experiments. Comparisons between animals receiving VLP with or with adjuvant are shown, where “*” indicates 0.01 < p < 0.05 (Kruskal–Wallis test).