T cell mediated immunity induced by the live-attenuated Shigella flexneri 2a vaccine candidate CVD 1208S in humans

Abstract

Background: Shigellosis persists as a public health problem worldwide causing ~ 165,000 deaths every year, of which ~ 55,000 are in children less than 5 years of age. No vaccine against shigellosis is currently licensed. The live-attenuated Shigella flexneri 2a vaccine candidate CVD 1208S (S. flexneri 2a; ΔguaBA, Δset, Δsen) demonstrated to be safe and immunogenic in phase 1 and 2 clinical trials. Earlier reports focused on humoral immunity. However, Shigella is an intracellular pathogen and therefore, T cell mediated immunity (T-CMI) is also expected to play an important role. T-CMI responses after CVD 1208S immunization are the focus of the current study.

Methods: Consenting volunteers were immunized orally (3 doses, 108 CFU/dose, 28 days apart) with CVD 1208S. T-CMI to IpaB was assessed using autologous EBV-transformed B-Lymphocytic cell lines as stimulator cells. T-CMI was assessed by the production of 4 cytokines (IFN-γ, IL-2, IL-17A and TNF-α) and/or expression of the degranulation marker CD107a in 14 volunteers (11 vaccine and 3 placebo recipients).

Results: Following the first immunization, T-CMI was detected in CD8 and CD4 T cells obtained from CVD 1208S recipients. Among CD8 T cells, the T effector memory (TEM) and central memory (TCM) subsets were the main cytokine/CD107a producers/expressors. Multifunctional (MF) cells were also detected in CD8 TEM cells. Cells with 2 and 3 functions were the most abundant. Interestingly, TNF-α appeared to be dominant in CD8 TEM MF cells. In CD4 T cells, TEM responses predominated. Following subsequent immunizations, no booster effect was detected. However, production of cytokines/expression of CD107a was detected in individuals who had previously not responded. After three doses, production of at least one cytokine/CD107a was detected in 8 vaccinees (73%) in CD8 TEM cells and in 10 vaccinees (90%) in CD4 TEM cells.

Conclusions: CVD 1208S induces diverse T-CMI responses, which likely complement the humoral responses in protection from disease. Trial registration This study was approved by the Institutional Review Board and registered on ClinicalTrials.gov (identifier NCT01531530).

Keywords: CVD 1208S; IpaB; Nanoparticles; Oral vaccine; Shigella flexneri 2a; T cell mediated immunity.

Figures

Fig. 1

Delivery of IpaB to B-LCL cells by LPS-nanoparticles. a Induction of necrosis/apoptosis in B-LCL by IpaB. Top panels: B-LCL exposed to media for 2 h. Center panels: in B-LCL exposed for 2 h to IpaB (Pacific Blue) two populations were identified (IpaB+ and IpaB++), IpaB+ B-LCL showed an increased in the percentage of cells undergoing necrosis (YeVID+, Anexin V+) (~ twofold compared to media), but no changes in the percentage of cells undergoing apoptosis (YeVID−, Anexin V+). IpaB++ cells showed an important increase in necrosis (~ tenfold compared to media), but not in apoptosis. Bottom panels: in B-LCL exposed for 2 h to to IpaB incorporated into the LPS-nanoparticle (LPS-IpaB-nanoparticles) the same two populations were identified (IpaB+ and IpaB++). However, no induction of necrosis was seen in the IpaB+ population. Also, the IpaB++ population showed a lower degree of necrosis than when exposed to IpaB alone. The data shown is from one representative volunteer of 4 evaluated. b After 18 h of incubation with the LPS-IpaB-nanoparticles, IpaB can be identified in 50–60% of B-LCL and that these cells (c) display upregulation of activation markers. Data in b is from one representative volunteer (#28) and data in c is a composite from four different vaccinees assayed in different days (#39, 24, 30 and 40)

Fig. 2

Gating strategy used for the identification of cytokine producing T cells. a The gating strategy used to identify CD4 and CD8 T cells. b displays the memory subsets defined by CCR7 and CD45RA in CD4 and CD8 T cells. c, d Examples of cytokine upregulation (Pre and post vaccination, days 0 and 28 respectively) in CD8 and CD4 T effector memory (TEM) cells of a representative vaccinated volunteer

Fig. 3

CD107a upregulation or cytokine production by TEM cells after CVD 1208S immunizations. a Displays CD107a upregulation [% net change (day 28-day0)] (degranulation marker) or cytokine production by CD8 TEM cells in CVD 1208S (left panel) and placebo (right panel) recipients 28 days after the first immunization. In b similar data is presented for CD4 TEM cells. c A summary of CD107a expression or cytokine production 28 days after each immunization (days 28, 56 and 84) in each vaccinated individual. The data is shown for CD8 and CD4 TEM cells. Volunteers who upregulated CD107a or exhibited increases in particular cytokines are indicated with a + sign in a green box (data derived from % net changes relative to day 0). Indicated in the blue boxes are the number (and percentage) of vaccinated volunteers that exhibited increases in at least one cytokine or upregulated CD107a after each immunization. The gray boxes show the number and corresponding percentage (%) of vaccinated volunteers that exhibited increases in at least one cytokine or upregulated CD107a after receiving the three vaccine doses

Fig. 4

Kinetics of cytokine production or CD107a expression in TEM cells. a Kinetics of CD107a expression (degranulation marker) or cytokine production by CVD1208S (left panels) and placebo (right panels) recipients in CD8 TEM cells. Filled triangles at the bottom indicate vaccination days. In the vaccinated group only the data of the individuals that showed increase in CD107a or cytokine expression relative to pre-vaccination (Fig. 3c) were used to generate these graphs. In the placebo group all the available data was used. The data is presented as the difference between the percentage of B-LCL cells loaded with IpaB and unstimulated B-LCL cells [LCL-IpaB − LCL (%)]. b Similar data from CD4 TEM cells. Arithmetic mean ± SD are displayed in the plots

Fig. 5

Multifunctional responses induced by CVD 1208S in TEM cells 28 days after the first immunization. a, e The comparison of cell that showed a single function (S+) (e.g., cells producing one cytokine or upregulating CD107a alone) versus multifunctional (MF) cells (e.g., cells exhibiting more than one function) in CD8 and CD4 TEM cells, respectively. b, f The comparison of cells that showed 2–5 simultaneous functions (2+, 3+, 4+ and 5+). For each cytokine, S+ and MF cells were compared. c CD8 TEM MF cells that produced TNF-α (in any combination) were more abundant than those producing TNF-α alone. These results were not observed in CD4 TEM cells (g). The arithmetic mean ± SD is indicated in all the groups. d, h The relative frequency (expressed as percentage) of the 5 most prevalent MF populations in CD8 TEM and CD4 TEM cells, respectively. The remaining MF populations were pooled in a single group indicated as other. *P < 0.05 (Mann–Whitney)

Fig. 6

Upregulation of CD107a and cytokine production by TEMRA and TCM cells after CVD 1208S immunizations. a CD107a upregulation [% net change (day 28 minus day0)] or cytokine production by CD8EMRA (left panel) and CD4TEMRA (right panel) cells in CVD 1208S recipients 28 days after the first immunization. b Similar data are presented for CD8 and CD4 TCM cells. c, d A summary of CD107a upregulation or cytokine production 28 days after each immunization (days 28, 56 and 84) in each vaccinated individual. The data shown corresponds to CD8 and CD4 TEMRA, as well as CD8 and CD4 TCM cells. Volunteers who upregulated CD107a or produced cytokines are indicated with a + sign in a green box (data are derived from % net changes relative to day 0). In the blue boxes the number (and percentage) of vaccinated volunteers that produced at least one cytokine or upregulated CD107a after each immunization are indicated. The gray boxes indicate the number (and percentage) of vaccinated volunteers that exhibited increases in at least one cytokine or upregulated CD107a after receiving the three vaccine doses

Fig. 7

TEMRA and TCM cells responses to CVD 1208S, 28 days after first immunization. a, c, e, g The comparison of single function (S+) versus multifunctional (MF) CD8 TEMRA, CD4 TEMRA, CD8 TCM, and CD4 TCM cells respectively, in response to CVD1208S immunization. b, d, f, h display the comparison of cells that showed more than one simultaneous function (e.g., increased cytokine production or CD107a upregulation) in CD8 TEMRA, CD4 TEMRA, CD8 TCM, and CD4 TCM cells, respectively. Means are indicated in each graph. *P < 0.05 (Mann–Whitney). i The relative frequency (expressed as percentage) of the 5 most prevalent MF populations in CD8 TEMRA, CD4 TEMRA, CD8 TCM, and CD4 TCM cells. The remaining MF populations were pooled in a single group indicated as other

Fig. 8

Increased cytokine production by CD8 TEM cells expressing integrin α4β7 among CVD 1208S vaccinees. a Identification of CD8 TEM cells expressing integrin α4β7. b Gating example to evaluate the expression of IFN-γ and TNF-α in integrin α4β7− and integrin α4β7+ CD8 TEM cells among individuals who exhibited increased production of these cytokines after the first immunization (day 28). The data shown in b are from volunteer #24. c Formula used to calculate the ratio (R) of cytokine production (IFN-γ and TNF-α) among integrin α4β7− and integrin α4β7+ CD8 TEM cells. c Also displays an example of the calculations using data from volunteer #24, i.e., each of the 3 quadrants (Q1, Q2 and Q3) containing single or double positive cells). d A summary of the data in integrin α4β7− and integrin α4β7+ in CD8 TEM cells from the vaccinated individuals that increased TNF-α and IFN-γ 28 days after the first vaccination