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. 2022 Mar 29:13:823652.
doi: 10.3389/fimmu.2022.823652. eCollection 2022.

Adult Memory T Cell Responses to the Respiratory Syncytial Virus Fusion Protein During a Single RSV Season (2018-2019)

Brittani N Blunck  1 , Laura S Angelo  1 , David Henke  1 , Vasanthi Avadhanula  1 , Matthew Cusick  2 , Laura Ferlic-Stark  1 , Lynn Zechiedrich  1   3   4 , Brian E Gilbert  1 , Pedro A Piedra  1   5
Affiliations

Adult Memory T Cell Responses to the Respiratory Syncytial Virus Fusion Protein During a Single RSV Season (2018-2019)

Brittani N Blunck et al. Front Immunol. .

Abstract

Respiratory Syncytial Virus (RSV) is ubiquitous and re-infection with both subtypes (RSV/A and RSV/B) is common. The fusion (F) protein of RSV is antigenically conserved, induces neutralizing antibodies, and is a primary target of vaccine development. Insight into the breadth and durability of RSV-specific adaptive immune response, particularly to the F protein, may shed light on susceptibility to re-infection. We prospectively enrolled healthy adult subjects (n = 19) and collected serum and peripheral blood mononuclear cells (PBMCs) during the 2018-2019 RSV season. Previously, we described their RSV-specific antibody responses and identified three distinct antibody kinetic profiles associated with infection status: uninfected (n = 12), acutely infected (n = 4), and recently infected (n = 3). In this study, we measured the longevity of RSV-specific memory T cell responses to the F protein following natural RSV infection. We stimulated PBMCs with overlapping 15-mer peptide libraries spanning the F protein derived from either RSV/A or RSV/B and found that memory T cell responses mimic the antibody responses for all three groups. The uninfected group had stable, robust memory T cell responses and polyfunctionality. The acutely infected group had reduced polyfunctionality of memory T cell response at enrollment compared to the uninfected group, but these returned to comparable levels by end-of-season. The recently infected group, who were unable to maintain high levels of RSV-specific antibody following infection, similarly had decreased memory T cell responses and polyfunctionality during the RSV season. We observed subtype-specific differences in memory T cell responses and polyfunctionality, with RSV/A stimulating stronger memory T cell responses with higher polyfunctionality even though RSV/B was the dominant subtype in circulation. A subset of individuals demonstrated an overall deficiency in the generation of a durable RSV-specific adaptive immune response. Because memory T cell polyfunctionality may be associated with protection against re-infection, this latter group would likely be at greater risk of re-infection. Overall, these results expand our understanding of the longevity of the adaptive immune response to the RSV fusion protein and should be considered in future vaccine development efforts.

Keywords: fusion protein; infection; memory T cell; peptide library; polyfunctionality; respiratory syncytial virus (RSV); viral immunity.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Total T cell responses to the RSV F protein peptide libraries as a function of RSV infection status and study visit. (A–D) Individual functional marker expression following stimulation with the RSV/A F (RSV FA) protein peptide library by RSV infection status: uninfected (n = 12), acutely infected (n = 4), and recently infected (n = 3). PBMCs from adult volunteers were stimulated with the RSV FA protein peptide library and the expression of CD107a, IFNγ, TNFα, and PD-1 were measured relative to the unstimulated controls by flow cytometry. These values are reported as percent positive of total CD3+ T cells. Each symbol represents the response from a single individual. The thick horizontal bar indicates the mean of all responses within each group at that visit. A significant pairwise comparison of mean percentage difference between visits within a group is denoted by a thin horizontal bar with *P ≤ 0.05, **P ≤ 0.01. (E) Polyfunctional T cell responses to RSV FA protein peptide library by RSV infection status. Simplified Presentation of Incredibly Complex Evaluations (SPICE) software was used for the identification of total T cells expressing the various activation markers. Pie charts show the frequency in which PBMCs produced the various combinations of the activation markers CD107a, IFNγ, and TNFα; or expressed PD-1 alone. Background (determined from the media-stimulated negative controls) was subtracted from all samples and negative values were set to zero. Surrounding arcs denote the specific markers produced by the cells in each pie segment. Representative negative and positive controls across all study visits are boxed.
Figure 2
Figure 2
CD4+ Memory T cell responses to the RSV F protein peptide libraries by RSV infection status and study visit. (A–D) Individual functional marker expression by RSV infection status: uninfected (n = 12), acutely infected (n = 4), and recently infected (n = 3). PBMCs were stimulated in vitro with RSV FA (A, B) or RSV FB (C, D) F protein peptide libraries. Marker expression is shown as percentage of total CD4+ memory T cells. symbol represents the response from a single individual. The thick horizontal bar indicates the mean of all responses within each group at that visit. A significant pairwise comparison of mean percentage difference between visits within a group is denoted by a thin horizontal bar with *P ≤ 0.05. (E, F) Polyfunctional CD4+ memory T cell responses to RSV FA or RSV FB protein peptide libraries as a function of RSV infection status and study visit. Simplified Presentation of Incredibly Complex Evaluations (SPICE) analysis was performed for the identification of CD4+ memory T cells expressing multiple activation markers. Pie charts show the frequency in which PBMCs produced the various combinations of the activation markers CD107a, IFNγ, and TNFα; or expressed PD-1 alone. Background (determined from the media-stimulated negative controls) was subtracted from all samples and negative values were set to zero. Surrounding arcs denote the specific markers produced by the cells in each pie segment. Representative negative and positive controls across all study visits are boxed.
Figure 3
Figure 3
CD8+ Memory T cell responses to RSV F protein peptide libraries as a function of RSV infection status and study visit. Polyfunctional CD8+ Memory T cell responses to (A) RSV FA or (B) RSV FB protein peptide libraries by RSV infection status: uninfected (n = 12), acutely infected (n = 4), and recently infected (n = 3). Simplified Presentation of Incredibly Complex Evaluations (SPICE) analysis was performed for the identification of CD8+ memory T cells expressing multiple cytokines. Pie charts show the frequency in which PBMCs produced the various combinations of the activation markers CD107a, IFNγ, and TNFα; or expressed PD-1 alone. Background (determined from the media-stimulated negative controls) was subtracted from all samples and negative values were set to zero. Surrounding arcs denote the specific markers produced by the cells in each pie segment. Representative negative and positive controls across all study visits are boxed.
Figure 4
Figure 4
RSV subtype-specific differences in total T cell responses to RSV F protein peptide libraries for all study subjects. (A–D) Individual functional marker expression following stimulation with either peptide library (n = 19). PBMCs were stimulated with either RSV FA or RSV FB peptide library and expression of CD107a, IFNγ, TNFα, and PD-1 were measured by flow cytometry and reported as a percentage of total T cells. Each symbol represents the response from a single individual. The thick horizontal bar indicates the mean of all responses within each group at that visit. A significant pairwise comparison of mean percentage difference between visits within a group is denoted by a thin horizontal bar with *P ≤ 0.05, **P ≤ 0.01. V1, Visit 1; V2, Visit 2; V3, Visit 3. (E) Polyfunctionality of activation markers in total T cell responses as a function of stimulation type and study visit. Pie charts show the frequency in which PBMCs produced the various combinations of the activation markers CD107a, IFNγ, and TNFα; or expressed PD-1 alone. Background (determined from the media-stimulated negative controls) was subtracted from all samples and negative values were set to zero. Surrounding arcs denote the specific markers produced by the cells in each pie segment. Representative negative and positive controls across all study visits are boxed. (F) Total polyfunctionality of total T cells by stimulation and study visit. Pie segments indicate frequency of cells producing combinations of all four functional markers CD107a, IFNγ, and TNFα and PD-1. Background (determined from the media-only negative controls) was subtracted from all samples and negative values were set to zero.
Figure 5
Figure 5
RSV subtype-specific differences in CD4+ Memory T cell responses to RSV F protein peptide libraries for all study participants. (A–D) Individual functional marker expression. PBMCs were stimulated in vitro with RSV FA (A, B) or RSV FB (C, D) peptide libraries (n = 19). Expression of CD107a, IFNγ, TNFα, and PD-1 was measured by flow cytometry and reported as a percentage of CD4+ memory T cells. Each symbol represents the response from a single individual. The thick horizontal bar indicates the mean of all responses within each group at that visit. A significant pairwise comparison of mean percentage difference between visits within a group is denoted by a thin horizontal bar with *P ≤ 0.05. V1, Visit 1; V2, Visit 2; V3, Visit 3. (E) Polyfunctionality of activation markers in CD4+ memory T cell responses by stimulation and study visit. Pie charts represent the mean frequencies of responding CD4+CD45RO+ T cells following stimulation with RSV FA or RSV FB protein peptide library. Pie charts indicate frequency of cells producing combinations of the activation markers CD107a, IFNγ, and TNFα or expressing PD-1 alone. Background (determined from the media-only negative controls) was subtracted from all samples and negative values were set to zero. (F) Total polyfunctionality of CD4+ memory T cells by stimulation and study visit. Pie segments indicate frequency of cells producing combinations of all four functional markers CD107a, IFNγ, and TNFα and PD-1. Background (determined from the media-only negative controls) was subtracted from all samples and negative values were set to zero.
Figure 6
Figure 6
RSV subtype-specific differences in CD8+ Memory T cell responses to RSV F protein peptide libraries for all study participants. (A–D) Individual functional marker expression. PBMCs were stimulated in vitro with RSV FA (A, B) or RSV FB (C, D) peptide libraries (n = 19). Expression of CD107a, IFNγ, TNFα, and PD-1 was measured by ICS and reported as a percentage of CD8+ memory T cells. Each symbol represents the response from a single individual. The thick horizontal bar indicates the mean of all responses within each group at that visit. A significant pairwise comparison of mean percentage difference between visits within a group is denoted by a thin horizontal bar with *P ≤ 0.05. V1, Visit 1; V2, Visit 2; V3, Visit 3. (E) Polyfunctionality of activation markers in CD8+ memory T cell responses by stimulation and study visit. Pie charts represent the mean frequencies of responding CD8+CD45RO+ T cells following stimulation with RSV FA or RSV FB peptide library. Pie charts indicate frequency of cells producing combinations of the activation markers CD107a, IFNγ, and TNFα or expressing PD-1 alone. Background (determined from the media-only negative controls) was subtracted from all samples and negative values were set to zero. (F) Total polyfunctionality of CD8+ memory T cells by stimulation and study visit. Pie segments indicate frequency of cells producing combinations of all four functional markers CD107a, IFNγ, and TNFα and PD-1. Background (determined from the media-only negative controls) was subtracted from all samples and negative values were set to zero.
Figure 7
Figure 7
Correlation between RSV FA T cell and neutralizing antibody responses. A significant linear relationship between T cell score and neutralizing antibody score is denoted by a correlation coefficient (r) with *P ≤ 0.05. (A) Correlation between RSV FA T cell score with neutralizing antibody score by study visit (n = 19). (B) Correlation between RSV FA T cell scores with neutralizing antibody score by RSV infection status and study visit. Uninfected (n = 12), acutely infected (n = 4), or recently infected (n = 3) individuals are shown.
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