Yellow Fever Vaccination Elicits Broad Functional CD4 ⁺ T Cell Responses That Recognize Structural and Nonstructural Proteins

Abstract

bYellow fever virus (YFV) can induce acute, life-threatening disease that is a significant health burden in areas where yellow fever is endemic, but it is preventable through vaccination. The live attenuated 17D YFV strain induces responses characterized by neutralizing antibodies and strong T cell responses. This vaccine provides an excellent model for studying human immunity. While several studies have characterized YFV-specific antibody and CD8 ؉ T cell responses, less is known about YFV-specific CD4 ؉ T cells. Here we characterize the epitope specificity, functional attributes, and dynamics of YFV-specific T cell responses in vaccinated subjects by investigating peripheral blood mononuclear cells by using HLA-DR tetramers. A total of 112 epitopes restricted by seven common HLA-DRB1 alleles were identified. Epitopes were present within all YFV proteins, but the capsid, envelope, NS2a, and NS3 proteins had the highest epitope density. Antibody blocking demonstrated that the majority of YFV-specific T cells were HLA-DR restricted. Therefore, CD4؉ T cell responses could be effectively characterized with HLA-DR tetramers. Ex vivo tetramer analysis revealed that YFV-specific T cells persisted at frequencies ranging from 0 to 100 cells per million that are detectable years after vaccination. Longitudinal analysis indicated that YFV-specific CD4 ؉ T cells reached peak frequencies, often exceeding 250 cells per million, approximately 2 weeks after vaccination. As frequencies subsequently declined, YFV-specific cells regained CCR7 expression, indicating a shift from effector to central memory. Cells were typically CXCR3 positive, suggesting Th1 polarization, and produced gamma interferon and other cytokines after reactivation in vitro. Therefore, YFV elicits robust early effector CD4؉ T cell responses that contract, forming a detectable memory population. Members of the family Flaviviridae such as Yellow fever virus (YFV) are important causes of illness both historically and at present, causing a significant health burden in areas where yellow fever is endemic (1). Yellow fever (YF) produces symptoms ranging from a mild flu-like illness to hemorrhagic fever and organ failure, but infection is preventable through vaccination (2, 3). The YF vaccine uses a live attenuated virus (17D) and is safe and extremely effective, generating robust antibody responses that persist for decades (4, 5). The vaccine is known to elicit neutralizing antibodies and strong T cell responses in nearly all recipients (6, 7). Therefore, the YFV 17D vaccine is an excellent and important model for studying human antiviral immunity. Multiple studies have investigated the attributes and dynamics of CD4 ϩ and CD8 ϩ T cell responses following YF vaccination (8-12). By using a variety of readouts, these studies demonstrated that YFVspecific CD8 ϩ T cell responses are polyfunctional, exhibit distinct surface phenotypic markers, and peak approximately 2 to 4 weeks after vaccination. Blom et al. evaluated both CD4 ϩ and CD8 ϩ T cell responses by examinin...

Dynamics of the Cytotoxic T Cell Response to a Model of Acute Viral Infection

“…Interestingly, almost all of the clones that were markedly expanded in the total PBMC sample from day 14 postvaccination (compared to the corresponding day 0 prevaccination total PBMC sample from the same individual) were classified as YFV-induced CD8 ϩ T cells by the combination of flow cytometry and statistical analysis. In fact, we observed very few clonally expanded T cells in the periphery that were not identified as YFV-induced clones, in agreement with previous reports showing that while CD8 ϩ T cells greatly expand in response to vaccination with YFV, the CD4 ϩ expansion is much less dramatic (6,30,32,33). It is important to consider that the sampling depth used in this study limits the detection of bystander CD8…”

“…ϩ -, and CD8 ϩ -based antiviral immune response (6,9,10,12,16,(30)(31)(32)(33)(34). Although some of these studies used either peptide pools or tetramer-based approaches to address the response to a limited number of viral epitopes (6,9,33), or calculated the percentage of different immune cellular compartments that represented clones induced by vaccination with YFV (reviewed in reference 16), the clonal breadth and complexity of the response to a viral infection has been beyond the reach of available methods.…”

“…ϩ T cells specific for a limited number of YFV epitopes in peripheral blood (from 0 to 100 cells per million CD4 ϩ T cells) and established that YFV-specific T cells, which display a predominant Th1-like memory phenotype, occur at ϳ10-to 100-foldhigher frequencies in vaccinated versus unvaccinated individuals, depending on the time point considered (33).…”

“…Some analyses have revealed that cytokineproducing YFV-specific CD4 ϩ T cells can be detected as early as day 2 postvaccination and that they return to baseline by day 28, suggesting that the kinetics of CD4 ϩ T cells precede those of CD8 ϩ T cells (12,32). Recently, James et al used class II HLA-DR restricted, YFV-specific tetramers to characterize the CD4 ϩ response to YFV in more depth, showing that all 10 proteins in the YF virus genome contain antigenic epitopes recognized by CD4 ϩ T cells (33). This study also revealed a wide range of frequencies of…”

Initial viral load determines the magnitude of the human CD8 T cell response to yellow fever vaccination

“…Because yellow fever is not endemic to the United States, immunization with YFV-17D induces a primary immune response (30,31). Previous work with YFV-17D has identified CD8 T cells specific for some of the YFV epitopes and defined the stages of expansion, contraction, and memory maintenance (32)(33)(34)(35)(36)(37)(38). We now know that YFV stimulates a polyfunctional, broadly targeting, and long-lasting CD8 T-cell response.…”