Making memories with Shingrix

Shingles, caused by reactivation of the varicella-zoster virus (VZV) that also causes chickenpox in children, can lead to debilitating nerve pain, particularly in older adults. The Recombinant Zoster vaccine (RZV, commercially known as SHINGRIX^TM) offers strong protection against shingles, but there’s still a lot we don’t understand about how the immune system prevents reactivation of VZV after administration of Shingrix.

In a new study in Nature Communications, UW/Fred Hutch Cancer Consortium Members Drs. Emily Ford and David Koelle collaborated with Drs. Xiaomin Wen and Bill Kwok at the Benaroya Research Institute to perform a longitudinal investigation into memory CD4 T cell responses to RZV vaccination. “RZV stands out as the first licensed therapeutic vaccine – it prevents reactivation of an endogenous virus that has been latent and hiding in patients for several decades after childhood chickenpox – and as having extraordinary efficacy of > 90% and remarkable durability of at least 10 years,” says Koelle, whose lab at UW studies T cell responses to viral pathogens.

RZV is made of a fragment of the extracellular domain of single protein on the surface of VZV called glycoprotein E (gE) attached to a specialized adjuvant system that helps stimulate innate immune cells, a critical step in developing long term immunity. When RZV is first administered, those members of the innate immune response teach T cells, specifically CD4 T helper cells, to recognize the gE protein as foreign, activating a cascade of events that additionally produces the high-affinity antibodies that make the vaccine so effective. As shingles is most severe in patients with a CD4 T cell deficiency, and RZV is known to increase gE-specific CD4 T helper cells in the blood, the investigators collected blood from patients immediately prior to and 14 days after each of their two doses of Shingrix and studied their CD4 T cells.

A tetramer-guided epitope mapping (TGEM) approach tested 68 possible epitopes from gE and identified 14 that were MHC class II-restricted, meaning that they are recognized by CD4⁺ T cells in the context of MHC class II molecules, with the four most dominant epitopes selected for further analysis. Epitope restriction is very similar to the shape-sorting toy given to infants, where they match different shapes (like circles, squares, triangles) to their corresponding holes in a box or container. In this case, the goal is to search for the hole/shape pair that causes the largest increase in CD4 T cells, which is determined by finding the conditions with the greatest amount of fluorescence from the loaded tetramer.

Next up, they wanted to see how many of these antigen-specific T cells could be found in their seven healthy participants using a tetramer activation assay, which they optimized for the best four combinations of HLA-DRB1 and the chosen ~30 amino acid peptides of gE. “The team not only observed large increases in the abundance of shingle virus-specific CD4 T cells over the course of vaccination, but also unexpected and persistent changes in the mRNA and protein patterns in these cells,” stated Koelle. Ford added that they “showed that most of the boosted cells were memory cells from primary infection and maintained persistent transcriptional changes a year after the vaccine series.”

Using single-cell sequencing of just the tetramer-bound activated CD4 T cells, the group next asked whether there where any differences in T Cell Receptor (TCR) usage and diversity as a result of vaccination. Ford further elaborated, “This method, which incorporated short ex vivo stimulation with tetramers and CD154 detection, provided high-quality single-cell RNA sequencing data, enabling a detailed characterization of vaccine-induced T cell activation and memory formation.”

While genetic variance in MHC molecules plays a critical role in the diversity of immune activation, TCR genes are also highly variable, which is a necessary feature for them to be able to recognize so many different pathogens. The diversity in TCRs is generated through a process called V(D)J recombination, which mixes and matches different gene segments to create a wide variety of TCRs.

The TCR is made up of two chains, called α and β chains in most T cells. The genes for these chains are located in the TCRα and TCRβ loci on the genome, and while sharing a TCRαβ clonotype was common within an individual, clonotype sharing between individuals was common (although it was observed). Using their single-cell data and a computational program called TCRdist, they also investigated TCR sequence similarity and found 37 clusters of biochemically similar clonotypes with identical epitope specificity, with 24 of the clusters representing multiple individuals. Interestingly, it became apparent that the less dominant clonotypes expanded more than the predominant clonotypes, although both sets remained after a year. Koelle comments, “computational analyses of the sequences of T cell receptors showed sequence convergence between individuals, allowing some simplicity to emerge from the ‘noise’ inherent in this system and potentially enabling future use of T cell receptor sequencing as an immune monitoring tool for vaccination studies.”

The group also took a deep dive into the gene expression profiles for the isolated T cells. They compared T cells with identical clonotypes between time points and found that 2 weeks after the first dose, genes related to T cell activation, trafficking and differentiation were increased. Speaking on the transcriptional changes, Ford speculates about whether “there’s a potential self-regulatory aspect, which is very interesting,” which arose from an apparent increase in negative regulation of T cell responses in addition to activation.

Thinking forward, Koelle wonders “Can we translate these findings to other therapeutic vaccines for permanent viral infections such as HSV, EBV, CMV, and others?” The findings from this collaboration with the Benaroya Research Institute not only deepen our understanding of the immune response to Shingrix but also provide valuable insights that could inform the development of more effective vaccines for other persistent viral infections.

The spotlighted research was funded by the National Institute of Health.

Fred Hutch/University of Washington/Seattle Children's Cancer Consortium members Drs. Emily Ford and David Koelle contributed to this work.

Wen X, Hu AK, Presnell SR, Ford ES, Koelle DM, Kwok WW. 2025. Longitudinal single cell profiling of epitope specific memory CD4+ T cell responses to recombinant zoster vaccine. Nature Communications. https://doi.org/10.1038/s41467-025-57562-7.