Reconsidering how to assess HSV-2 vaccine responses

Vaccines are one of the most important medical inventions of modern times that have saved and enhanced innumerable lives around the world. Despite the technological advances in vaccine design and formulation over the decades, there are still many infectious diseases for which effective vaccines have still not been developed. There are many reasons for this, but the lack of reliable methods for detecting strong and protective responses to some vaccines may contribute to this delayed progress. One of the pathogens for which vaccine design has continued to stall has been Herpes Simplex Virus-2 (HSV-2), which infects mucosal tissue. HSV-2 can lie dormant in infected individuals for long periods of time, occasionally reactivating to cause lesions. Some vaccine candidates have been tested for HSV-2 but with little success, despite indications of activation in circulating immune cells. It was this discrepancy which Dr. Emily Ford, a research associate in the lab of Lawrence Corey in the Vaccine and Infectious Disease Division at Fred Hutch, was interested in exploring in a recent publication in JCI Insight.

Compared to the relatively rare populations of HSV-2-specific T cells that can be identified in peripheral blood mononuclear cells (PBMCs) in those infected with HSV-2, skin-resident HSV-2-specific memory T cells at lesion sites may be more abundant and are likely important for anti-viral responses. The research team wanted to ask whether T cell responses in the skin could, therefore, be an alternative method of assessing vaccine responsiveness. Dr. Ford explained that “one of the hypotheses was that the skin T cells are just very different from what's happening in the blood.” To approach this, the team utilized the vaccine candidate HSV529, a replication-deficient HSV-2 whole-virus vaccine, administered intramuscularly. In a previous phase I study of this vaccine, almost half of the participants had detectable CD4 T cell responses in the blood, yet the vaccine has since been shelved due to poor immunogenicity. Dr. Ford and colleagues enrolled nine participants who had histories of HSV-2 reactivation and administered three HSV529 doses at 0, 30 and 180 days. Additionally, they collected biopsies of either a control site (the contralateral arm to the vaccine) or a site of a previous lesion at days of vaccination and at intervals between vaccinations.

The team found that, after vaccination began, there was no change in the amount or spatial distribution of either CD4 or CD8 T cells found at the lesion site by fluorescent imaging. Interestingly, they did observe an increase in CD4 T cells in the control-arm biopsy, even though this was not the arm which the vaccine was delivered to. In addition to the evaluation of the visible T cell responses in the biopsies, the research team also wanted to sequence the T cell receptors (TCRs) from these cells to assess clonotypes, i.e. cells which arose from the replication of one precursor. When they performed the TCR sequencing on these biopsy cells, they observed that there was no increase in the number of clonotypes at the lesion site after vaccination; however, there were generally more clonotypes found in the lesion site compared to the arm, likely reflective of the presence of stimulating antigen (i.e. reactivating virus). Looking next at the circulating PBMCs, the research team isolated HSV-2 reactive CD4 T cells (omitting CD8 T cells, as these were not seen to be responsive from the previous phase I trial) and performed TCR sequencing on these cells as well. In contrast to the response in the skin, following vaccination there was a striking increase in the number of clonotypes they were able to identify in the blood. Next, the team wanted to see whether they could identify overlapping HSV-2-specific TCRs between the PBMCs and the skin biopsies. From the 205 identified HSV-2-reactive clonotypes in the PBMCs at day 0, only 16 (7.8%) could be found in the skin biopsies at any point. After the first vaccine dose, they had identified 2,212 HSV-2-reactive clones in the PBMCs, of which 447 (20%) were then found in the skin, with as much as 45% of those 447 clonotypes found at day 0. “It's not like this vaccine was stimulating the development of or expansion of novelty cells,” explained Dr. Ford, commenting on the excitement of this data, “these are T cells that have already been there."

Since there was no bulk method to detect bona fide HSV-2-specific T cells in the skin biopsies, the team approached this problem by looking for exceptionally expanded clonotypes after the first vaccine dose. They found that the lesion sites—compared to the arm—had significantly more expanding prevalent and elicited clonotypes, but that few of these clonotypes could be detected in the blood. They also noticed that the majority of the expanding clones were the ones they identified as prevalent, or having been present prior to vaccination. From all the expanded clones of interest they found in the skin, only a small fraction of them were detected in the pool of HSV-2-specific PBMCs, and from only 2 participants. Commenting on the importance of this finding, Dr. Ford explained, “the [T cells] that expanded in blood were not the ones that expanded in skin,” adding that “the behavior in blood and skin was very different.” Lastly, they wanted to see how V-J gene usage changed in their participants over time. V-J chains are components of the TCR which are less variable between clonotypes and can provide higher-order perspective on T cell dynamics. The team saw that there was a dramatic shift in the blood after vaccination, but this was less striking in the lesion biopsies. Interestingly, for one participant who they had historical data for, there was a complete shift from a previously stable dominant V-J usage to an entirely new combination of dominant V-J chains. Because of the surprise of this shift, they even confirmed it was an HSV-2-specific TCR via cloning into a T cell line and seeing that it was reactive to HSV-2 antigen stimulation.

When it comes to designing an effective HSV-2 vaccine, we may still have a ways to go, but the data presented in this study emphasizes the need for diverse approaches to studying vaccine responses. Instead of focusing on circulating T cell responses—the standard in the field—there is a strong case to look more into tissue-resident-based responses in the skin. Additionally, considering much of the observed skin-based T cell responses were from cells previously present, the team recommended focusing on recruiting and stimulating these cells when designing new vaccine approaches. Luckily, as Dr. Ford emphasized, people living with HSV-2 are extremely altruistic when it has come to the development of treatments. “These are some of the most dedicated people you have ever met,” she stressed, adding that their willingness to participate in research studies is a “heroic volunteer effort.” There are still efforts underway to develop an HSV-2 vaccine, potentially utilizing mRNA platforms, but there will need to be more care taken into how these are benchmarked at early stages. All in all, this study showed that a new perspective can incredibly valuable when traditional approaches no longer serve us.

Ford ES, Li AZ, Laing KJ, Dong L, Diem K, Jing L, Mayer-Blackwell K, Basu K, Ott M, Tartaglia J, Gurunathan S, Reid JL, Ecsedi M, Chapuis AG, Huang ML, Magaret AS, Johnston C, Zhu J, Koelle DM, Corey L. Expansion of the HSV-2-specific T cell repertoire in skin after immunotherapeutic HSV-2 vaccine. JCI Insight. 2024 Jun 18;9(14):e179010. doi: 10.1172/jci.insight.179010. PMID: 39133650.

This spotlighted work was funded by the National Institutes of Health and Sanofi.

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