Dissecting vaccinia virus and monkeypox virus-specific T cell responses in humans

In a recent study posted to the bioRxiv* preprint server, researchers studied different antigens targeted by vaccinia virus (VACV)- and monkeypox virus (MPXV)-specific T cells.

Study: Defining antigen targets to dissect vaccinia virus (VACV) and Monkeypox virus (MPXV)-specific T cell responses in humans. Image Credit: FOTOGRIN/ShutterstockStudy: Defining antigen targets to dissect vaccinia virus (VACV) and Monkeypox virus (MPXV)-specific T cell responses in humans. Image Credit: FOTOGRIN/Shutterstock


Global health agencies are monitoring the 2022 MPXV outbreak based on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic experience. It is crucial because studies have barely investigated the quality and duration of immune responses associated with natural MPXV infection in humans. There is a lack of data about the effectiveness of all MPXV vaccines, primarily based on VACV. As for VACV, T cells provide overall immunity against MPXV infections. However, studies have not investigated MPXV immunity induced by MPXV infection or whether the cellular immune response induced in humans by VACV vaccination is cross-reactive with MPXV epitopes.

In a previous study, the authors demonstrated how the pools of a large number of peptides, mega pools (MP) generated by sequential lyophilization, could be used to accurately measure cluster of differentiation (CD)4+ and CD8+ T cell responses against complex antigens. MPs could be the peptides spanning the entire sequence of an antigen or experimentally defined or predicted T cell epitopes. While this approach has worked for several allergens and bacterial and viral targets, studies have not tested and validated MPs for poxviruses.

About the study

In the present study, researchers retrieved the epitope information available on VACV vaccines in the immune epitope database (IEDB) to predict potential MPXV targets recognized by T cells. They developed MPs based on the most immunodominant MPXV ortholog proteins that could help assess T cell responses to VACV and predict MPXV T cell epitopes likely to be recognized by VACV-induced T cell responses. Finally, they validated the biological activity of these MPs by testing banked peripheral blood mononuclear cells (PBMCs) from subjects previously vaccinated with the Dryvax vaccine.

VACV vaccine, marketed as Dryvax, eradicated smallpox in the 1980s. Later in 2001, because it posed a risk of severe reactions and other safety issues, the Dryvax vaccine was discontinued and was replaced by the modified vaccinia Ankara (MVA) virus-based vaccine under the brand name JYNNEOS.

Study findings

The analysis of the epitope data from the IEDB revealed a remarkable breadth of CD4+ and CD8+ T cell immune responses. The authors noted that 19 and 40 open-reading frames (ORFs) covered 67% and 61% of CD4+ and CD8+ T cell responses, respectively. This finding highlighted that focusing on over 200 ORFs typically encoded in pox genomes could help capture the majority of T cell responses, thus, enabling the identification and defining of the MPXV-specific mega pool reagents. The definition of dominant ORFs provided insights into the mechanisms underlying the development of these responses showing that dominant antigens are predominantly early ORFs, enriched in structural proteins in the case of CD4+ T cell responses, confirming earlier studies.

Furthermore, the authors noted that MVA had conserved all dominant ORFs, and had orthologs in MPXV. Again, this is an important finding suggesting that MVA-induced T cell response directed to MPXV should ideally be able to mirror VACV vaccine-induced responses, with substantial clinical efficacy against MPXV in humans.

Two weeks from vaccination (early time points), all the subjects showed positive CD4+ T cell responses to the pools of predicted MPXV epitopes but only 63% CD8+ T cell response. These responses diminished substantially within five to seven months after vaccination. The validation of the epitope pools revealed some novel aspects of human T cell responses to VACV. The study confirmed that CD8+ T cell responses were less durable than their CD4+ counterpart. At the same time, the authors observed that human CD4+ T cell responses were associated with up to 53% of granzyme B-secreting antigen-specific cells. Previous studies in macaques have reported that CD4+ T cells offer more protection from VACV and MPXV infections than CD8+ T cells. The current data, adding to the previous findings, suggested their role in supporting the development of antibody responses. It pointed out that the longevity and the cytotoxic component of CD4+ T cells contributed to a sustained antiviral function of CD4+ T cells.


According to the authors, this is the first study demonstrating the use of epitope MPs to detect pox-specific T cell responses in humans, in particular, MPXV-specific sequences. This novel method alleviated all the interference by pox-virus expression of immune antagonizing genes when T cell responses are quantified using infected cells. In the case of MPXV, this method also raised biosafety concerns. Interestingly, over 60 studies have developed SARS-CoV-2 MPs using a similar approach.

The study identified immunodominant cross-reactive ORFs to generate MPs to measure T cell responses, benefitting the research community. The median degree of amino acid sequence conservation was 61 to 67%, implying that T cell responses induced by VACV vaccination should recognize ortholog protein sequences in the MPXV genome. The study enabled the design of MPs that could experimentally detect VACV-specific T cell responses and MPXV cross-reactive T cells in Dryvax -vaccinated individuals, which, in the future, could facilitate the monitoring of cellular immunity following MPXV infection and vaccination. Further, the study data could help evaluate specific regions conserved across several OPXV species, including MPXV, for vaccine development.

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
Neha Mathur

Written by

Neha Mathur

Neha is a digital marketing professional based in Gurugram, India. She has a Master’s degree from the University of Rajasthan with a specialization in Biotechnology in 2008. She has experience in pre-clinical research as part of her research project in The Department of Toxicology at the prestigious Central Drug Research Institute (CDRI), Lucknow, India. She also holds a certification in C++ programming.


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