Dec 15, 2021 / Immunology

One vaccine against multiple viruses: Single cell analysis of adjuvanted influenza vaccine reveals surprising results

Olivia Habern

Researchers from Stanford University School of Medicine have found that an adjuvant for the influenza vaccine primes the innate immune system to simultaneously provide protection against Dengue and Zika virus. Their discovery advances our knowledge of the role of epigenomic reprogramming in supporting the immune response to vaccination, and contributes to a growing case for the potential of vaccines that can protect us from more than one virus.

Universal vaccines and the value of trained immunity  

What makes an effective vaccine? The answer depends, in part, on the pathogen that vaccine is attempting to confer immunity against. While the traditional vaccination approach aims to provide protection against a single, specific pathogen (1), some viruses present unique challenges, such as a need to confer immunity to antigenically distinct serotypes of the same virus. This has led researchers to explore the potential of vaccines that can address multiple strains simultaneously. For example, in an article from the NIH in July of this year, evidence was shown for the potential of a “universal” COVID-19 vaccine that could confer protection against multiple coronaviruses, including SARS-CoV, bat viruses, and other SARS-CoV-2 variants. The research team’s approach leveraged mRNA vaccine technology to encode a spike protein chimera built from multiple coronaviruses to activate the adaptive immune response against each viral strain simultaneously.

Though still in its early stages of feasibility, this innovative vaccine approach shows us that vaccines can be designed to do more than what they were initially invented to do. There are different design approaches to generate such “heterologous” vaccines—vaccines that confer protection against unrelated pathogens in addition to the target pathogen. Scientists are now exploring the possibility of leveraging the innate immune system to generate a beneficial nonspecific immune response against multiple viruses. This approach depends on the concept of trained immunity.

“Heterologous effects of vaccination may alternatively be mediated by heterologous immune responses that are not specifically directed against the vaccine antigen. [...] These effects may persist as a result of ‘innate memory’ mechanisms involving macrophages or NK cells. For example, epigenetic reprogramming due to sustained changes in gene expression and cell physiology, without permanent genetic changes (mutations or recombination), underlies ‘trained immunity’” (2).

Typically after vaccination, innate immune cells, such as monocytes and macrophages, coordinate with adaptive immune cells, such as T and B cells, to drive antigen-specific reactions. This causes adaptive immune cells to sustain long-term protection from viral infection. Thus, the innate immune response is generally considered to be short lived. Trained immunity goes against this convention, enabling potentially long-term functional reprogramming of innate immune cells through epigenetic modification.

“Mechanistic studies are consistent with the possibility that at least some heterologous [vaccine] effects may be durable. Heterologous lymphocyte responses may persist for decades in memory cells, and innate immune memory may be maintained by epigenetic reprogramming of long-lived differentiated cells (including tissue-resident macrophages) or hematopoietic progenitors” (2).

How do these epigenetic changes occur? Previous studies suggest that innate immune cells  may develop immune memory through exposure to either the same pathogenic challenge presented in a vaccine or an unrelated stimulus (2). One example is lipopolysaccharide (LPS)-induced tolerance. LPS is a major antigen from gram-negative bacteria and, in a 2007 study, was shown to induce epigenetic changes in innate immune cells. After secondary exposures to LPS, monocytes and macrophages exhibited gene-specific chromatin modifications that turned off genes coding inflammatory molecules, but promoted expression of genes coding antimicrobial molecules (2).

So what’s the main takeaway? Epigenetic reprogramming of innate immune cells as a result of vaccination can influence their subsequent responses to related or unrelated pathogens, and thus shows promise to cause those cells to provide general protection against multiple pathogens, even over a sustained period.

Exploring AS03’s potential as an epigenetic adjuvant for the influenza vaccine

Scientists are now digging deeper into this epigenomic immune response to vaccination and testing different adjuvants that may be able to prime innate immune cells to provide this sustained, nonspecific immunity against multiple pathogens. In a recent publication from Cell, researchers from Stanford University School of Medicine shared findings on the immune response to the seasonal influenza vaccines (3). Using Chromium Single Cell Gene Expression and Chromium Single Cell ATAC (Assay for Transposase Accessible Chromatin), they constructed a single cell landscape of the innate immune response to traditional influenza vaccination at the epigenomic and transcriptional level before and after vaccination. PBMCs from vaccinated individuals were first isolated and enriched for dendritic cell subsets, including classic and non-classical monocytes, and myeloid (mDC) and plasmacytoid (pDC) cells, to focus on innate immune populations.

Assessing changes in chromatin accessibility at day 30 after vaccination compared to day 0, they identified more than 10,000 differentially accessible regions in CD14⁺ monocytes. In particular, they noted a transition to inaccessible chromatin at gene loci targeted by AP-1, a transcription factor to inflammatory genes and regions associated with the production of proinflammatory cytokines. This chromatin accessibility trend was accompanied by reduced cytokine production (3). This finding suggested that the epigenetic reprogramming event that occurred in innate immune populations following vaccination did not have the priming effect expected of trained immunity. Rather, the effect was trained tolerance, a quieting of the innate immune response. This led the research team to a different vaccination approach, this time leveraging an adjuvant, AS03.

Taking PBMCs from four subjects, two vaccinated with a new AS03-adjuvanted H5N1 pandemic influenza vaccine, and two with H5N1 alone, they assessed the epigenomic and transcriptional landscape of innate immune populations, again using single cell RNA-seq and single cell ATAC-seq. Similar results of reduced chromatin accessibility at gene loci targeted by AP-1 were observed. However, in subjects vaccinated with H5N1+AS03, they also saw an increase in chromatin accessibility at interferon response factor (IRF) loci in monocytes and mDCs, which was correlated with an elevated expression of antiviral genes (3).

To determine the impact of this epigenomic reprogramming on resistance to viral infections, PBMCs were infected with Dengue or Zika virus, which target innate immune cells. To their surprise, the research team observed a significant reduction in viral titers for both Dengue and Zika virus at day 21 among PBMCs from subjects vaccinated with H5N1+AS03. This was believed to likely be the result of vaccine-induced expression of antiviral genes, as IRF1 was among the top genes negatively correlating with Dengue and Zika titer (3). Reflecting the mixed impact characteristic of trained immunity, where some antiviral genes are upregulated while other inflammatory genes are downregulated, these findings suggest AS03 may be a viable epigenetic adjuvant for influenza vaccines. And they ultimately have implications for designing vaccines that can confer protection against multiple unrelated viruses.

Single cell resolution clarifies vaccination response 

These exciting findings demonstrate the importance of approaching our current understanding of infectious disease and vaccination, and any field of biology, with a skeptical lens in order to push the boundaries of science and discover the unexpected. Is there another explanation beyond adaptive immunity, another underlying mechanism at work, that enables vaccine efficacy? What is the role of innate immune populations, or of epigenomic reprogramming, in driving the immune response to vaccination? Answering these questions requires technology that can allow scientists to not only incorporate more analytes into their research, but refine the resolution of their readout in order to capture the full complexity of biology. We’re proud to see single cell tools supporting this research and clearing new paths for innovation in vaccine design that can ultimately benefit human health.


  1. Lee Y, et al. Non-specific Effect of Vaccines: Immediate Protection against Respiratory Syncytial Virus Infection by a Live Attenuated Influenza Vaccine. Front Microbiol 9: 83 (2018). doi: 10.3389/fmicb.2018.00083
  2. Goodridge H, et al. Harnessing the beneficial heterologous effects of vaccination. Nat Rev Immunol 16: 392–400 (2016). doi: 10.1038/nri.2016.43
  3. Wimmers F, et al. The single-cell epigenomic and transcriptional landscape of immunity to influenza vaccination. Cell 184: 1–21 (2021). doi: 10.1016/j.cell.2021.05.039