Vaccines are critical tools to combat diseases caused by viruses including influenza and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While existing vaccines against influenza make a substantial contribution to public health, the antibody responses that are induced generally offer limited protection against viral strains different from those found in seasonal vaccines. Now, writing in Nature Materials, Yin et al.1 report an immunostimulatory nanoparticle-based approach to induce strong immune responses that can protect against heterologous strains of the virus, that is, virus strains that express antigens that differ from those in the vaccine formulation.

While whole or split virus-based vaccines can promote strong immune responses due to the presence of immunostimulatory viral factors, ‘subunit’ vaccines are instead based on well-defined viral antigens with potential to promote protective immunity. These offer the advantages of enhanced safety and a well-characterized nature, but ‘subunit’ antigens alone are generally unable to trigger protective immune responses and, consequently, vaccine adjuvants, substances that enhance and modulate immune responses, are required. Aluminium salts (alum) remain the principal adjuvants in clinical use with a well-established record in enhancing protective immune responses against associated antigens2. However, their ability to enhance antibodies that mediate cross-protection against multiple viral strains (cross-reactivity) and the cell-mediated immunity, required to efficiently control infection and protect against disease, is more limited. Combining immunostimulatory Toll-like receptor (TLR) agonists, particularly TLR4 ligands3, with alum or emulsions has generated more potent adjuvant formulations. Imidazoquinolines target the endosomal TLR7 and their inclusion in vaccines enhanced protection in preclinical models of cancer and viral infection4, but their low molecular weight leads to rapid diffusion from the injection site. A potential solution is to associate the TLR ligand to a particulate carrier.

Based on this principle, Yin et al. describe a gardiquimod (TLR7 agonist) nanoparticle (TLR7-NP) adjuvant. Gardiquimod was conjugated to polylactide (PLA) by ring-opening polymerization of lactide, resulting in gardiquimod–PLA conjugates (TLR7–PLA). These were subsequently mixed with poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) (PEG–PLGA) and self-assembled by nanoprecipitation into TLR7-NP of 77 nm diameter (Fig. 1). This process results in more efficient association of the TLR7 agonist with the nanoparticle-based carrier than that seen when the agonist was mixed with alum (TLR7–Alum). TLR7 activation is facilitated through the increased release of the agonist at low pH levels, upon internalization into endosomes (pH = 5.0).

Fig. 1: Generation of TLR7-NP.
figure 1

In step 1, gardiquimod (TLR7 agonist) is conjugated through ester linkages to polylactide (PLA) by initiation of the ring-opening polymerization of lactide to produce gardiquimod–polylactide (TLR7–PLA). In step 2, TLR7–PLA is mixed with poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) (PEG–PLGA) in dimethylformamide and self-assembled, by nanoprecipitation, into hydrodynamic TLR7-nanoparticles (TLR7-NP) of 77 nm diameter.

One of the first described mechanisms of adjuvant action is the depot effect or ability of vaccine formulations to sustain the presence of antigen at the injection site. Accordingly, Yin et al. demonstrate a higher accumulation of TLR7-NP at the injection site and draining lymph nodes compared with TLR7–Alum. Antigen association with TLR7-NP results in the activation and mobilization of conventional type 1 dendritic cells (DCs) to draining lymph nodes, where they induce early and sustained effector and memory cytotoxic CD8+ T cells — a response that can enhance protective immunity against disease. In addition, TLR7-NP promotes preferential antigen internalization by follicular dendritic cells (FDCs) and B cells in draining lymph nodes and increases germinal centre formation, which are key for generating high-affinity antibodies. The induction of such responses, long-lived antibodies and B cell memory relies on the interaction of B cells with CD4+ follicular T cells. This T cell population can be divided into T follicular helper (Tfh) cells, which promote B cell responses, and T follicular regulatory (Tfr) (FOXP3+) cells, which restrain these responses5. Mice vaccinated with antigen and TLR7-NP show an increased ratio of Tfh to Tfr cells, correlating with an enhanced germinal centre response, higher diversity of B cell clones, and larger breadth and quality of antibodies (Fig. 2).

Fig. 2: Mechanism of action of TLR7-NP.
figure 2

a, TLR7-NP-based vaccine forms a depot at the injection site leading to sustained activation of innate immune cells, including dendritic cells (DCs), and rapid passive or active (via DCs) transport of vaccine components to draining lymph nodes (dLNs), stimulating adaptive immune responses. b, In dLNs, TLR7-NP and antigen are internalized by B cells, which upon activation induce the formation of a germinal centre (GC), and by FDCs, which select B cells with the strongest antigen affinities. In parallel, TLR7-NP enhance DC-mediated activation and differentiation of T cells into cytotoxic CD8+ T cells and CD4+ follicular T cells (increasing the ratio Tfh to Tfr cells). c, Collectively, this enhances GC responses, the diversity of B cell clones and, therefore, induces broader protection by the production of cross-reactive antibodies that target antigens from multiple viral strains.

Following promising results with the model antigen ovalbumin, the authors investigated whether TLR7-NP could enhance broadly reactive antibodies to influenza virus antigens. The head region of hemagglutinin, the major glycoprotein on the influenza virus surface, is the main target of neutralizing antibodies induced by vaccination or infection (immunodominant epitopes), but due to antigenic drift, antibodies lose their ability to protect, necessitating annual updating of vaccines6. Inducing antibodies that preferentially target the more conserved stem region of hemagglutinin could lead to a broader and more efficient protection against different strains of the influenza virus. Vaccination of mice with hemagglutinin and TLR7-NP induces germinal centre B cells with greater receptor diversity and increases broadly reactive antigen-specific antibodies. Furthermore, a vaccine formulation combining hemagglutinin adsorbed to alum and TLR7-NP results in significantly enhanced protection against challenge with a lethal dose of a different influenza strain compared with mice vaccinated with alum-adsorbed antigen and TLR7–Alum.

In the case of SARS-CoV-2, viral variants exhibit mutations in the spike protein — the main viral protein allowing it to gain access to cells, especially in the receptor-binding domain of the protein (immunodominant epitope)7. The authors demonstrate that vaccination with the full-length spike protein from SARS-CoV-2 virus combined with TLR7-NP induces higher antibody titres against the spike protein and receptor-binding domain of different SARS-CoV-2 variants, as well as antigen-specific long-lived plasma cells in the bone marrow, compared with mice receiving the antigen and TLR7–Alum. Further preclinical studies would be required to address the ability of this formulation to protect against challenge with heterologous strains of SARS-CoV-2.

Despite major advances in viral vaccine adjuvants, the key challenge of developing broadly protective influenza vaccines remains8. A lipid-nanoparticle-based vaccine formulation containing hemagglutinin mRNA from the 20 lineages of influenza A and B virus provided protection against homologous and heterologous viral challenges9. This highlights the progress being made in the development of universal vaccines. Several efforts to combat SARS-CoV-2 antigenic drift have also been made. Recently, a pan-spike-protein-based vaccine was developed by investigating viral evolution and identifying spike protein consensus sequences that could elicit broadly neutralizing antibodies against circulating strains of the virus and possible future variants. However, further studies to evaluate the efficacy and durability of the immune response are required10. An optimal vaccine will ultimately combine innovations in antigen discovery and design with advances in adjuvants and vaccine formulation. The latter are critical, as they can be widely implemented, thus potentially accelerating the development of universal viral vaccines. In this context, Yin et al. describe a promising TLR ligand-nanoparticle adjuvant for viral vaccines that promotes antigen-specific CD8+ T cell responses and cross-reactive humoral responses, providing an important contribution to the development of broadly applicable adjuvants for viral vaccines.