Peptides and mRNA have demonstrated significant potential for large-scale vaccine deployment due to their enhanced adaptability, making them suitable for combating rapidly mutating viruses or highly heterogeneous cancer types—this is made possible by the automated synthesis of diverse epitope sequences that has already been established for these platforms.

Compared with attenuated pathogens, proteins and peptides generally exhibit reduced immunogenicity. To achieve effective therapeutic outcomes with these subunit vaccines, it is crucial to carefully select the antigen and to precisely co-deliver it with adjuvants that potently stimulate immune cells. Toll-like receptor (TLR) agonists, which elicit robust innate and adaptive immune responses, can be employed. Such agonists include nucleic acid-based adjuvants—poly(I:C), a TLR3 agonist, and CpG oligodeoxynucleotides, a TLR9 agonist—as well as small-molecule agonists like imidazoquinolines, which activate TLR7 and TLR8. These agonists bind to TLRs expressed on the endosomal membranes of antigen-presenting cells (APCs), such as dendritic cells (DCs), thereby promoting APC maturation and upregulating the expression of MHC class I and II molecules. In addition, they enhance co-stimulatory signaling and the release of pro-inflammatory cytokines, which in turn drive the downstream activation of other immune cells. Given their ability to present exogenous antigens to T cells, the activation and maturation of APC-like dendritic cells are essential prerequisites for eliciting an effective immune response. This process, known as cross-presentation, leads to the induction of antigen-specific cytotoxic CD8+ T cells, which are critical for eliminating malignant tumor cells. Beyond CD8+ T-cell epitopes, peptide-based vaccines must also incorporate CD4+ T-cell epitopes to ensure the generation of antigen-specific T helper cells. The interaction between these T helper cells and antigen-specific CD8+ T cells is indispensable for mounting an effective cellular immune response.

Compared with whole-protein antigens, peptide antigens not only offer a more cost-effective and convenient production process but also enable sequence-selective tuning of specific epitopes, such as those arising from mutations. Particularly when targeting patient-specific tumor neoantigens, peptide antigens can be custom-designed to match an individual’s unique neoantigen repertoire. Moreover, their fully synthetic nature enhances drug safety by reducing the risk of contamination by pathogens and toxins.

Dendritic cells (DCs) play a pivotal role in eliciting adaptive immune responses by activating naïve T cells; moreover, they orchestrate these adaptive immune responses through the integration of diverse stimuli, including pathogen-associated molecular patterns and cytokine environments. Immature DCs are capable of phagocytosing protein antigens, whereas mature DCs serve as highly efficient antigen-presenting cells for naïve T cells. Human DCs can be readily cultured from peripheral blood monocytes, which are isolated from human blood. During vaccine development, it is essential to monitor the immunogenicity of candidate vaccines in a high-throughput manner. In addition, regulatory agencies mandate efficacy and safety testing prior to product release. Some vaccine assays require animal models, a process that can cause pain and distress and may also confound the results. To accelerate vaccine development and reduce the number of animals used in batch-release testing, it is recommended to more broadly employ human DCs in vaccine immunogenicity testing; DCs are increasingly being adopted for evaluating vaccine efficacy and enhancing immunogenicity.

Dendritic cells (DCs) are sentinel cells capable of initiating cellular immune responses. They manifest as immature DCs that efficiently uptake protein antigens and as mature DCs that can present peptides to naïve T cells. Compared with T and B cells, DCs are antigen-nonspecific; therefore, DC-based assays do not measure antigen-specific responses but rather responses to pathogen-associated molecular patterns (PAMPs) associated with the vaccine components. PAMPs are recognized by pattern-recognition receptors (PRRs) that are highly expressed on DCs. These PRRs can be located on the cell surface, within intracellular compartments, or in the cytoplasm. Importantly, the nature of the ensuing immune response is largely determined by the PRRs stimulated by the vaccine—either a single receptor or a combination thereof—and by the cytokine milieu. In this way, DCs establish a critical link between innate and adaptive immunity. There are several types of DCs, with the most common classification being myeloid DCs and plasmacytoid DCs. Myeloid DCs (mDCs) are specialized antigen-processing and antigen-presenting cells: like their immature counterparts, they exhibit robust phagocytic activity, while, like their mature counterparts, they possess a high capacity for cytokine production. They regulate T-cell responses both under homeostatic conditions and during infection. Plasmacytoid DCs (pDCs), on the other hand, are specifically adapted to respond to viral infections by producing type I interferons. They can also function as antigen-presenting cells and modulate T-cell responses. During vaccine development, it is crucial to monitor vaccine immunogenicity. For instance, in the case of allergy vaccines, numerous vaccine candidates (allergens) are currently under investigation, yet only a small fraction demonstrate sufficient immunogenicity. Testing all these candidate vaccines would require large numbers of experimental animals. Moreover, for batch release, regulatory authorities mandate the ability to distinguish among different vaccine components based on their underlying mechanisms.

The efficacy and safety of each vaccine batch must be rigorously tested. Vaccine testing requires large numbers of animals and often involves pain and distress. Dendritic cells (DCs) play a critical role in eliciting protective immunity; therefore, it is logical to employ DCs to evaluate vaccine immunogenicity by assessing DC maturation. Human DCs can be readily isolated from human blood, thereby eliminating the need for cross-species extrapolation and the use of laboratory animals. The successful application of human DC–based in vitro assays in vaccine immunogenicity testing—particularly assays that measure DC maturation—as well as more advanced approaches such as endolysosomal degradation assays and DC–T cell co-culture systems, supports the adoption of human DC–based assays for evaluating vaccine immunogenicity during vaccine development and as an alternative to animal studies for batch release testing.

Distinguish the various components of the Hib vaccine. By comparing various cell lines with monocyte-derived dendritic cells (moDC) using gene expression profiling, we found that the MUTZ-3 cell line most closely resembles moDC. Using this cell line, we were able to demonstrate differences in surface marker expression and cytokine production in vitro following treatment with a conjugate of Haemophilus influenzae type b (Hib) antigen—polyribosylribitol phosphate (PRP)—and Menactra outer membrane protein (OMP), as well as between PRP and OMP alone. This conjugate represents an actual vaccine; comparison of the conjugate with the final formulation (adjuvanted with aluminum) revealed no differences in surface marker expression or cytokine production. Although the conjugation process is routinely monitored by standard biochemical assays, implying that in vitro assays are not strictly necessary for its monitoring, this approach nonetheless demonstrates the utility of such assays. Similarly, moDC can be used to reveal differential responses among vaccine components. The MUTZ-3 cell line has the advantages of easy availability, safety, and minimal variability. In contrast, moDC offers a broader dynamic range and expresses functional Toll-like receptor 4 (TLR4), whereas the MUTZ-3 cell line exhibits attenuated TLR4-mediated signal transduction.

In summary, based on their immunogenicity, MUTZ-3 cells and moDCs can be used to distinguish the carbohydrate antigen PRP, the bacterial antigen OMP, and the conjugate PRP-OMP.

The efficacy of the YF-17D live attenuated vaccine. The live attenuated yellow fever vaccine YF-17D is a highly effective vaccine. YF-17D efficiently induces the expression of CD80 and CD86 and stimulates the production of IL-6, TNF-α, MCP-1 (CCL2), IP-10 (CXCL10), and IL-12p40. IL-12p70 is also induced, but this requires the presence of CD154. Furthermore, YF-17D prompts human pDCs to produce IFN-α. This study confirms the high efficacy of YF-17D. To investigate its underlying mechanisms, we examined the effects of YF-17D on DCs from Tlr-knockout mice. The absence of Tlr2, Tlr7, or Tlr9 resulted in a marked reduction in IL-12p40 production, indicating that these three TLRs function in a synergistic manner. In summary, the impact of YF-17D on surface marker expression and cytokine production substantiates its high efficacy.

Assess the immunogenicity of adjuvants. The CoVaccine HTTM adjuvant increased the expression of CD83 and CD86 on moDCs, but did not enhance the expression of CD11c, CD80, or MHC class II. Furthermore, incubation with the CoVaccine-HTTM adjuvant boosted IL-6 production while reducing IL-10 production, whereas the production of IL-1β, IL-12p70, and TNF-α remained unaffected. IL-6 production was almost completely abrogated by anti-TLR4 antibody treatment, but not by anti-TLR2 antibody treatment. Consistent with this observation, the reduction in IL-10 production was reversed by anti-TLR4 rather than anti-TLR2 treatment.

In this study, in vitro DC assays were shown to be valuable for screening adjuvants. Such screening would enable comparison of the efficacy of different adjuvants and identification of potential safety concerns associated with them.

IFN-β pretreatment can enhance the immunogenicity of BCG. Bacillus Calmette–Guérin (BCG) vaccination confers variable protective immunity, underscoring the need for new tuberculosis vaccines. Dendritic cells (DCs) infected with BCG exhibit increased expression of CD38, CD83, CD86, and HLA-DR, although this upregulation is less pronounced than that observed in DCs infected with Mycobacterium tuberculosis. Furthermore, BCG-infected DCs produce lower levels of IL-12p70 and display reduced expression of IL-12p35 and IFN-β compared with DCs infected with M. tuberculosis. The expression of IL-12p35 is regulated by interferon regulatory factor-3 (IRF-3); indeed, IRF-3 phosphorylation is induced by M. tuberculosis infection but not by BCG infection. In vitro pretreatment of DCs with IFN-β enhances the expression of CD38, CD83, and CD86 and increases IL-12p70 production. Finally, IFN-β pretreatment of DCs leads to enhanced production of IFN-γ and TNF-α by naïve allogeneic umbilical cord blood lymphocytes. This study demonstrates that in vitro DC assays can also be used to evaluate strategies for enhancing vaccine immunogenicity through cytokine supplementation, thereby providing insights for improving vaccine efficacy in humans.

Lysosomal degradation within cells serves as a metric for vaccine immunogenicity. The fundamental principle underlying the approach developed by Egger et al. is the correlation between a protein’s immunogenicity and its relatively low susceptibility to lysosomal proteolysis. By employing structurally related proteomes with varying capacities to elicit T-cell responses in vivo, the authors were able to demonstrate a link between in vivo T-cell priming and in vitro–simulated susceptibility to lysosomal proteases. Similar results were obtained using moDCs and mouse bone-marrow–derived DCs. Consequently, the authors also utilized the murine DC cell line JAWSII.13, which yielded comparable findings. This represents a highly promising method for assessing immunogenicity. A side-by-side comparison between this approach and measurements of DC maturation would appear particularly informative.

Vaccine immunogenicity was assessed using co-cultures of purified DCs and naïve CD4+ T cells. In large volumes of peripheral blood mononuclear cells (PBMCs), the number of DCs may be too low, or the DCs may be in an activation state that is not conducive to eliciting a primary T-cell response. Therefore, a combination of purified DCs and CD4+ T cells was employed. This protocol involves pulsing DCs with YF-VAX® (the commercial formulation of YF-17D), co-culturing the DCs with CD4+ cells in vitro for 14 days, and then stimulating the cultures with YF-VAX for 7 hours before determining the percentage of CD4+CD154+IFNγ+ cells. Using this approach, the proportion of positive cells increased from 0.4% to 3.2%. Notably, following YF-VAX® vaccination, the percentage of positive cells increased only 1.2- to 2.6-fold between 6 and 12 weeks post-vaccination. Furthermore, the in vitro protocol induced CD4+CD154+ cells to produce cytokines other than IFN-γ, including TNF-α, IL-5, IL-17, and IL-21. This protocol has been shown to exhibit substantially higher sensitivity than conventional PBMC-based assays for priming naïve T cells, even after the addition of DCs to the PBMC preparation.

Although this approach has been validated for highly effective vaccines, its performance in low-efficiency vaccines remains to be determined; nonetheless, it holds great promise as a method for assessing vaccine immunogenicity. A key advantage is that it does not rely on DC maturation per se, but rather on the functional outcome—namely, CD4+ T-cell expansion. Similarly, a side-by-side comparison between this approach and measurements of DC maturation would appear highly informative.

In vitro assays using human moDCs provide an excellent platform for evaluating vaccine immunogenicity; moreover, assessing the effects on DCs in vitro—relative to established in vivo outcomes—facilitates a deeper understanding of the mechanisms underlying immune response induction. Such assays can significantly reduce the need for animal testing and streamline batch release during vaccine development. In vitro testing also has the potential to shorten the duration and lower the cost of immunogenicity assessments, which is particularly important in the early stages of development. To ensure accurate evaluation of DC-based assays, it is essential to test a diverse panel of bacterial and viral vaccines, including live attenuated vaccines, inactivated vaccines, and adjuvanted formulations. These tests should encompass the ability to detect inter-batch variability in immunogenicity as well as the potential for high-throughput screening. More advanced approaches, such as measuring endolysosomal degradation and employing DC–T cell co-cultures, should be pursued in parallel with conventional DC maturation assays.

In summary, we recommend more extensive evaluation of in vitro immunogenicity assays using human dendritic cells, with the goals of enhancing our understanding of vaccine-induced immune responses, reducing the use of laboratory animals, and increasing assay throughput.