Cancer vaccination addressing neo-antigens based on the mutanome of an established tumor is the most personalized immunotherapeutic approach so far. First clinical results in melanoma and pancreatic cancer indicate the induction of a long-lasting and de novo-generated response caused by an individually formulated mRNA vaccine. The composition of these vaccines includes predicted human leukocyte antigen (HLA) class II and class I epitopes to induce a robust mixed CD4⁺ and CD8⁺ T cell response, which is reported to be a crucial factor for success.

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Recently, a research team from the German Cancer Research Center published an article titled "Neo-antigen tumor vaccination depends on CD4-licensing conveyed by adeno-associated virus like particles" in Molecular Therapy. The article focuses on neo-antigen vaccine research based on the AAVLP platform, emphasizing the significance of the licensing process of CD4⁺ T cells toward CD8⁺ T cells and the significant anti-tumor efficacy it provides, thereby establishing a new paradigm for next-generation neoantigen vaccine development.

Virus-like particles (VLPs) have a long-standing history in vaccine research. They are multi-protein structures that resemble intact viruses but do not contain a viral genome. VLPs are intentionally used to induce strong antibody responses and have been clinically applied in cancer prevention, viral infections, and both acute and chronic inflammatory diseases. The adeno-associated virus (AAV) is a non-enveloped, single-stranded DNA virus. AAV replication depends on the presence of helper viruses, such as adenovirus or herpesvirus, which provide genes for genome replication and viral assembly. Owing to its non-pathogenicity in humans and ability to transduce a broad spectrum of cells, AAV became an established vector for gene therapy. At present, many studies have also explored its potential use as a vaccine.

SIINFEKL-displaying AAVLPs induce CD8⁺ T cell responses and prevent tumor development

The researchers have engineered capsid-modified AAV-2 particles by genomic insertion of the DNA sequence encoding the SIINFEKL peptide in either the VR-VIII or the VR-IV loop of the VP1 protein (Figure 1A). A vaccination scheme was established in immunocompetent C57BL/6 mice by comparison of different injection routes, doses, and adjuvants. Vaccine responses were determined by the frequency of CD8⁺ T cells secreting interferon (IFN)-γ and tumor necrosis factor (TNF)-α derived from splenocytes after vaccination and after co-incubation with peptide-pulsed autologous antigen-presenting cell (APCs). Subcutaneous hock injection induced T cell responses more frequently compared with tailbase and intramuscular injections, and the use of Montanide ISA51 as an adjuvant was more effective than c-di-AMP, CpG, and combinations thereof. The researchers also defined 5.0E+11 VLPs to be an effective dose, which induced a significant response to the AAVLP-SIINFEKL vaccine, which peaked at 3 weeks after vaccination, after which it gradually decreased (Figure 1B). At 3 weeks, the AAVLP vaccine outperforms short peptide vaccination using SIINFEKL alone (Figure 1C). H2-Kb-SIINFEKL tetramer staining showed that specific CD8⁺ T cells are already generated in blood at 2 weeks after vaccination, although T cells from spleens are not responsive to pulsed APCs at that time (Figure 1D). To test the functional consequences of the AAVLP vaccine, the researchers challenged mice by subcutaneous (s.c.) injection of B16F10 melanoma cells stably expressing the OVA protein at 3 weeks and 10 weeks after vaccination. In both cases, the preventive effect of the AAVLP-SIINFEKL vaccine in six of seven animals (85%) is observed, and it led to abrogated tumor formation and consequently longer survival compared with unvaccinated or non-engineered AAV2-wild-type (WT)-vaccinated mice (Figures 1F and 1G). To test the broad applicability and means to boost potency, the researchers also made use of the AAVLP vaccination strategy for the viral epitope LCMV and showed the feasibility of double insertions at loop IV and VII in parallel with SIINFEKL and the T helper type 1-stimulating peptide J-ICBL29.

 

Figure 1: AAVLP-SIINFEKL vaccines prevent tumor formation by CD8⁺-mediated rejection

(Source: Neukirch L, et al, Mol Ther., 2025)

Neoantigen-AAVLPs delay tumor formation

The researchers used B16F10 melanoma cells as a model cell line due to their aggressiveness and the frequent occurrence of mutations that can be exploited as neo-antigens. They have chosen the peptide sequences of Kif18b(K739N), Ddb1(L438I), Golgb1(E2855D), and Snx5(R373Q) to serve as vaccine candidates based on their predicted NetMHCPan 4.1 eluted ligand rank position in the H2-Kb allele (Figure 2A) and their ability to form intact particles when inserted into the AAV2 framework. The research adhered to the prophylactic vaccination scheme. The researchers have compared a composite of all four neo-antigen AAVLPs with an equimolar mixture of the respective 21mer peptides, followed by tumor cell injection 3 weeks after vaccination. Although the induction of CD8⁺ responses in the spleen was significantly altered for only one AAVLP-neoantigen, in comparison with PBS-injected mice, they observed a significant delay of tumor growth in the AAVLP-Neo group but not in the peptide vaccination group (Figure 2B), which consequently led to a significantly longer survival of AAVLP-vaccinated mice. At the point of euthanization, the grown tumors were explanted, and the cellular composition was analyzed by fluorescence-activated cell sorting (FACS). The researchers detected a significant enrichment of CD3⁺ T cells in the AAVLP treatment group when compared with the sham injection group, but no change when the peptide-neo-antigen vaccine was administered.

 

Figure 2: Reduced tumor growth after neo-antigen-AAVLP vaccination

(Source: Neukirch L, et al, Mol Ther., 2025)

CD4⁺ T cell epitopes in AAVLP vaccines are required for antigen-specific CD8⁺ T cell responses

To investigate the influence of CD4⁺ T cell responses on the outcome of AAVLP vaccination, the researchers treated mice with a CD4-depleting antibody before administering the vaccine (Figure 3A). After CD4⁺ T cell depletion, the AAVLP vaccination scheme did not induce detectable CD8⁺ T cell responses in the spleen upon SIINFEKL stimulation as observed before (Figure 3B). Depletion of B cells in turn did not impede the induction of CD8⁺ T cell responses. In a tumor challenge experiment, the CD4⁺ T cell depletion setting also abrogated the AAVLP vaccination outcome, as CD4⁺ T cell-depleted mice showed the same survival rate as untreated littermates, while antibody isotype pre-treated mice responded to the AAVLP immunization as seen before (Figure 3C). All animals in this experiment remained in a healthy condition while being CD4⁺ depleted until tumor challenge. Signs of autoimmune disease were not observed, as it may be imputed by a possible CD4⁺ regulatory T cell depletion in this setting.

 

Figure 3 Role of CD4+ T cells in induction of antigen-specific CD8⁺ T cell responses and identification of AAVLP helper epitopes

(Source: Neukirch L, et al, Mol Ther., 2025)

Implementation of MHC II epitopes improves therapeutic vaccination

Next, the researchers investigated whether the deconstruction of AAVLP capsids into CD4⁺ response-inducing peptides can improve the outcome when added in trans to the previously unsuccessful SIINFEKL peptide vaccination. They chose four potential MHC class II epitope peptides (i.e., p6, p7, p8, and p11) from the validation experiment (Figures 3D and 3E) and spiked them into SIINFEKL peptides at an equimolar ratio to the AAVLP capsid epitopes. Within a preventive tumor formation setting, a significant improvement in success by SIINFEKL + AAVLP helper peptide vaccination was observed compared with SIINFEKL alone. Interestingly, the SIINFEKL + helper peptide vaccine performed similarly to the AAVLP-SIINFEKL vaccine, reflected by longer survival of the animals. This effect seems to be CD8⁺ T cell-mediated; a significantly greater number of SIINFEKL-specific T cells were found in the spleens of surviving mice compared with non-survivors. Moreover, among them, the rate of SIINFEKL T cells exhibiting a memory phenotype is higher in survivors vs. non-survivors as well. Prompted by these observations, the researchers then scrutinized the effect of spiked helper peptides (SHPs) on vaccination in a therapeutic setting by application of the vaccine 4 days after melanoma cell injection. They observed reduced tumor growth after treating mice with AAVLP-SIINFEKL, leading to significantly prolonged survival (Figure 4A), which was outperformed by the SHP vaccine (Figure 4B). In the SHP setting, 3 of 10 mice developed no tumors, while SIINFEKL treatment alone was ineffective.

To investigate the effect of helper peptide substitution on neo-antigen vaccination, the researchers mixed SHP with the four top-ranked MHC class I peptides to capsid-epitope equimolar amounts and vaccinated mice in a treatment setting (Figure 5A). Results showed significantly delayed tumor growth and extended survival of mice vaccinated with neo-antigen peptides alone; the addition of SHP to the neo-antigen peptides yielded a slightly better outcome, although the difference was not statistically significant (Figure 5B). As seen before, the SHP vaccine mix induced a higher T cell infiltration into tumors (Figure 5C) that also invaded deeper into the tumor tissue (Figure 5D).

 

Figure 4: Therapeutic effects of AAVLP vaccination and a substituted peptide vaccine

(Source: Neukirch L, et al, Mol Ther., 2025)

 

Figure 5: Reduced tumor growth after therapeutic helper epitope-substituted neo-antigen vaccination

(Source: Neukirch L, et al, Mol Ther., 2025)

Patient response to neo-antigen vaccination depends on MHC class II epitopes

To test the clinical relevance of MHC class II epitopes in a neo-antigen vaccine formulation, the researchers retrospectively analyzed samples from six patients from their department (Table 1) who received a personalized tumor vaccine as a last line of therapy within an individualized treatment/compassionate use setting and after providing informed consent. Blood draws were carried out before and after vaccination on a regular basis for functional enzyme-linked immunospot assay (ELISpot) assays on the vaccinated peptides. A response was determined as positive when the IFN-γ counts for at least one mutated peptide exceeded the internal assay control and were undetectable in samples before vaccination (Figure 6C). Post-vaccination responses in three of the six patients were observed; among them, the number of responses varied frequently (Figure 6B). The researchers retrospectively analyzed the vaccine peptides for the occurrence of MHC class II (HLA-DRB1) epitopes of each patient and only found them in responding patients. The number of responses did not correlate with the number of detected epitopes, however. A detailed analysis of the individual patients (Figure 6C) revealed an initially high level of neo-antigen-specific T cell responses that were detected up to the 10th round of vaccination, but were declining in the number of positive IFN-γ counts over time. The majority of responses were induced de novo, with the exception of one peptide in patient 3, where reactive T cells were frequent pre-vaccination and a response was detectable to the non-mutated control peptide. Interestingly, the neo-antigen-specific responses measured in patient 1 after the third and eighth vaccinations and for patient 2 throughout treatment affected peptide sequences that did not bear an MHC class II epitope. Thus, it may be concluded that these responses were CD8⁺ mediated (Figure 6D). To support the hypothesis, the researchers performed a more detailed analysis on in vitro stimulated PBMCs derived from patient 2, who received an adapted vaccine due to declining measurable response and finally disease relapse. The clinical dataset supports the finding that sequence-independent CD4⁺ help is needed to evoke neo-antigen-specific T cell responses.

Table 1: Patient characteristics

(Source: Neukirch L, et al, Mol Ther., 2025)

 

 

Figure 6: Patient response analysis after neo-antigen vaccination.

(Source: Neukirch L, et al, Mol Ther., 2025)

Conclusion

This study proposes and experimentally demonstrates a novel neo-antigen vaccine strategy based on the AAVLP platform, highlighting the importance of CD4⁺ T cells in promoting CD8⁺ T cell functionality. The research showed that co-incorporating CD4 and CD8 neo-antigen epitopes into AAVLPs could achieve efficient antigen delivery, robust T cell activation, and significant anti-tumor efficacy. CD4⁺ T cells not only enhance the expansion and cytotoxicity of CD8⁺ T cells but also indirectly improve antigen presentation efficiency by modulating dendritic cell function. The study further proves that the disruption of the CD4⁺ pathway markedly compromises vaccine potency, underscoring the indispensable role of helper signals in vaccine-induced immunity. In summary, AAVLP, as a highly engineerable and immunogenic vaccine platform, provides a new paradigm for the development of next-generation neo-antigen vaccines.