Purpose:Aspartate β-hydroxylase (ASPH) contributes to carcinogenesis by promoting tumor cell proliferation, migration, and invasion. The enzymatic activity of ASPH can be inhibited by small molecule inhibitors that have been shown to have anti-metastatic activity in rodent models. ASPH has also been shown to inhibit the activation of natural killer (NK) cells. Therefore, this study aimed to investigate the effect of ASPH inhibition on the induction of anti-tumor immunity and to analyze the immune cells involved. Methods:In the mouse TC-1/A9 model characterized by reversible downregulation of major histocompatibility class I (MHC-I) molecules, ASPH inhibition was combined with stimulation of innate and/or adaptive immunity, and the anti-tumor response was analyzed by evaluation of tumor growth, in vivo depletion of immune cell subpopulations, and ELISPOT assay. Characteristics of immune cells in the spleen and tumor were determined by flow cytometry and single-cell RNA sequencing (scRNA-seq). Results:ASPH inhibition did not reduce tumor growth or promote the anti-tumor effect of innate immunity stimulation with the synthetic oligonucleotide ODN1826, but it significantly enhanced tumor growth reduction induced by DNA vaccination. In vivo immune cell depletion suggested that CD8+ T cells played a critical role in this immunity stimulated by combined treatment with ASPH inhibition and DNA vaccination. ASPH inhibition also significantly enhanced the specific response of CD8+ T cells induced by DNA vaccination in splenocytes, as detected by ELISPOT assay, and reduced the number of regulatory T cells in tumors. scRNA-seq confirmed the improved activation of CD8+ T cells in tumor-infiltrating cells after combined therapy with DNA vaccination and ASPH inhibition. It also showed activation of NK cells, macrophages, and dendritic cells in tumors. Conclusion:ASPH inhibition stimulated T-cell-mediated adaptive immunity induced by DNA vaccination. Different types of lymphoid and myeloid cells were likely involved in the activated immune response that was efficient against tumors with MHC-I downregulation, which are often resistant to T-cell-based therapies. Due to different types of activated immune cells, ASPH inhibition could improve immunotherapy for tumors with various MHC-I expression levels.

Background : Cancer development involves alterations in key cellular pathways, with aspartate beta -hydroxylase (ASPH) emerging as an important player in tumorigenesis. ASPH is upregulated in various cancer types, where it promotes cancer progression mainly by regulating the Notch1 and SRC pathways. Methods : This study explored the responses of various human cervical, pharyngeal, and breast tumor cell lines to second- and third-generation ASPH inhibitors (MO -I-1151 and MO -I-1182) using proliferation, migration, and invasion assays; western blotting; and cell cycle analysis. Results : ASPH inhibition significantly reduced cell proliferation, migration, and invasion and disrupted both the canonical and noncanonical Notch1 pathways. The noncanonical pathway was particularly mediated by AKT signaling. Cell cycle analysis revealed a marked reduction in cyclin D1 expression, further confirming the inhibitory effect of ASPH inhibitors on cell proliferation. Additional analysis revealed G0/G1 arrest and restricted progression into S phase, highlighting the regulatory impact of ASPH inhibitors on the cell cycle. Furthermore, ASPH inhibition induced distinctive alterations in nuclear morphology. The high heterogeneity in the responses of individual tumor cell lines to ASPH inhibitors, both quantitatively and qualitatively, underscores the complex network of mechanisms that are regulated by ASPH and influence the efficacy of ASPH inhibition. The effects of ASPH inhibitors on Notch1 pathway activity, cyclin D1 expression, and nuclear morphology contribute to the understanding of the multifaceted effects of these inhibitors on cancer cell behavior. Conclusion : This study not only suggests that ASPH inhibitors are effective against tumor cell progression, in part through the induction of cell cycle arrest, but also highlights the diverse and heterogeneous effects of these inhibitors on the behavior of tumor cells of different origins.

BACKGROUND:SARS-CoV-2, which causes COVID-19, has killed more than 7 million people worldwide. Understanding the development of postinfectious and postvaccination immune responses is necessary for effective treatment and the introduction of appropriate antipandemic measures. OBJECTIVES:We analysed humoral and cell-mediated anti-SARS-CoV-2 immune responses to spike (S), nucleocapsid (N), membrane (M), and open reading frame (O) proteins in individuals collected up to 1.5 years after COVID-19 onset and evaluated immune memory. METHODS:Peripheral blood mononuclear cells and serum were collected from patients after COVID-19. Sampling was performed in two rounds: 3-6 months after infection and after another year. Most of the patients were vaccinated between samplings. SARS-CoV-2-seronegative donors served as controls. ELISpot assays were used to detect SARS-CoV-2-specific T and B cells using peptide pools (S, NMO) or recombinant proteins (rS, rN), respectively. A CEF peptide pool consisting of selected viral epitopes was applied to assess the antiviral T-cell response. SARS-CoV-2-specific antibodies were detected via ELISA and a surrogate virus neutralisation assay. RESULTS:We confirmed that SARS-CoV-2 infection induces the establishment of long-term memory IgG+ B cells and memory T cells. We also found that vaccination enhanced the levels of anti-S memory B and T cells. Multivariate comparison also revealed the benefit of repeated vaccination. Interestingly, the T-cell response to CEF was lower in patients than in controls. CONCLUSION:This study supports the importance of repeated vaccination for enhancing immunity and suggests a possible long-term perturbation of the overall antiviral immune response caused by SARS-CoV-2 infection.

Tumor-associated macrophages (TAMs) plentifully infiltrate the tumor microenvironment (TME), but their role in anti-tumor immunity is controversial. Depending on the acquired polarization, they can either support tumor growth or participate in the elimination of neoplastic cells. In this study, we analyzed the TME by RNA-seq and flow cytometry and examined TAMs after ex vivo activation. Tumors with normal and either reversibly or irreversibly decreased expression of major histocompatibility complex class I (MHC-I) molecules were induced with TC-1, TC-1/A9, and TC-1/dB2m cells, respectively. We found that combined immunotherapy (IT), composed of DNA immunization and the CpG oligodeoxynucleotide (ODN) ODN1826, evoked immune reactions in the TME of TC-1- and TC-1/A9-induced tumors, while the TME of TC-1/dB2m tumors was mostly immunologically unresponsive. TAMs infiltrated both tumor types with MHC-I downregulation, but only TAMs from TC-1/A9 tumors acquired the M1 phenotype upon IT and were cytotoxic in in vitro assay. The anti-tumor effect of combined IT was markedly enhanced by a blockade of the colony-stimulating factor-1 receptor (CSF-1R), but only against TC-1/A9 tumors. Overall, TAMs from tumors with irreversible MHC-I downregulation were resistant to the stimulation of cytotoxic activity. These data suggest the dissimilarity of TAMs from different tumor types, which should be considered when utilizing TAMs in cancer IT.

Cluster of differentiation (CD) 80 is mainly expressed in immune cells but can also be found in several types of cancer cells. This molecule may either activate or inhibit immune reactions. Here, we determined the immunosuppressive role of CD80 in the tumor microenvironment by CRISPR/Cas9-mediated deactivation of the corresponding gene in the mouse oncogenic TC-1 cell line. The tumor cells with deactivated CD80 (TC-1/dCD80-1) were more immunogenic than parental cells and induced tumors that gained sensitivity to cytotoxic T-lymphocyte antigen 4 (CTLA-4) blockade, as compared with the TC-1 cells. In vivo depletion experiments showed that the deactivation of CD80 switched the pro-tumorigenic effect of macrophages observed in TC-1-induced tumors into an anti-tumorigenic effect in TC-1/dCD80-1 tumors and induced the pro-tumorigenic activity of CD4+ cells. Moreover, the frequency of lymphoid and myeloid cells and the CTLA-4 expression by T helper (Th)17 cells were increased in TC-1/dCD80-1- compared with that in the TC-1-induced tumors. CTLA-4 blockade downregulated the frequencies of most immune cell types and upregulated the frequency of M2 macrophages in the TC-1 tumors, while it increased the frequency of lymphoid cells in TC-1/dCD80-1-induced tumors. Furthermore, the anti-CTLA-4 therapy enhanced the frequency of CD8+ T cells as well as CD4+ T cells, especially for a Th1 subset. Regulatory T cells (Treg) formed the most abundant CD4+ T cell subset in untreated tumors. The anti-CTLA-4 treatment downregulated the frequency of Treg cells with limited immunosuppressive potential in the TC-1 tumors, whereas it enriched this type of Treg cells and decreased the Treg cells with high immunosuppressive potential in TC-1/dCD80-1-induced tumors. The immunosuppressive role of tumor-cell-expressed CD80 should be considered in research into biomarkers for the prediction of cancer patients’ sensitivity to immune checkpoint inhibitors and for the development of a tumor-cell-specific CD80 blockade.

Programmed cell death protein 1 (PD-1)/PD-1 ligand 1 (PD-L1) blockade is a promising therapy for various cancer types, but most patients are still resistant. Therefore, a larger number of predictive biomarkers is necessary. In this study, we assessed whether a loss-of-function mutation of the interferon (IFN)-γ receptor 1 (IFNGR1) in tumor cells can interfere with anti-PD-L1 therapy. For this purpose, we used the mouse oncogenic TC-1 cell line expressing PD-L1 and major histocompatibility complex class I (MHC-I) molecules and its TC-1/A9 clone with reversibly downregulated PD-L1 and MHC-I expression. Using the CRISPR/Cas9 system, we generated cells with deactivated IFNGR1 (TC-1/dIfngr1 and TC-1/A9/dIfngr1). In tumors, IFNGR1 deactivation did not lead to PD-L1 or MHC-I reduction on tumor cells. From potential inducers, mainly IFN-α and IFN-β enhanced PD-L1 and MHC-I expression on TC-1/dIfngr1 and TC-1/A9/dIfngr1 cells in vitro. Neutralization of the IFN-α/IFN-β receptor confirmed the effect of these cytokines in vivo. Combined immunotherapy with PD-L1 blockade and DNA vaccination showed that IFNGR1 deactivation did not reduce tumor sensitivity to anti-PD-L1. Thus, the impairment of IFN-γ signaling may not be sufficient for PD-L1 and MHC-I reduction on tumor cells and resistance to PD-L1 blockade, and thus should not be used as a single predictive marker for anti-PD-1/PD-L1 cancer therapy.

Tumor‐associated macrophages (TAMs) are essential components of the tumor microenvironment involved in the progression and metastasis of cancer. They are intimately involved in angiogenesis and immunosuppression in normal and malignant tissues, as well as pro‐fibrotic activities. With the development of immunotherapy, eradication of cancer cells through activation of the innate immune system has achieved inspiring results, whereas only a handful of patients show a durable response. The tumor‐suppressive environment has been investigated with respect to playing a vital role in cancer relapse. In this review, we uncover the heterogeneity of the origin of TAMs, as well as the functions of TAMs in tumor progression associated with intricate regulatory networks in the tumor microenvironment, aiming to inspire therapeutic insight for tumor immunotherapy.

Monitoring immune responses to solid cancers may be a better prognostic tool than conventional staging criteria, and it can also serve as an important criterion for the selection of individualized therapy. Multiparametric phenotyping by mass cytometry extended possibilities for immunoprofiling. However, careful optimization of each step of such method is necessary for obtaining reliable results. Also, with respect to procedure length and costs, sample preparation, staining, and storage should be optimized. Here, we designed a panel of 31 antibodies which allows for identification of several subpopulations of lymphoid and myeloid cells in a solid tumor and peripheral blood simultaneously. For sample preparation, disaggregation of tumor tissue with two different collagenases combined with DNase I was compared, and removal of dead or tumor cells by magnetic separation was evaluated. Two possible procedures of barcoding for single-tube staining of several samples were examined. While the palladium-based barcoding affected the stability of several antigens, the staining with two differently labeled CD45 antibodies was suitable for cells isolated from a patient's blood and tumor. The storage of samples in the intercalation solution for up to two weeks did not influence results of the analysis, which allowed the measurement of samples collected within this interval on the same day. This procedure optimized on samples from patients with head and neck squamous cell carcinoma enabled identification of various immune cells including rare subpopulations.

In the majority of human tumors, downregulation of major histocompatibility complex class I (MHC‑I) expression contributes to the escape from the host immune system and resistance to immunotherapy. Relevant animal models are therefore needed to enhance the efficacy of cancer immunotherapy. As loss of β‑2 microglobulin expression results in irreversible downregulation of surface MHC‑I molecules in various human tumors, the β‑2 microglobulin gene (B2m) was deactivated in a mouse oncogenic TC‑1 cell line and a TC‑1/dB2m cell line that was negative for surface MHC‑I expression was derived. Following stimulation with interferon γ, MHC‑I heavy chains, particularly the H‑2Db molecules, were found to be expressed at low levels on the cell surface, but without β‑2 microglobulin. B2m deactivation in TC‑1/dB2m cells led to reduced proliferation and tumor growth. These cells were insensitive to DNA vaccination and only weakly responsive to combined immunotherapy with a DNA vaccine and the ODN1826 adjuvant. In vivo depletion demonstrated that NK1.1+ cells were involved in both reduced tumor growth and an antitumor effect of immunotherapy. The number of immune cells infiltrating TC‑1/dB2m‑induced tumors was comparable with that in tumors developing from TC‑1/A9 cells characterized by reversible MHC‑I downregulation. However, the composition of the cell infiltrate was different and, most importantly, infiltration with immune cells was not increased in TC‑1/dB2m tumors after immunotherapy. Therefore, the TC‑1/dB2m cell line represents a clinically relevant tumor model that may be used for enhancement of cancer immunotherapy.

The current diagnostic algorithm for beta-lactam allergy is based on skin and provocation tests, both of which carry a certain risk of inducing hypersensitivity reactions. Thus, non-invasive in vitro tests reliable enough to replace skin and provocation tests at least in a portion of patients are desirable. We aimed to verify the utility of IFN-γ ELISPOT as a first-line test in patients with suspected non-immediate hypersensitivity reaction to amoxicillin (AMX) and penicillin (PNC). The prospective observational study included 24 patients with recent, suspected non-immediate hypersensitivity reaction to AMX or PNC and 6 recently-exposed healthy subjects. In vitro tests were performed in all patients and healthy subjects: a) IFN-γ ELISPOT with PNC, AMX and amoxicillin plus clavulanic acid (AMX-CL); b) penicillin specific IgE; c) basophil activation test (BAT). Skin and provocation tests followed only in certain patients. IFN-γ ELISPOT results with PNC and AMX stimulation did not differ from the unstimulated condition. The highest IFN-γ responses to AMX-CL were close to previously published criteria in three patients; one of which had true hypersensitivity according to drug provocation tests. Five patients with confirmed hypersensitivity by skin tests showed no response to the culprit antibiotic on IFN-γ ELISPOT assay. Our results did not support the utility of IFN-γ ELISPOT in the diagnosis of mild, non-immediate hypersensitivity to amoxicillin and penicillin.

Combined immunotherapy constitutes a novel, advanced strategy in cancer treatment. In this study, we investigated immunotherapy in the mouse TC-1/A9 model of human papillomavirus type 16 (HPV16)-associated tumors characterized by major histocompatibility complex class I (MHC-I) downregulation. We found that the induction of a significant anti-tumor response required a combination of DNA vaccination with the administration of an adjuvant, either the synthetic oligodeoxynucleotide ODN1826, carrying immunostimulatory CpG motifs, or α-galactosylceramide (α-GalCer). The most profound anti-tumor effect was achieved when these adjuvants were applied in a mix with a one-week delay relative to DNA immunization. Combined immunotherapy induced tumor infiltration with various subsets of immune cells contributing to tumor regression, of which cluster of differentiation (CD) 8+ T cells were the predominant subpopulation. In contrast, the numbers of tumor-associated macrophages (TAMs) were not markedly increased after immunotherapy but in vivo and in vitro results showed that they could be repolarized to an anti-tumor M1 phenotype. A blockade of T cell immunoglobulin and mucin-domain containing-3 (Tim-3) immune checkpoint had a negligible effect on anti-tumor immunity and TAMs repolarization. Our results demonstrate a benefit of combined immunotherapy comprising the activation of both adaptive and innate immunity in the treatment of tumors with reduced MHC-I expression.

Aurora kinase A (AURKA) is a centrosomal protein that is overexpressed in a number of human malignancies and can contribute to tumor progression. As we used this protein as a target of DNA immunization, we increased its immunogenicity by the addition of the PADRE helper epitope and decreased its potential oncogenicity by mutagenesis of the kinase domain. For in vitro analysis of induced immune responses in mice, we identified the Aurka 220–228 nonapeptide representing an H-2K b epitope. As DNA vaccination against the Aurka self-antigen by a gene gun did not show any antitumor effect, we combined DNA immunization with anti-CD25 treatment that depletes mainly regulatory T cells. Whereas 1 anti-CD25 dose injected before DNA vaccination did not enhance the activation of Aurka-specific splenocytes, 3 doses administered on days of immunizations augmented about 10-fold immunity against Aurka. However, an opposite effect was found for antitumor immunity—only 1 anti-CD25 dose combined with DNA vaccination reduced tumor growth. Moreover, the administration of 3 doses of anti-CD25 antibody alone accelerated tumor growth. Analysis of tumor-infiltrating cells showed that 3 anti-CD25 doses not only efficiently depleted regulatory T cells but also activated helper T cells and CD3 − CD25 + cells. Next, we found that blockade of the PD-1 receptor initiated 1 week after the first immunization was necessary for significant inhibition of tumor growth with therapeutic DNA vaccination against Aurka combined with depletion of CD25 + cells. Our results suggest that combined cancer immunotherapy should be carefully evaluated to achieve the optimal antitumor effect.

The asparaginyl endopeptidase legumain that is overexpressed in M2-polarized tumor-associated macrophages has been identified as a suitable target for elimination of these cells supporting tumor progression. To enhance the efficacy of DNA immunization against legumain, we performed several modifications in this protein that could improve induction of immune responses. First, we mutated the RGD motif into GGD or RGG sequences. This alteration resulted in diminished maturation of legumain and impaired cellular localization. Then, as tolerance to self-antigens can be broken by the activation of CD4+ T-cell help, we tried to enhance the immunogenicity of legumain by the insertion of a foreign helper epitope, namely the p30 epitope from the tetanus toxin. Finally, the 2 modifications were combined. After gene gun DNA immunization of C57BL/6 mice with these constructs, we identified the Lgmn111-119 CD8+ T-cell epitope that binds to H-2Db molecules. Furthermore, we showed that mutagenesis in the RGD motif significantly enhanced the immune response against legumain. The addition of the p30 helper epitope induced the specific production of IFN-&ggr; by T cells, but did not significantly increase legumain-specific immunity activated after mutagenesis in the RGD motif which might be caused by simultaneous activation of a Th2 response demonstrated by the production of IL-4. However, the beneficial effect of the helper epitope on legumain-specific response was proved after the depletion of regulatory T cells by antibody against CD25 that preferentially stimulated Th1 immunity. The antitumor effect of the modified legumain gene was shown in the immunization against tumors induced by MK16 cells.

In DNA vaccination, CD4+ T-cell help can be enhanced by fusion of a gene encoding an immunization protein with a foreign gene or its part providing Th epitopes. To study the effect of helper epitope localization in a protein molecule, the influence of the vicinity of the helper epitope, and the impact of chimeric protein cellular localization, we fused the helper epitope p30 from tetanus toxin (TT, aa 947–967) with the N- or C-terminus of the mutated E7 oncoprotein (E7GGG) of human papillomavirus type 16, enlarged the p30 epitope with the flanking residues containing potential protease-sensitive sites and altered the cellular localization of the fusion constructs by signal sequences. The p30 epitope enhanced the E7-specific response, but only in constructs without added signal sequences. After localization of the fusion proteins into the endoplasmic reticulum and endo/lysosomal compartment, the TT-specific Th2 response was increased. The synthetic Pan DR epitope (PADRE) induced a stronger E7-specific response than the p30 epitope and its stimulatory effect was not limited to nuclear/cytoplasmic localization of the E7 antigen. These results suggest that in the optimization of immune responses by adding helper epitopes to DNA vaccines delivered by the gene gun, the cellular localization of the antigen needs to be taken into account.

As cancer stem cells (CSCs) are resistant to chemotherapy, radiotherapy and targeted molecular therapy, immunotherapy of tumors could be aimed at their elimination. Markers specific for CSCs have not been identified to date, but microarray analyses have shown that CSCs and embryonic stem cells use similar transcriptional programs, thus suggesting the production of shared transcription factors. In this study, we developed an experimental DNA vaccine against the transcription factor Sox2 that is important for self-renewal of stem cells and is overexpressed in numerous human cancers. The Sox2 gene was codon optimized for the expression in human cells, its sequences encoding two nuclear localization signals (NLSs) were mutagenized, and the sequence coding for the PADRE helper epitope was fused with its 5' terminus. While codon optimization did not increase Sox2 production and mutagenesis in NLSs only partially reduced nuclear localization of Sox2, the addition of the PADRE epitope was crucial for the enhancement of Sox2 immunogenicity. The antitumor effect was shown after immunization against mouse oncogenic TC-1/B7 cells derived from the lung cancer cell line TC-1 and characterized by high Sox2 production. Sox2-specific reactivity in an ELISPOT assay was further augmented by the depletion of regulatory T (Treg) cells, but this depletion did not enhance the antitumor effect. These data demonstrated the induction of immune responses against the Sox2 self-antigen, but did not confirm the usefulness of Treg depletion when combined with antitumor vaccination.

Infection with Human papillomaviruses (HPVs) is linked to cervical cancer, which is one of the most common cancers found among women worldwide. Despite preventive immunization, a therapeutic vaccine, targeting infected individuals, is required. Vaccine strategies for treatment of HPV—induced cervical cancer are based on E7 and E6 oncoproteins. In this work we report the transient expression of chimeric particles containing the E6 oncoprotein from Human papillomavirus type 16 (HPV-16) in plants. We fused a mutagenized coding sequence of the E6 oncoprotein (E6GT) with the coding sequence of Potato virus X coat protein (PVX CP) both with the 5′- and 3′-terminus using linkers of different length (0, 4 or 15 amino acids). The expression in E. coli was performed to assess the characteristics of the recombinant protein prior to plant expression. The yield and immunological reactivity of the expressed proteins were screened with anti-PVX CP and E6 antibodies. The highest yields of chimeric particles were observed in the transgenic N. benthamiana expressing Potato virus A HC-Pro protein and the Tobacco mosaic virus movement protein. When inoculated on host plants, these recombinant viruses were not able to spread systemically. The obtained results revealed the new relations for design of expression cassettes for plant-based vaccine production.

DNA vaccines showed great promise in preclinical models of infectious and malignant diseases, but their potency was insufficient in clinical trials and is needed to be improved. In this study, we tested systemic administration of two conventional adjuvants, synthetic oligodeoxynucleotide carrying immunostimulatory CpG motifs (CpG-ODN) and levamisole (LMS), and evaluated their effect on immune reactions induced by DNA vaccines delivered by a gene gun. DNA vaccination was directed either against the E7 oncoprotein of human papillomavirus type 16 or against the BCR-ABL1 oncoprotein characteristic for chronic myeloid leukemia. High doses of both adjuvants reduced activation of mouse splenic CD8+ T lymphocytes, but the overall antitumor effect was enhanced in both tumor models. High-dose CpG-ODN exhibited a superior adjuvant effect in comparison with any combination of CpG-ODN with LMS. In summary, our results demonstrate the benefit of combined therapy with gene-gun-delivered antitumor DNA vaccines and systemic administration of CpG-ODN or LMS.

Since its discovery, DNA vaccination has become an effective strategy for the development of vaccines against cancer including cervical carcinoma (CC). The formation of CC is associated with human papillomavirus (HPV) infection. Viral E6 and E7 oncoproteins are suitable targets for therapeutic vaccination. To adapt the HPV16 E6 oncogene for DNA immunisation, we performed several modifications. First we fused the E6 gene with the 5′ or 3′-terminus of the Escherichia coli β-glucuronidase (GUS) gene and showed enhanced immunogenicity of the 3′ fusion (GUS.E6). Then, as the E6 oncogene contains two alternative introns that result in the production of truncated forms of the E6 protein, we abolished the 5′ splice site in the E6 gene. This modification completely eliminated the expression of the truncated E6 transcripts and thus increased the production of the full-length E6 protein. At the same time, it moderately reduced the immunogenicity of the modified non-fused (E6cc) or fused (GUS.E6cc) genes, probably as a consequence of the substitution in the immunodominant E6 epitope following the abolishment of the splice site. Furthermore, we reduced the oncogenicity of the E6 protein by two point mutations (E6GT) that, together, prevented E6-mediated p53 degradation. Finally, we constructed the GUS.E6GT gene characterized by enhanced safety and immunogenicity when compared with the wild-type E6 gene.

Tattooing has been shown to be very efficient at inducing immunity by vaccination with DNA vaccines. In this study, we examined the usability of tattooing for delivery of peptide vaccines. We compared tattooing with subcutaneous (s.c.) needle injection using peptides derived from human papillomavirus type 16 (HPV16) proteins. We observed that higher peptide-specific immune responses were elicited after vaccination with the simple peptides (E7(44-62) and E7(49-57)) and keyhole limpet hemocyanin-(KLH)-conjugated peptides (E7(49-57), L2(18-38) and L2(108-120)) with a tattoo device compared to s.c. inoculation. The administration of the synthetic oligonucleotide containing immunostimulatory CpG motifs (ODN1826) enhanced the immune responses developed after s.c. injection of some peptides (E7(44-62), KLH-conjugated L2(18-38) and L2(108-120)) to levels close to or even comparable to those after tattoo delivery of identical peptides with ODN1826. The highest efficacy of tattooing was observed in combination with ODN1826 for the vaccination with the less immunogenic E6(48-57) peptide and KLH-conjugated and non-conjugated E7(49-57) peptides which form the visible aggregates that could negatively influence the development of immune responses after s.c. injection but probably not after tattooing. In summary, we first evidenced that tattoo administration of peptide vaccines that might be useful in some cases efficiently induced both humoral and cell-mediated immune responses.