1. DISSOLVING OF HPV-DERIVED PEPTIDES FOR IMMUNIZATION PURPOSES Dana Pokorná, Martina Kindlová, Ingrid Poláková, Michal Šmahel Institute of Hematology and Blood Transfusion, U nemocnice 1, 128 20 Prague, Czech Republic, (danap@uhkt.cz)

2. Abstract INTRODUCTION: Aggregation of peptides often results in loss of their biological activity. The immunization with aggregated peptides is generally believed to enhance the development of antibody responses. However, the impact of aggregation on the induction of cellular immune responses may be negative. METHODS: For one immunization dose, we dissolved 100  g of HPV-derived non- conjugated or keyhole limpet hemocyanin-(KLH)-conjugated peptides with or without 50  g of CpG motifs (ODN 1826) in 20  l (tattoo) or 200  l (subcutaneous injection) PBS buffer. Mice were immunized using tattoo device or subcutaneous (s.c.) needle injection, respectively, and development of peptide-specific immune responses was examined. RESULTS: We observed that both KLH-conjugated and non-conjugated E7 49-57 peptides (RAHYNIVTF) were precipitated in the presence of CpG motifs in PBS buffer. The specific immune responses induced after s.c. delivery of the KLH-conjugated and non-conjugated E7 49-57 peptides were poor, in contrast, the tattoo delivery of either the KLH-conjugated or non-conjugated E7 49-57 peptide in combination with CpG motifs induced a very high activation of E7-specific CD8 + T cells. CONCLUSIONS: We suppose that the tattoo procedure might mechanically disrupt the aggregates of the peptide and the CpG motifs. More experiments are needed for evaluation of tattoo procedure for immunization with aggregated peptides.

3. T-cell mediated immune responses (aggregated E7 49-57 and dissolved E7 44-62 peptides) After 3 peptide immunizations of mice, splenocytes were isolated, incubated with or without the E7 49-57 peptide for 7 days and stained with anti-CD8 and tetramers of MHC class I molecules bound with the E7 49- 57 peptide. Higher rates of E7-specific CD8 + T cells were found in mice vaccinated by tattoo than s.c. needle-injection. CpG motifs enhanced overall CD8 + T cell responses with the exception of s.c. delivery of the E7 49-57 peptide. We suppose that the observed effect might be caused by formation of the E7 49-57 peptide aggregates after the addition of CpG motifs and furthermore that tattoo procedure could mechanically disrupt peptide aggregates.

4. Formation of TC-1-induced-tumors (after vaccination with aggregated E7 49-57 and dissolved E7 44-62 peptides) Mice (C57BL/6) were vaccinated 3 times with 100 micrograms of E7 peptides with or without 50 micrograms of CpG motifs using tattoo device or s.c. needle injection. Two weeks after the last vaccination 30 000 TC-1 tumor cells that express the E7 oncogene were injected and tumor growth was monitored. Vaccines delivered by tattooing protected mice against tumor onset more efficiently than identical vaccines injected subcutaneously by needle injection.

5. Humoral responses (dissolved E7 44-62 peptide) The production of E7-specific antibodies was detected after 3 immunizations with 100 micrograms of the E7 44-62 peptide with or without 50 micrograms of CpG motifs. Vaccination with the E7 44-62 peptide containing a B-cell epitope (aa 44-48), Th epitope and CTL epitope induced E7-specific antibody responses. Immunization with the minimalistic CTL epitope, the E7 49-57 peptide, did not elicit E7-specific antibodies (not shown). Tattoo delivery of the E7 44-62 peptide induced higher titers of E7-specific antibodies than s.c. administration. The addition of CpG motifs further enhanced antibody production after both modes of peptide delivery.

6. T-cell mediated immune responses (aggregated KLH-conjugated E7 49-57 peptide) After 3 peptide immunizations of mice, splenocytes were isolated, incubated with or without the E7 49-57 peptide and stained with anti-CD8 and anti-IFN  antibodies. Higher rates of E7-specific CD8 + T cells were found in mice vaccinated by tattoo than s.c. needle-injection. CpG motifs enhanced overall CD8 + T cell responses. Freund adjuvant (FA) did not substantially influence peptide-specific CTL responses. We suppose that tattoo procedure could mechanically disrupt aggregates of KLH- conjugated peptide, which could be advantageous for induction of peptide- specific CTL responses.

7. Humoral responses (aggregated KLH protein) The production of KLH-specific antibodies was detected after 3 immunizations with 50 micrograms of the KLH-conjugated E7 49-57 peptide with or without 50 micrograms of CpG motifs, mixed or unmixed (1:1) with Freund adjuvant (FA). Immunization with the KLH-conjugated E7 49-57 peptide induced antibodies against carrier KLH protein. Higher titers of KLH-specific antibodies were surprisingly observed after s.c. administration than after tattoo delivery. The addition of CpG motifs and Freund adjuvant (FA) further enhanced antibody production after both modes of protein delivery. We suppose that hypothetical disruption of protein aggregates after tattooing was disadvantageous for induction of protein- specific humoral responses.

8. Humoral responses (dissolved KLH-conjugated L2 18-38 peptide) The L2 18-38 peptide is supposed to contain the B-cell epitope and no Th epitope. The immunization with non-conjugated L2 18-38 peptide either by tattooing or subcutaneously with Freund adjuvant (FA) did not elicit production of L2-specific antibodies. Three immunizations with 50 microgram of the L2 18-38 peptide conjugated with the KLH protein induced L2- and KLH- specific antibodies. Higher titers of L2- and KLH- specific antibodies were observed after tattoo delivery than s.c. administration. However, the combination of adjuvants (CpG motifs and Freund adjuvant) enhanced induced humoral responses after s.c. delivery to the level comparable or even higher than that after tattoo administration supported by CpG motifs. Legend: white bars – after the 1st immunization light grey bars – after the 2nd immunization dark grey bars – after the 3rd immunization

9. Summary The tattoo delivery is more advantageous than s.c. injection for induction of cellular responses after immunization with both aggregated and dissolved peptides and aggreated proteins (non aproved for dissolved proteins). Furthermore, tattoo delivery is more beneficial than s.c. injection for induction of humoral responses after immunization with dissolved peptides and dissolved proteins. We suppose that tattoo delivery is not convenient for induction of humoral responses by aggregated proteins (non aproved for aggregated peptides) because tattoo procedure might disrupt aggregates of proteins.

10. Publication: Dana Pokorná, Ingrid Poláková, Martina Kindlová, Martina Dušková, Viera Ludvíková, Pavel Gabriel, Luďa Kutinová, Martin Müller, Michal Šmahel. Vaccination with human papillomavirus type 16-derived peptides using a tattoo device. Vaccine, 2009, 27:3519-29.