Immune responses, therapeutic anti-tumor effects, and tolerability upon therapeutic HPV16/18 E6/E7 DNA vaccination via needle-free biojector

ABSTRACT Intramuscular vaccination of mice with the naked pBI-11 DNA plasmid targeting E6 and E7 of HPV16 and HPV18 via a conventional syringe and needle generates human papillomavirus (HPV) antigen-specific CD8+ T cell-mediated immune responses and therapeutic effects against the TC-1 tumor model. However, delivery of DNA vaccines by this method is less effective in patients, likely due to poor transduction of host tissues. Needle-free biojectors show great promise for DNA vaccination because of their simplicity of administration and high patient acceptability and also, we hypothesize, because of greater efficiency of cell transduction in host tissues. Here, we compared the kinetics of transgene expression from a plasmid DNA using intramuscular injection with a conventional needle administration to intradermal or intramuscular delivery with a customized Tropis biojector. Delivery using the customized Tropis biojector leads to enhanced transgene expression compared to intramuscular needle injection. In addition, we characterized the HPV antigen-specific CD8+ T cell-mediated immune responses and anti-tumor effects generated by pBI-11 DNA vaccination by each route of administration, as well as by split-dose multi-site injection. Intradermal, but not intramuscular, vaccination with pBI-11 DNA vaccine via customized Tropis biojector enhanced HPV antigen-specific CD8+ T cell-mediated immune responses over needle injection. Intradermal, but not intramuscular, vaccination via customized needle-free Tropis biojector elicited greater HPV antigen-specific CD8+ T cell-mediated immune responses as well as anti-tumor effects compared to intramuscular injection of pBI-11 with a needle. Good manufacturing practices (GMP) grade pBI-11 DNA vaccine delivered intradermally or intramuscularly via customized Tropis biojector was well tolerated by mice. IMPORTANCE Respectively, HPV16 and HPV18 cause 50% and 20% of cervical cancer cases globally. Viral proteins E6 and E7 are obligate drivers of oncogenic transformation. We recently developed a candidate therapeutic DNA vaccine, pBI-11, that targets HPV16 and HPV18 E6 and E7. Single-site intramuscular delivery of pBI-11 via a needle elicited therapeutic anti-tumor effects in mice and is now being tested in high-risk human papillomavirus+ head and neck cancer patients (NCT05799144). Needle-free biojectors such as the Tropis device show promise due to ease of administration, high patient acceptability, and the possibility of improved delivery. For example, vaccination of patients with the ZyCoV-D DNA vaccine using the Tropis device is effective against COVID19, well tolerated, and licensed. Here we show that split-dose, multi-site administration and intradermal delivery via the Tropis biojector increase the delivery of pBI-11 DNA vaccine, enhance HPV antigen-specific CD8+ T-cell responses, and improve anti-tumor therapeutic effects, suggesting its translational potential to treat HPV16/18 infection and disease.

mice/group) were divided into 3 groups.The mice of the first group (Naïve) were left unvaccinated.The mice of the second group were vaccinated with a total of 50 µg of pBI-11 DNA (25 µg/50 µl/mouse per injection with two separate injections) through I.D. injection on the back using a customized needle-free biojector under anesthesia with ketamine.The mice were boosted twice with the same regimen at a 1-week interval.
The mice of the third group were vaccinated with a total of 50 µg of pBI-11 DNA (25 µg/50 µl/mouse per injection with two separate injections on each hind leg) through I.M. injection using a customized needle-free biojector under anesthesia with ketamine.The mice were boosted twice with the same regimen at a 1-week interval.7 days after the last vaccination, splenocytes were prepared and stimulated with either HPV16 E6aa50-57 peptide (5 µg/ml), HPV16 E7aa49-57 peptide (1 µg/ml), or HPV18 E6aa67-75 peptide (1 µg/ml) in the presence of GolgiPlug (1 µl/ml) overnight.The cells were stained with PE-conjugated anti-mouse CD8a (clone 53.6.7) at 4 °C for 30 minutes.After washing, the cells were permeabilized and fixed with perm/fix buffer (from eBioscience) at 4 °C for 30 minutes.After washing, the cells were further stained with FITCconjugated anti-mouse IFN-g (clone XMG1.2) at 4 °C for 45 minutes.The cells were resuspended in PBS + 0.5% BSA after washing.The cells were acquired with FACSCalibur flow cytometer and data were analyzed with CellQuest Pro software.
Abbreviation: P(KW) -p-value based on Kruskal-Wallis test for global difference.
All other pair-wise p-values were from Wilcoxon test with multiplicity adjustment using Holm Method. 5. Weight of organs of female C57BL/6 mice after vaccination.Female C57BL/6 mice (11-to 12-week-old, 5 mice/group) were divided into 3 groups.The mice of the first group (Naïve) were left unvaccinated.The mice of the second group were vaccinated with a total of 50 µg of pBI-11 DNA (25 µg/50 µl/mouse per injection with two separate injections) through I.D. injection on the back using a customized needle-free biojector anesthesia with ketamine.The mice were boosted twice with the same regimen at a 1-week interval.The mice of the third group were vaccinated with a total of 50 µg of pBI-11 DNA (25 µg/50 µl/mouse per injection with two separate injections on each hind leg) through I.M. injection using a customized needle-free biojector under anesthesia with ketamine.The mice were boosted twice with the same regimen at a 1-week interval.7 days after the last vaccination, mice were sacrificed and lung with trachea (A), heart (B), liver (C), spleen (D), left kidney (E) and right kidney (F) were collected and weight was measured with a digital scale (from Mettler Toledo, Columbus, OH, USA, model AB104-S).

Supplementary Figure
Abbreviation: P(KW) -p-value based on Kruskal-Wallis test for global difference.Table S1:
typical behavior.
typical behavior.

Table S3 :
Assessment of edema formation at vaccinated site 139

Table S4 :
Within normal limits / No significant findings (unremarkable) 140 141 142 Complete blood count of vaccinated mice.

Table S5 :
Summary of the biochemistry study in vaccinated mice