Clin Exp Vaccine Res.  2013 Jul;2(2):128-134. 10.7774/cevr.2013.2.2.128.

Intranasal immunization with a flagellin-adjuvanted peptide anticancer vaccine prevents tumor development by enhancing specific cytotoxic T lymphocyte response in a mouse model

• Nguyen CT
• Hong SH
• Ung TT
• Verma V
• Kim SY
• Rhee JH
• Lee SE

• KMID: 2167723
• DOI: http://doi.org/10.7774/cevr.2013.2.2.128

Abstract

PURPOSE
Human papillomavirus (HPV) is a significant cause of cervical cancer-related deaths worldwide. Because HPV is a sexually transmitted mucosal pathogen, enhancement of antigen-specific mucosal immune response likely serves good strategy for vaccination. However, mucosal vaccines generally do not induce strong enough immune responses. Previously we proved that a bacterial flagellin, Vibrio vulnificus FlaB, induce strong antigen-specific immune responses by stimulating the Toll-like receptor 5. In this study, we tested whether FlaB could serve as an effective mucosal adjuvant for a peptide-based HPV preventive cancer vaccine.
MATERIALS AND METHODS
Mice were intranasally administered with a mixture of FlaB and E6/E7 protective peptides in 5-day interval for a total of two times. Five-days after the last vaccination, cellular immune responses of the vaccinated mice were analyzed. Tumor growth was also observed after a subcutaneous implantation of TC-1 cells bearing E6/E7 antigens.
RESULTS
Intranasal administration of the E6/E7 peptide mixture with FlaB elicited a strong antigen-specific cytotoxic T lymphocyte activity and antigen-specific interferon-gamma production from splenocytes and cervical lymph node cells. Furthermore, FlaB, as a mucosal adjuvant, conferred an excellent protection against TC-1 tumor challenge with high survival rates in E6/E7 immunized animals.
CONCLUSION
These results indicate that FlaB can be a promising mucosal adjuvant for nasal HPV vaccine development.

Keyword

Flagellin; Adjuvant; Neoplasms; Vaccines

MeSH Terms

Administration, Intranasal
Animals
Flagellin
Humans
Immunity, Cellular
Immunity, Mucosal
Immunization
Interferon-gamma
Lymph Nodes
Lymphocytes
Mice
Peptides
Survival Rate
Toll-Like Receptor 5
Ursidae
Vaccination
Vaccines
Vibrio vulnificus
Flagellin
Interferon-gamma
Peptides
Toll-Like Receptor 5
Vaccines

Figure

• 

  Fig. 1 FlaB enhances antigen-specific cytotoxic T lymphocyte (CTL) responses. Each group of vaccinated mice (n = 5) was sacrificed at day 10. The splenocytes (SPL) were prepared to determine the CTL activity in vitro. To prepare the effector cells, SPLs were stimulated in vitro with E6/E7 peptides (1 µg/mL each) in the presence of interleukin-2 (25 IU/mL) for 5 days. The TC-1 cells were treated with cisplatin (1 µg/mL) for 24 hours and then used as the target cells. The effector and target cells were co-cultured at various effector:target (E:T) cell ratios. After 5-hour incubation at 37℃, 50 µL of the cell supernatant was collected to measure the lactate dehydrogenase activity in the culture medium. The ANOVA was used to compare CTL responses. *p < 0.05.

• 

  Fig. 2 Interferon-γ (IFN-γ) production from splenocytes and cervical lymph node cells. Each group of vaccinated mice (n = 5) was sacrificed at day 10. The splenocytes (A) and cervical lymph node cells (B) were prepared to determine IFN-γ production. Immune cells were stimulated in vitro with the E6/E7 peptides (1 µg/mL each) for 3 days. The number of E6/E7 peptide-specific IFN-γ producing cells generated in vaccinated mice was determined by group by ELISPOT assay. The Student's t-test was used to compare the IFN-γ production between two groups. *p < 0.05 and **p < 0.01.

• 

  Fig. 3 Prophylactic antitumor effect of flagellin-adjuvanted peptide based vaccine. Groups of 5 vaccinated mice were subcutaneously challenged with 5 × 105 TC-1 tumor cells at 5 days after the last vaccination. Changes in tumor sizes (A) and mouse survival rates (B) were monitored at 3-day intervals. The statistical significance of tumor volume at 21 days after TC-1 cell challenge was determined by the Student's t-test. The survival curve was constructed according to the Kaplan-Meier method, and the statistical significance between P and P + F group was determined by the log-rank test. *p < 0.05 and **p < 0.01.

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