Immune Netw.  2009 Oct;9(5):169-178. 10.4110/in.2009.9.5.169.

Enhancement of DNA Vaccine-induced Immune Responses by Influenza Virus NP Gene

• Choi SY
• Suh YS
• Cho JH
• Jin HT
• Chang J
• Sung YC

• KMID: 1474578
• DOI: http://doi.org/10.4110/in.2009.9.5.169

Abstract

DNA immunization induces B and T cell responses to various pathogens and tumors. However, these responses are known to be relatively weak and often transient. Thus, novel strategies are necessary for enhancing immune responses induced by DNA immunization. Here, we demonstrated that co-immunization of influenza virus nucleoprotein (NP) gene significantly enhances humoral and cell-mediated responses to codelivered antigens in mice. We also found that NP DNA coimmunization augments in vivo proliferation of adoptively transferred antigen-specific CD4 and CD8 T cells, which enhanced protective immunity against tumor challenge. Our results suggest that NP DNA can serve as a novel genetic adjuvant in cocktail DNA vaccination.

Keyword

DNA vaccine; influenza virus NP; adjuvant

MeSH Terms

Animals
DNA
Immunization
Influenza, Human
Mice
Nucleoproteins
Orthomyxoviridae
T-Lymphocytes
Vaccination
DNA
Nucleoproteins

Figure

• 

  Figure 1 Increased antigen-specific antibody and CTL responses by NP DNA coimmunization. The plasmid vectors encoding the influenza viruses HA (A/Jap/57), HIV Env (HXBc2) and HCV E2 (genotype 1b) were mixed with NP (A/PR/8/34) or mock DNA, and then intramuscularly injected twice, at four-week interval, into female BALB/c mice. Antibody (A) and CTL responses (B) to HA, HIV Env, and HCV E2 were analyzed 4 weeks after the boost immunization. (A) Sera from the immunized mice were diluted to 1/100 and antibody responses were determined by ELISA assay. Circles represent O.D. at 405 nm and the bars are the means of four mice per group. Data are representative of two independent experiments. *p<0.05, **p<0.005 (B) Splenocytes from two mice per group were pooled and the specific killing of HA peptide-loaded P815 target cells was determined by standard 51Cr release assay. Killing of P815 target cells without HA peptide was <2%. rVV Env-infected naive syngeneic splenocytes and P815 cells were used as stimulator and target cells, respectively, for determining HIV Env-specific CTL response. An E2-expressing CT26 cell line was used as stimulator and target cells for measuring HCV E2-specific CTL activity. Non-specific killing of target cells without Env or E2 was <5%. Similar data were obtained in three separate experiments. (C) HIV Env-specific T-cell responses were measured by ELISPOT assay. Each group of mice were immunized twice with pTV-GE in the absence or presence of pTV-NP at 0 and 6 weeks. At the indicated time points, pooled splenocytes were prepared and Env-specific IFN-γ responses to V3 peptide were analyzed. Values represent the means±s.d. of triplicate cultures in one experiment. Similar data were obtained in three separate experiments. **p<0.005, ***P<0.001, Statistical significance was determined using the Student t test.

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  Figure 2 Increased frequency and proliferation of OVA-specific CD4 and CD8 T cells by coimmunization of NP DNA. (A) Plasmid DNA encoding OVA was mixed with different doses of NP DNA(5, 20, 50, and 100 µg) or mock DNA, and then intramuscularly injected into female C57BL/6 mice. At 4 weeks after booster DNA immunization, pooled splenocytes were prepared from two mice per group. Cells were stimulated with OVA (257-264: H-2Kb-restricted epitope) or OVA (323-339: class II-restricted epitope) peptides and analyzed for IFN-γ-producing T cells by ELISPOT assay. Values represent the means ± s.d. of triplicate cultures in one experiment. The experiment was repeated three times and produced similar results. (B, C) CFSE-labeled naive OT-I and OT-II cells (2×106) were adoptively transferred into C57BL/6 mice and then one day later OVA DNA was intramuscularly injected with or without NP DNA. At day 9 post-immunization, pooled draining inguinal lymph nodes from two mice per group were analyzed by measuring the in vivo proliferation of (B) OT-I and (C) OT-II cells by flow cytometry. Live lymphocytes were gated according to forward and side light scatter profiles. Data are representative of three independent experiments that produced similar results.

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  Figure 3 NP DNA coinjection enhances protection against tumor challenge. Naive. OT-I cells (2×106) were adoptively transferred into C57BL/6 mice and the DNA encoding OVA was intramuscularly injected with or without NP DNA one day later (n=12). At 28 days post-immunization, mice were s.c. inoculated with 5×105 EG-7 tumor cells. Tumor sizes were measured using microcaliper in two dimensions. The populations of mice bearing palpable tumors (tumor volume>40 mm3) are presented as the percentage of tumor incidence. Coimmunization of OVA DNA and NP DNA showed the reduced level of tumor incidence (p<0.01, Student t test).

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