The study of novel DNA vaccines against tuberculosis

Figures & data

Figure 1. HVJ-Envelope exerts strong adjuvant activity by the induction of CTL/NK and repression of regulatory T cells via the activation of dendritic cells.

Table 1. ELISPOT assay for IFN-γ antigen-specific responses in the spleens of vaccinated mice following stimulation with HSP65 protein

Vaccination		Number of IFN-γ secreting cells/ (10^6splenocytes)
	(1st/2nd/3rd)
G1	−/−/-	3	±	1
G2	−/−/BCG	13	±	6
G3	DNA* / DNA / DNA	16	±	4
G4	BCG / DNA / DNA	115	±	12

*DNA means HVJ-Envelope/HSP65 DNA + IL-12 DNA vaccine. Spleen cell cultures were stimulated with rHSP65 protein for 20 h. The number of IFN-γ-secreting cells specific for rHSP65 protein per million cells was determined individually by ELISPOT assay. Results are expressed as the mean ± S.D. of 6 wells of 3 mice per group. The statistically significant differences of the G1 (naïve) group compared with the G2 (BCG alone group), G3 (DNA / DNA / DNA) or G4 (BCG / DNA / DNA) were observed (p < 0.01). The statistically significant difference between G2 group and G4 was observed (p < 0.01). The statistically significant difference between G3 group and G4 was observed (p < 0.01).

Figure 2. Induction of CD8⁺ CTL specific for HSP65 protein and M. tuberculosis by vaccination with HVJ-Envelope/HSP65 DNA + IL-12 DNA. Spleen cells from the naïve, BCG-, and HVJ-Envelope/HSP65 DNA + IL-12 DNA vaccinated mice were obtained eight weeks after the final vaccination. Cytotoxicity was assayed as release of radioactivity from ⁵¹Cr-labeled P815 target that had been transfected with HSP65 DNA using conventional ⁵¹Cr release assay as described in Materials and Methods.

Figure 3. Induction of CD8 positive CTL against TB by HVJ-Envelope/HSP65 DNA + IL-12 DNA vaccine, and differentiation of CTL.

Table 2. The in vivo necessity CD8 positive T cells and CD4 positive T cells for prophylactic efficacy of the HVJ-Envelope/HSP65 DNA + IL-12 DNA vaccine

Vaccination				Antibody	Log10 CFU of TB in the lung
	1st	2nd	3rd
G1	-	-	-	-	6.6 ± 0.3
G2	BCG	DNA	DNA	-	5.3 ± 0.3
G3	BCG	DNA	DNA	αCD8 Ab	5.9 ± 0.1
G4	BCG	DNA	DNA	αCD4 Ab	7.1 ± 0.5
G5	BCG	DNA	DNA	αCD8 Ab+αCD4 Ab	8.4 ± 0.2

*p < 0.05 G2vsG3, G4; **p < 0.01. G2vsG5, Anti-CD8 antibody and/or Anti-CD4 antibody were injected i.p. every 5 d after the challenge of TB. BCG was used as a prime vaccine and the DNA vaccine was immunized twice (HVJ-Envelope/HSP65 DNA 50 µg + IL-12 DNA50 µg) as boost vaccine. Four weeks after last immunization, 5 × 10⁵ H37Rv were challenged i.v. into mice. (G2-G3: p < 0.05); (G2-G4: p < 0.05); (G2-G5: p < 0.01)

Figure 4. IFN-γ and IL-2 production efficacy of HSP65+IL-12/HVJ and BCG using prime-boost method against TB challenged cynomologus monkeys. Group of animals were vaccinated three times (every three weeks) with (1^○) BCG Tokyo, (2^○) HSP65 +IL-12/HVJ, (3^○) HSP65+IL-12/HVJ = G1(■) BCG prime-HVJ/DNA boost group; (1^○) HSP65+IL-12/HVJ, (2^○) HSP65 +IL-12/HVJ, (3^○)BCG = G2(□) HVJ/DNA prime-BCG boost group; (1^○) HSP65+IL-12/HVJ, (2^○) HSP65 +IL-12/HVJ, (3^○) HSP65+IL-12/HVJ = G3(▩); (1) BCG, (2^○)saline, (3^○) saline = G4(▥) G4 group animals were vaccinated with BCG once.; (1^○) saline, (2^○)saline, (3^○) saline = G5 (▤). (A) PBL from vaccinated monkeys on 56 d or 112 d after 1st immunization, were cultured for three days. IFN-γ activity on 56 d and IL-2 activity (B) on 112days were assessed by ELISA.

Table 3. IL-2 production from PBL in the cynomolgus monkeys

(A)	(monkeys) Responder cell	H37 Ra Ag	IL-2 (u/ml)
	vaccine-treated	(-)	2 ± 3
	control	(-)	0 ± 0
	vaccine-treated	(+)	13 ± 4
	control	(+)	3 ± 5
(B)	G4 (saline control)	IL-2 production (U/ml) (HSP65 stimulation)		Survival
	ID of monkeys	Baseline	Day53 Post-challenge
	PR 6847 D	0.0	21.1	Survival
	PR 8018 B	0.0	43.2	Survival
	PR 5368 B	0.0	43.2	Survival
	PR PbB2–4F	0.0	0.0	Death (after 41 d)
	PrZ7–51 AC	0.0	0.0	Death (after 55 d)

(A)Augmentation of IL-2 production from PBL by H37Ra stimulation in the monkeys treated with HVJ-Envelope/HSP65 DNA + IL-12 DNA vaccine. (B) IL-2 production from PBL in the survived monkeys and the non-survived monkeys. PBL from monkeys on day 53 after TB challenge were stimulated with HSP65 protein for 3 d. ID (PR PbB2–4F) monkey and ID (PrZ7–51 AC) were died after 41 d and 55 d, respectively.

Table 4. In vitro and in vivo CTL induction by 15K granulysin

		15K Granulysin	% Specific Cytotoxicity
(A)	In vitro (5day MLC)
		-	8.0	±	1.0
		+	23.0	±	0.5
(B)	In vivo CTL
	spleen	-	1.0	±	0.2
		+	11.0	±	0.3
	LN	-	7.2	±	2.5
		+	22.5	±	2.8
	PEC	-	0.5	±	2.0
		+	28.5	±	1.0

(A)In vitro induction of human cytotoxic T cell by the stimulation with recombinant 15K granulysin protein. T cells from human PBL were obtained by nylon-wool column method. An amount of 1 × 10⁶ T cells were cultured with CESS_MMC cells (Mitomycic C treated CESS tumor cells) in the presence of 15K granulysin for five days. CTL activity of effector cells was assayed using ⁵¹Cr-labbelled CESS cells. Results are expressed as % Specific cytotoxicity ± S.D. (B) In vivo induction of CTL by 15K granulysin. C57BL/6 were injected i.p. with 1 × 10⁷ syngeneic FBL-3 tumor cells and then treated with recombinant 15K granulysin i.p. (5 µg/mouse) six times. Twenty-one days after FBL-3 inoculation, mice were sacrificed, and spleen cells, lymph node (LN) cells and peritoneal exudates cells (PEC) were used as effector cells for CTL. Cytotoxic activity against FBL-3 cells were assessed using ⁵¹Cr release method.

Figure 5. Differentiation of CTL by 15K granulysin and positive feedback loop of 15K granulysin in CTL induction. Precursor CTL are activated by IL-2 in the early stage of CTL induction of five day MLC. On the other hand, IL-6 acts, as a cytotoxic T cell differentiation factor, on the late stage of CTL induction as shown by Okada, et al. (J.I. 1988). 15K granulysin is produced from effector CTL, and induced the differentiation of CTL as a CTL differentiation factor. (Positive feedback loop by 15K granulysin.)

Figure 6. Induction of CTL differentiation by killer-specific secretory protein of 37Kd (Ksp37). Splenic T cell from C57BL/6 mice (H-2^b) were obtained as described in (PNAS Okada et al. 1981), and cultured with uv-treated BALB/c (H-2^d) spleen cells (Mitomycin C treated) in the presence of 100ng/ml rKsp37 and/or an anti-Ksp37 antibody for 5 d. CTL activity against P815 tumor cells (H-2^d) was assessed by using ⁵¹Cr assay.

Figure 7. Augmentation of cytokines (IL-2, IFN-γ and IL-6) production by Ksp37 was also observed. (A) Augmentation of IL-2 production from T cells or spleen cells by Ksp37. An amount of 5 × 10⁶ splenic T cells or spleen cells from C57BL/6 mice were cultured with 5 × 10⁵ BALB/c spleen cells (Mitomycin-C treated) in the presence of rKsp37 for two days. IL-2, IFN-γ and IL-6 activities in the supernatants were assessed by ELISA. (B) Augmentation of IFN-γ production from spleen cells by Ksp37. (C) Augmentation of IL-6 production from spleen cells by Ksp37.

Figure 8. Augmentation of CTL differentiation in vivo by the treatment with Ksp37 DNA was demonstrated. C57BL/six mice were challenged with killed H37Ra antigen (1,000 µg/mouse) in vivo (i.p.) and then treated with Ksp37 DNA (100 µg/mouse, six times) i.m. Twenty-one days after challenge of killed TB antigen, and PEC (peritoneal exudate cells) from these mice were harvested. CTL activity againt TB antigen (HSP65 antigen) was assessed by using ⁵¹Cr EL-4 which had been transfected with HSP65 DNA.

Figure 9. Synergistic efficacy of Ksp37 and granulysin on the in vitro CTL induction. Splenic T cells were cultured with uv-treated BALB/c spleen cells (Mytomycin C treated) in the presence or absence of 100ng/ml Ksp37 protein and 500ng/ml 15K granulysin protein for 5 d. Five days after culture, CTL activity against P815 tumor cells was assessed by using ⁵¹Cr release assay.