Activation of HIV-1-specific immune responses to an HIV-1 vaccine constructed from a replication-defective adenovirus vector using various combinations of immunization protocols

Abstract

We constructed a recombinant replication defective adenovirus vector containing the env gene (Ad-Bal) derived from macrophage-trophic HIV-1 (HIV-1 Bal). We then immunized mice with this vector using several administration routes and protocols, and examined the immune response. When the Ad-Bal viral vector (over 1 x 10(7) pfu) was injected subcutaneously, both humoral and cell-mediated immunities were induced. However, immune response induced by the Ad-Bal vector alone was weaker than that induced by the recombinant vaccinia viral vector. We then employed the following three immunization protocols: (l) DNA vaccination followed by immunization with the Ad-Bal; (2) vaccination using the Ad-Bal vector followed by DNA vaccination; and (3) DNA vaccination followed by Ad-Bal infection and passive transfer of dendritic cells (DCs) infected with the Ad-Bal. Among the three protocols, the last gave the strongest humoral and cell-mediated immunity. These results suggest that the combination of DNA vaccination, Ad-Bal vector infection and passive transfer of Ad-Bal-infected DCs can induce strong immunity against HIV-1 Bal.

Fig. 1

Serum antibody titres of mice administered Ad-Bal or Ad-β Gal in various concentrations via various routes of immunization: subcutaneous (s.c.), intranasal (i.n.), intramuscular (i.m.) and intraperitoneal (i.p.). After 4 weeks an antigen-specific IgG response induced by Ad-Bal immunization was observed in the test groups, but in the control vector group (Ad-β Gal) the titre was below the detection limit. Among the four administration routes, s.c. immunization produced the best antibody response. Although there was no statistical significance, the i.n. immunization gave a better response than i.m. or i.p. Myoglobin peptide was used as a control, but there was no significant rise in titre even after 4 weeks. ▪, s.c. Ad-Bal; ▴, i.n. Ad-Bal; □, i.m. Ad-Bal; •;, i.p. Ad-Bal; *, s.c. Ad-βGal; ▵, i.n. Ad-βGal; ◊–-, i.m. Ad-βGal; ○, i.p. Ad-βGal; —, non-immunized; ◊, s.c. Vac-env.

Fig. 2

Serum antibody titres of mice administered Ad-Bal. After 4, 8 and 16 weeks, an antigen-specific IgG response induced by Ad-Bal immunization was observed. Myoglobin peptide was used as a control, but there was no significant rise in titre even after 4 weeks. •;, Ad-Bal 5 × 10⁷ pfu; ▪, Ad-Bal 5 × 10⁶ pfu; □, Ad-Bal bGal 5 × 10⁷ pfu.

Fig. 3

CTL activity of spleen cells from mice immunized with Ad-Bal or Vac-env. On day 0, 5 × 10⁷ pfu of Ad-Bal, Ad-β Gal or Vac-env were injected s.c. into BALB/c mice. After 3 weeks, splenic lymphoid cells were collected and co-cultured with peptide-pulsed P815 cells (H-2^d) for 5 days. These effector cells were reacted with ⁵¹Cr-labelled target cells. Target cells (P815 cells (H-2^d) or EL-4 (H-2^b)) were pulsed with V3 peptide. Data represent means ± s.e. of 4–5 mice. Data from two other separate experiments showed a similar tendency.

Fig. 4

CTL activity of spleen cells from mice immunized with various combinations of vaccine using Ad-Bal, pCMV160Bal or Vac-env. On day 0, 5 × 10⁷ pfu of Ad-Bal, Ad-beta Gal or Vac-env were injected s.c. In other groups, 30 μg of pCMV160Bal was injected i.m. on day 0. After 2 weeks, each immunized group was boosted using the same dose and the same techniques as described in the legend of Fig. 2. CTL activity was assayed after another 3 weeks. The method for evaluation of CTL responses was the same as in Fig. 2. Data represent means ± s.e. of 4–6 mice. Data from two other separate experiments showed a similar inclination. Immunization with: ○, DNA vaccine→Vac-enc; ▪, DNA vaccine→Ad-Bal; •;, Ad-Bal→DNA vaccine; □, DNA vaccine→Ad-βGal; ▵, DNA vaccine→Ad-Bal (EL-4 target); ▴, non-immunization.

Fig. 5

ELISPOT analysis of lymphocytes from mice immunized with Ad-Bal, Ad-Bal-infected DCs and pCMV160 Bal.On day 0, Ad-Bal or Ad-β Gal was injected s.c. In other groups, 30 μg of pCMV160Bal was injected i.m. on days 0. After 2 and 4 weeks, each immunized group was boosted using the same dose and the same techniques as described in Tables 2 and 6. ELISPOT analysis was performed after another 2 weeks using immune spleen cells. Procedures for immunization: 1, Ad-Bal; 2, DNA vaccine →Ad-Bal; 3, Ad-Bal→DNA vaccine→Ad-Bal-inf DCs; 4, DNA vaccine →Ad-Bal→Ad-Bal-inf DCs; 5, DNA vaccine→Ad-Bal→Ad-Bal-inf DCs (target, myoglobin peptide-pulsed P815); 6, Ad-beta Gal. Data from another experiment showed a similar tendency. With the exception of 5, the targets were Bal peptide-pulsed P815 cells. Data represent means ± S.E. of 4–6 mice. *Significant difference between six and other groups (P < 0·05).