Live attenuated Listeria monocytogenes expressing HIV Gag: immunogenicity in rhesus monkeys

Abstract

Induction of strong cellular immunity will be important for AIDS vaccine candidates. Natural infection with wild-type Listeria monocytogenes (Lm), an orally transmitted organism, is known to generate strong cellular immunity, thus raising the possibility that live attenuated Lm could serve as a vaccine vector. We sought to examine the potential of live attenuated Lm to induce cellular immune responses to HIV Gag. Rhesus macaques were immunized with Lmdd-gag that expresses HIV gag and lacks two genes in the D-alanine (D-ala) synthesis pathway. Without this key component of the bacterial cell wall, vaccine vector replication critically depends on exogenous D-ala. Lmdd-gag was given to animals either solely orally or by oral priming followed by intramuscular (i.m.) boosting; D-ala was co-administered with all vaccinations. Lmdd-gag and D-ala were well tolerated. Oral priming/oral boosting induced Gag-specific cellular immune responses, whereas oral priming/i.m. boosting induced systemic as well as mucosal anti-Gag antibodies. These results suggest that the route of vaccination may bias anti-Gag immune responses either towards T-helper type 1 (Th1) or Th2 responses; overall, our data show that live attenuated, recombinant Lmdd-gag is safe and immunogenic in primates.

Fig. 1

Primary immunization schedule for Lmdd-gag administration to rhesus macaques. Animals were enrolled into 3 groups and given Lmdd-gag or Lmdd at 0, 6 and 19 weeks (vertical arrows) as noted. The number of organisms administered at each time point is shown in parentheses for each group. All animals were given D-ala (640 mg/kg) i.v. at 15 min prior to and 2.5 hrs after each immunization.

Fig. 2

Pharmacokinetics of D-ala in rhesus macaques. Serum D-ala levels are shown for two representative animals receiving oral Lmdd-gag and accompanying i.v. and oral D-ala as noted in Fig. 1.

Fig. 3

Gag-specific IFN-γ-secreting T cells by ELISPOT assay in Lmdd-gag-immunized macaques. A. PBMC from individual monkeys were tested at the indicated time points for Gag-specific IFN-γ secreting T cells after in vitro stimulation with overlapping HIV Gag peptides. B. Gag-specific IFN-γ-secreting T cells in Lmdd-gag-boosted macaques following prolonged rest (the interval between the week 19 boost and the final boost ranged from 27–64 weeks because of staggered enrollment).

Fig. 4

Gag-specific T-cell proliferative responses in Lmdd-gag-immunized macaques. A, PBMC from individual monkeys were tested for Gag-specific T-cell proliferative responses at the indicated time points during and after vaccination. Stimulation indices (SI) were calculated as described. B, Gag-specific T-cell proliferative responses in Lmdd-gag boosted macaques following prolonged rest. The dotted horizontal line at SI of 4 denotes the cut-off for a positive response.

Fig. 5

Gag-specific antibody titers in vaginal and rectal fluids. Vaginal and rectal fluid samples were obtained from animals REg-8 (circles) and RMg-8 (triangles) at the indicated time points following the final i.m. inoculation with Lmdd-gag. Fluid samples were tested for anti-Gag IgG levels by ELISA. A, Vaginal fluid samples. B, Rectal fluid samples. The background OD readings (determined as 2 SD above the mean OD from samples of 6 naïve animals) are indicated by the horizontal dotted line.