A live attenuated Listeria monocytogenes vaccine vector expressing SIV Gag is safe and immunogenic in macaques and can be administered repeatedly

Abstract

Listeria monocytogenes (Lm) is known to induce strong cellular immune responses. We constructed a live-attenuated Lm vector, Lmdd-BdopSIVgag, which encodes SIVmac239 gag. Intragastric (i.g.) administration of 3 × 10(12) bacteria to rhesus macaques was safe and induced anti-Gag cellular but no humoral immune responses. Boosting of Gag-specific cellular responses was observed after i.g. administration of Lmdd-BdopSIVgag to previously vaccinated RM despite preexisting anti-Lm immunity shown by lymphoproliferative responses. Surprisingly, anti-Lm cellular responses were also detected in non-vaccinated controls, which may reflect the fact that Lm is a ubiquitous bacterium. The novel, live-attenuated Lmdd-BdopSIVgag may be an attractive platform for oral vaccine delivery.

Fig. 1

Construction of Lmdd-BdopSIVgag and analysis of SIV gag expression by Lmdd-BdopSIVgag. The recombinant Listeria monocytogenes (Lm) vector Lmdd-BdopSIVgag was obtained by allelic exchange between the Lm dal dat vector and pKSV-7 carrying a Bacillus subtilis dal gene and Lm-codon-optimized SIV gag (A). Culture supernatants from control Lmdd-Bdop (lanes 1, 2) and from gag-expressing Lmdd-BdopSIVgag (lanes 3–6) were analyzed by Western blot. SIV Gag was detected using the anti-SIV Gag monoclonal antibody 2F12 (AIDS Research & Reference Reagent Program, NIH). Cultures contained approximately 7 ng/ml p27 antigen as determined by ELISA. The multiple bands reflect proteolytic processing of Gag within the Lm and following secretion into the medium.

Fig. 2

Hematological profiles of RM following Lm administration. The percent lymphocytes (left panels) and neutrophils (right panels) are shown. No neutrophilia was seen; one Group C RM had an absolute neutrophil count on day 4 after the last dose of Lmdd-BdopSIVgag of 14,280 cells/mm³ (normal range for RM, 0.2–14.6 × 10³ cells/mm³) without bands or concomitant changes in the percent neutrophils and lymphocytes. This RM also did not show any clinical signs of illness. The normal percentages of lymphocytes and neutrophils range between 8% to 92% and 5% to 88%, respectively, as wide fluctuations have been reported.

Fig. 3

The frequency of SIV Gag-specific cells measured by IFN-γ ELISPOT assay at the time points indicated. Arrows, intragastric administration of either Lmdd-BdopSIVgag (L in dark circle) or Lmdd-Bdop (L in open circle); *, statistically significant difference (P = 0.031) between Groups B and C at week 6 in Phase II.

Fig. 4

Frequency of CD3+CD8+ Gag p11C tetramer+ cells among Mamu-A*001⁺ animals during Phases I and II. The results are shown only for responder RM. Results were considered positive if >0.03%, a cut-off that was determined from analysis of naïve monkeys. No positive cells were detected among Mamu-A*001 negative vaccinated monkeys.

Fig. 5

Proliferation of PBMC after stimulation with whole heat-inactivated Lm (A) or SIV Gag protein (B). Percent CD4+ (left panels) and CD8+ (right panels) proliferating cells are shown. Dashed line, mean frequency of proliferating cells shown by control (Group D) animals.