Haloarchaeal gas vesicle nanoparticles displaying Salmonella SopB antigen reduce bacterial burden when administered with live attenuated bacteria

Abstract

Innovative vaccines against typhoid and other Salmonella diseases that are safe, effective, and inexpensive are urgently needed. In order to address this need, buoyant, self-adjuvating gas vesicle nanoparticles (GVNPs) from the halophilic archaeon Halobacterium sp. NRC-1 were bioengineered to display the highly conserved Salmonella enterica antigen SopB, a secreted inosine phosphate effector protein injected by pathogenic bacteria during infection into the host cell. Two highly conserved sopB gene segments near the 3'-coding region, named sopB4 and B5, were each fused to the gvpC gene, and resulting GVNPs were purified by centrifugally accelerated flotation. Display of SopB4 and B5 antigenic epitopes on GVNPs was established by Western blotting analysis using antisera raised against short synthetic peptides of SopB. Immunostimulatory activities of the SopB4 and B5 nanoparticles were tested by intraperitoneal administration of recombinant GVNPs to BALB/c mice which had been immunized with S. enterica serovar Typhimurium 14028 ΔpmrG-HM-D (DV-STM-07), a live attenuated vaccine strain. Proinflammatory cytokines IFN-γ, IL-2, and IL-9 were significantly induced in mice boosted with SopB5-GVNPs, consistent with a robust Th1 response. After challenge with virulent S. enterica serovar Typhimurium 14028, bacterial burden was found to be diminished in spleen of mice boosted with SopB4-GVNPs and absent or significantly diminished in liver, mesenteric lymph node, and spleen of mice boosted with SopB5-GVNPs, indicating that the C-terminal portions of SopB displayed on GVNPs elicit a protective response to Salmonella infection in mice. SopB antigen-GVNPs were found to be stable at elevated temperatures for extended periods without refrigeration in Halobacterium cells. The results all together show that bioengineered GVNPs are likely to represent a valuable platform for the development of improved vaccines against Salmonella diseases.

Keywords: Antigen display; Enteric disease; Haloarchaea; Nanoparticle; Salmonella.

Fig. 1

Halobacterium sp. NRC-1 gas vesicle nanoparticles (GVNPs). A. Phase contrast micrograph of purified GVNPs, which appear as phase bright dots (bar is 112.5 μm). B. Transmission electron micrograph of negatively stained GVNPs (bar is 500 nm) [17].

Fig. 2

Aligned partial sequences of SopB proteins from Salmonella enterica serovar Typhimurium, Typhi and Paratyphi. The neighbor-joining alignment is shown for the B4 (underlined) and B5 (broken underlined) regions from S. enterica serovar Typhimurium LT2 (S. Typhimurium LT2), S. enterica serovar Typhi CT18 (S. Typhi CT18), S. enterica serovarParatyphi A ATCC 9150 (S. Paratyphi A), and S. enterica serovar Paratyphi B SPB7 (S. Paratyphi B). A six amino acid region of overlap is indicated by the heavy underline.

Fig. 3

Western blotting analysis of Halobacterium GVNPs. A. Blot probed with SopB4 antiserum is shown between molecular weight markers 70 KDa and 100 KDa (lane labeled MW) for Halobacterium wild-type (lane labeled NRC-1) and Halobacterium SD109 (pSD104dsopB4) (lane labeled B4) GVNPs. B. Blot probed with SopB5 antiserum is shown between molecular weight markers 70 KDa and 100 KDa (lane labeled MW) for Halobacterium wild type (lane labeled NRC-1) and Halobacterium SD109 (pSD104dsopB5) (lane labeled B5) GVNPs.

Fig. 4

ELISAs for proinflammatory cytokines, IFN-γ, IL-2 and IL-9 in GVNP immunized mouse sera. Concentrations of cytokines (pg/ml) are indicated on the vertical axes and the types of GVNPs used for immunizations indicated in the horizontal axes. Dark gray bars denote preimmune sera and light gray bars, sera postimmunization. NRC: Wild type Halobacterium sp. NRC-1 GVNPs; B4: Halobacterium SD109 (pSD104dsopB4) GVNPs; B5: Halobacterium SD109 (pSD104dsopB5) GVNPs. Standard deviations are indicated by error bars.

Fig. 5

Bacterial burden in the liver, MLN and spleen of immunized and challenged mice. The cfu numbers are plotted on the vertical axes. GVNP identities are plotted on the horizontal axes. Cohorts of 5 mice were used in each case, with average values for surviving mice indicated by “X” and with standard deviations indicated by error bars. All mice were initially immunized with attenuated S. enterica serovar Typhimurium 14028 ΔpmrG-HM-D and challenged with virulent S. enterica serovar Typhimurium 14028 orally.

Fig. 6

CD4+ T cell assay for immunized mice. Flow cytometric analysis of spleenocytes are shown with CD4+ T cell-specific monoclonal antibodies of animals immunized with wild-type (WT), SopB4-GVNPs (B4), and SopB5-GVNPs (B5) (on the horizontal axis), with percent cell population plotted (on the vertical axis). Standard deviations are indicated by error bars.