The Thomas Jefferson University Institutional Animal Care and Use Committee has approved these studies. Mice were housed in micro-isolator cages with free access to food and water and were maintained in a specific pathogen-free facility. C57BL/6J (stock no. 000664), 129S1/SvImJ (stock no. 002448), A/J (stock no. 000646), and C3H/HeOuJ (stock no. 000635) were purchased from The Jackson Laboratory (Bar Harbor, ME). CD-1 [Crl : CD1(ICR); Strain code 022] outbred mice were purchased from Charles River Laboratories (Wilmington, MA). Age-matched mice of both sexes were used for all experiments.

The adjuvant named Turbo was prepared by mixing 1 mg phosphorylated hexaacyldisaccharide (PHAD^®), a synthetic monophosphoryl lipid A (MPLA) and 2 mg of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids, Alabaster, AL) in chloroform. Following chloroform evaporation, the contents were suspended in 1% polyethylene glycol sorbitan monooleate (Tween^® 80; Sigma-Aldrich) to a concentration of 500 μg/ml of MPLA and homogenized by sonication. The homogenate was mixed 50 times using two syringes connected via a 25G needle and passed five times through a polyethersulfone membrane filter with pore size of 0.22 μm (Millipore), and the filter-sterilized adjuvant was stored at 4°C. Nanoparticle tracking analysis using NanoSight NS300 instrumentation (Malvern, Instruments Ltd., Worcestershire) revealed that the size distribution and concentration of liposomes in the adjuvant formulation were 130 ± 40 nm and 4×10¹⁰/ml, respectively. The physical characteristics and adjuvant activity were stable for at least 1 year at 4°C.

ViPS (lot 5, PDMI 158299) was obtained from the US Food and Drug Administration (Silver Spring, MD) (21). A portion of this ViPS preparation was subjected to phenol extraction to eliminate TLR4 ligands/immunostimulatory compounds as described previously (12). Typhim Vi^® (Sanofi Pasteur), an FDA-approved unconjugated typhoid vaccine, was purchased, while Typbar TCV^® was obtained from Bharat Biotech India Limited, Hyderabad, India.

Mice (3 weeks of age and above) were immunized i.m. with 2.5 μg of ViPS [i.e., Typbar TCV^®, Typhim Vi^®, ViPS preparation (US FDA, lot 5, PDMI 158299) without or with phenol extraction] admixed with or without Turbo adjuvant (containing 5 μg MPLA) in 50 μl volume in the thigh region of the hind limb. Infant mice (9 days old) were immunized s.c. with 1.25 μg of ViPS admixed with or without Turbo adjuvant (containing 2.5 μg MPLA) in 25 μl volume in the scruff area between the neck and shoulder. Blood samples were obtained 0, 7, 14, 21, and 28 days (or as indicated) following immunization and stored at −20°C.

ViPS-specific IgM, IgG, IgG1, IgG2b, IgG2a, IgG2c, and IgG3 were measured by coating 96-well microtiter plates (Nunc MaxiSorp™; Invitrogen, Carlsbad, CA) with 2 µg/ml of ViPS purified from S. Typhi clinical isolate C652464 (22) in DPBS overnight at room temperature. All plates were washed and blocked with 1% bovine serum albumin in PBS pH 7.2 (blocking buffer) for 2 h at room temperature. Blood from ViPS or Typhim Vi^® and Typbar TCV^® immunized mice was diluted to 1:25 and 1:200, respectively, for IgM and IgG detection. These dilutions were based on a linear range of detection. ViPS-specific mouse IgM, IgG, IgG1, IgG2b, IgG2a, IgG2c, and IgG3 levels were interpreted as ng/μl “equivalents” using normal mouse serum standards (Bethyl Laboratories, Montgomery, TX), mouse isotype-specific capture antibodies, and HRPO-conjugated anti-mouse IgM, IgG, IgG1, IgG2b, IgG2a, IgG2c, and IgG3 as described previously (12, 21).

To test the relative protection conferred by immunization with Typbar TCV^® or Typhim Vi^® admixed with and without Turbo adjuvant, mice were infected with a chimeric strain of S. Typhimurium (strain RC60) that expresses the S. Typhi genes necessary for ViPS synthesis, export, and regulation as in S. Typhi (23). This protection model was previously validated in several mouse systems (21, 24–26). In brief, Strain RC60 was grown to an OD⁶⁰⁰ of ~1.0 in Luria–Bertani broth containing 10 mM NaCl. The expression of ViPS was assessed by slide agglutination test using a commercial Vi monoclonal antibody reagent (Statens Serum Institute Diagnostica A/S, Denmark; Lot 188L-8). Bacteria were washed twice in DPBS, and 100 μl of DPBS containing ~3×10⁴ CFUs was injected i.p. Three days post-infection, the liver and spleen were collected, and the tissues were processed using a Minilys tissue homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France). Blood was collected into anti-coagulant and bacterial burden in the blood, and tissue homogenates were measured by counting CFUs on LB agar plates.

Serum bactericidal assay (SBA) was performed as previously described (27). In brief, log-phase cultures (OD₆₀₀ of 0.5 at 37°C) of S. Typhi strain Ty2 were prepared in LB broth with 10 mM NaCl. Bacterial cells were washed in DPBS, and the bacterial cell density was adjusted to 2.5–5.0 × 10⁴ colony forming units (CFU) per milliliter in DPBS. The expression of ViPS was assessed by slide agglutination test using a commercial Vi monoclonal antibody reagent (Statens Serum Institute diagnostica A/S, Denmark; Lot 188L-8). Serum samples were heat inactivated by incubating at 56°C for 30 min prior to use in the assay. A volume of 10 μl of S. Typhi strain Ty2 in DPBS (250–500 CFU) was added to each well of a round-bottom polypropylene 96-well plate containing 50 µl of heat-inactivated serum in serial dilutions, 12.5 µl baby rabbit complement (Pel-Freeze, Rogers, AR), and 27.5 µl DPBS. Triplicate samples of each dilution were incubated for 120 min at 37°C with gentle rocking, and 10 µl of this mixture was plated on LB agar plates for counting CFU. Serum bactericidal antibody titers are defined as the reciprocal of the highest dilution that produced 50% killing in relation to control wells containing complement but no mouse serum. Naive mouse serum served as a negative control and serum from either mice immunized with heat-killed E. coli strain W3110 expressing pDC5 plasmid, which contains the genes necessary for the synthesis and export of ViPS (28), or S. Typhi anti-Vi human IgG standard (Lot R1, 2011; US Food and Drug Administration, Silver Spring, MD 20993) as two independent positive controls.

Data presented throughout depict pooled data from at least two independent experiments. Statistics were performed using the Prism 10 software program (GraphPad Software, Inc., La Jolla, CA), and the statistical tests are indicated in the figure legends.

Although Typbar TCV^® contains TLR4 ligands or low levels of endotoxin (12), when admixed with Turbo, significantly enhanced anti-ViPS IgM, IgG1, IgG2b, IgG2a/c, and IgG3 responses were observed in male and female C57BL6/J mice ( Figure 1 ). Recently, the immunogenicity of meningococcal conjugate vaccine has been shown to vary among inbred and outbred strains (29). To test the rigor of the immunogenicity promoted by Turbo as an adjuvant, we have used outbred strain CD-1 and inbred strains such as C3H and A/J, which can also serve as a model to test vaccine-mediated protection against Salmonella in vivo (30). We found that Turbo adjuvanticity occurs in all mouse strains tested, but is strikingly more pronounced in outbred CD-1 strain ( Figure 2 ).

Aged individuals do not respond efficiently to vaccines due to immune senescence (31), and the anti-ViPS response is lower in 82-week-old mice compared to that in 16-week-old adult mice ( Figures 3A vs. B ). However, when adjuvanted with Turbo, aged mice showed a significant improvement in anti-ViPS IgM and IgG responses to Typbar TCV^® ( Figures 3A vs. B ). The lack of an efficient antibody response early in life is due to an incomplete development of the immune system (32). Nevertheless, when Typbar TCV^® is adjuvanted with Turbo, infant mice produced a rapid IgM response and an order of magnitude higher IgG response that is sustained for at least 120 days post-immunization compared to mice that were immunized with Typbar TCV^® alone ( Figure 3C ). Since S. Typhi does not infect commonly used mouse strains (33), we have used S. Typhimurium strain RC60 expressing ViPS antigen to test ViPS-mediated protection in vivo, as described previously (21, 24–26). We found that infant mice vaccinated with Turbo-adjuvanted Typbar TCV^®, when challenged 120 days post-immunization with S. Typhimurium strain RC60, exhibited more significant reduction in bacterial load in the blood, liver, and spleen, compared to mice immunized without Turbo ( Figure 3D ).

Compared to conjugated vaccines such as Typbar TCV^®, the antibody responses to Typhim Vi^®, is an order of magnitude lower (12). Despite the presence of TLR4 ligands in Typhim Vi^® (12), when this vaccine was adjuvanted with Turbo, all IgG isotypes were enhanced ( Figure 4A ). Moreover, seroconversion with the adjuvanted Typhim Vi^® was 100%, unlike the unadjuvanted Typhim Vi^®, and IgG responses were sustained for at least 90 days ( Figure 4B ), and the control of bacteremia was significantly improved ( Figure 4C ).

Unconjugated polysaccharide vaccines such as Typhim Vi^® do not induce an efficient response in young (3-week-old) compared to adult mice due to a restricted V_H gene usage early in life and a paucity of antigen-specific B-cell precursors (26). To test whether admixing Turbo with ViPS (US FDA, lot 5, PDMI 158299) overcomes the poor antibody responses in the young, 3-week-old C57BL/6J mice were immunized. We found that Turbo-adjuvanted ViPS dramatically improved the IgG response in young mice ( Figure 5A ). We showed that this ViPS preparation contains TLR4 ligands that play an important role in the immunogenicity of this ViPS (12) and that the elimination of these immunostimulatory components by phenol extraction results in a dramatic loss of immunogenicity (12). Remarkably, when phenol extracted ViPS preparation was admixed with Turbo, immunogenicity was restored in young mice ( Figure 5B ). Furthermore, we found that native ViPS and phenol-extracted ViPS preparation when adjuvanted with Turbo induced efficient IgM and all four IgG isotypes in adult inbred and outbred strains of mice compared to mice immunized without Turbo ( Figure 6 ). These data suggest that Turbo can serve as an adjuvant for unconjugated ViPS vaccines in young and adult mice.

A characteristic of adjuvants is their ability to promote antigen-specific responses even at very low concentrations of a given antigen. To test the impact of Turbo adjuvanticity on dose sparing of the antigen, we immunized mice with decreasing amounts of ViPS over a 100-fold range (2.5 μg to 0.02 μg). We found that admixing Turbo promoted immune responses even at a concentration (0.02 μg) that failed to induce a detectable anti ViPS-IgG response without Turbo ( Figure 7A ). We also found that at a constant concentration of ViPS (2.5 μg), Turbo containing 5 μg of MPLA induced the highest IgM and IgG response, suggesting that this amount of MPLA is optimal in mice.