Cross-neutralization Abs elicited by the RG1-VLP vaccine were demonstrated by the presence of titers specific to HPV18 and HPV39. consisting of novel bacterial 4E2RCat enzymatic combinatorial chemistry (BECC)-derived toll-like receptor 4 (TLR4) agonists and the aluminum hydroxide adjuvant Alhydrogel. In the presence of BECC molecules, consistent improvements in the magnitude of Ab responses to both HPV16-L1 and the L2 RG1 epitope were observed compared to Alhydrogel alone. Furthermore, neutralizing titers to HPV16 as well as cross-neutralization of pseudovirion (PsV) types HPV18 and HPV39 were augmented in the presence of BECC agonists as well. Levels of L1 and L2-specific Abs were achieved after two vaccinations with BECC/Alhydrogel adjuvant that were equivalent to or greater than levels achieved with 3 vaccinations with Alhydrogel alone, indicating that the presence of BECC molecules resulted in accelerated immune responses that could allow for a decreased dose schedule for VLP-based HPV vaccines. In addition, dose-sparing studies indicated that adjuvantation with BECC/Alhydrogel allowed for a 75% reduction in antigen dose while still retaining equivalent magnitudes of responses to the full VLP dose with Alhydrogel. These data suggest that adjuvant optimization of HPV VLP-based vaccines can lead to rapid immunity requiring fewer boosts, dose-sparing of VLPs expensive to produce, and the establishment of a longer-lasting humoral immunity. antigen rF1-V to increase anti-rF1-V IgG and IgG2c titers and to protect from lethal challenge (15). In BALB/c mice, the novel RG1-VLP vaccine when combined with Alhydrogel has demonstrated strong induction of HPV16-L1- and HPV16-L2-specific Abs as well as cross-neutralization activity against other HPV types (11). In an attempt to further optimize the RG1-VLP vaccine and strengthen its ability for cross-type-specific neutralization, this study investigates the combination of BECC438 (15) or BECC470 with Alhydrogel for the ability of the vaccine to induce higher magnitude humoral responses as well as accelerated and longer-lasting immunity. MATERIALS AND METHODS BECC molecule synthesis The previously described BECC438 and BECC470 KIM6+ strains (14C15) (Fig. 1) were grown in shaking culture at 26 C for 18 h, within which time the culture reached an OD600 of 1 1.0C1.4. After pelleting bacteria from the liquid culture, lipooligosaccharide was extracted from the pellet as previously described using a double hot phenol method followed by three 2:1 (vol/vol) chloroform-methanol washes (14). Mass spectrometry was used to confirm the extracted lipid A structures (Bruker Microflex MALDI-TOF, norharmane matrix, negative ion mode). Open in a separate window Fig. 1: Chemical structures of BECC470 and BECC438. Both molecules are hexa-acylated with one modification in the acyl-chain arrangement. BECC470 has a secondary C12 addition at the 3 position while BECC438 has a secondary C16:1 addition at the 2 2 position. The 4 phosphate group is removed in BECC470 making it mono-phosphorylated while BECC438 remains bis-phosphorylated. Measurement of BECC Binding to Alhydrogel A reversed-phase high performance liquid chromatography method was developed to analyze the amount of BECC470 in a solution. A standard curve was created from 0.01 mg/ml to 0.9 mg/ml BECC470 (Sup. Fig. 1). Binding isotherms for BECC470 were conducted in PBS at 25 C. Samples were prepared by mixing BECC470 (nine different concentrations) and Alhydrogel (at 0.2 or 1.0 g/L). Samples were vortexed for 5 sec and equilibrated 4E2RCat for at least 4 h at r.t. after mixing. After equilibration, samples were centrifuged (4000 g for 10 min), the supernatant collected, and the concentration of BECC470 determined from the standard curve. Three independent isotherms MULK were conducted for each Alhydrogel concentration. Physicochemical analysis of BECC/alum formulations The size distribution of Alhydrogel (InvivoGen) was determined using a Malvern Mastersizer 3000 (Malvern Instruments). First, 6 mL of dH2O was added to 4E2RCat the sample chamber and a background measurement was taken. 300 L of Alhydrogel sample was injected for each measurement (Alhydrogel at 1 g/L) for a total light obscuration of ~4%. A refractive index of 1 1.57 was used for Alhydrogel in the Mie scattering calculations for the determination of size. Size distributions were reported by number. Each sample is an average of ten instrumental replicates..