Colocalized Delivery of Adjuvant and Antigen Using Nanolipoprotein Particles Enhances the Immune Response to Recombinant Antigens

Subunit antigen-based vaccines can provide a number of important benefits over traditional vaccine candidates, such as overall safety. However, because of the inherently low immunogenicity of these antigens, methods for colocalized delivery of antigen and immunostimulatory molecules (i.e., adjuvants) are needed. Here we report a robust nanolipoprotein particle (NLP)-based vaccine delivery platform that facilitates the codelivery of both subunit antigens and adjuvants. Ni-chelating NLPs (NiNLPs) were assembled to incorporate the amphipathic adjuvants monophosphoryl lipid A and cholesterol-modified CpG oligodeoxynucleotides, which can bind His-tagged protein antigens. Colocalization of antigen and adjuvant delivery using the NiNLP platform resulted in elevated antibody production against His-tagged influenza hemagglutinin 5 and Yersinia pestis LcrV antigens. Antibody titers in mice immunized with the adjuvanted NLPs were 5–10 times higher than those observed with coadministration formulations and nonadjuvanted NiNLPs. Colocalized delivery of adjuvant and antigen provides significantly greater immune stimulation in mice than coadministered formulations

Lipid Cross-Linking of Nanolipoprotein Particles Substantially Enhances Serum Stability and Cellular Uptake

Nanolipoprotein particles (NLPs) consist of a discoidal phospholipid lipid bilayer confined by an apolipoprotein belt. NLPs are a promising platform for a variety of biomedical applications due to their biocompatibility, size, definable composition, and amphipathic characteristics. However, poor serum stability hampers the use of NLPs for in vivo applications such as drug formulation. In this study, NLP stability was enhanced upon the incorporation and subsequent UV-mediated intermolecular cross-linking of photoactive DiynePC phospholipids in the lipid bilayer, forming cross-linked nanoparticles (X-NLPs). Both the concentration of DiynePC in the bilayer and UV exposure time significantly affected the resulting X-NLP stability in 100% serum, as assessed by size exclusion chromatography (SEC) of fluorescently labeled particles. Cross-linking did not significantly impact the size of X-NLPs as determined by dynamic light scattering and SEC. X-NLPs had essentially no degradation over 48 h in 100% serum, which is a drastic improvement compared to non-cross-linked NLPs (50% degradation by ∼10 min). X-NLPs had greater uptake into the human ATCC 5637 bladder cancer cell line compared to non-cross-linked particles, indicating their potential utility for targeted drug delivery. X-NLPs also exhibited enhanced stability following intravenous administration in mice. These results collectively support the potential utility of X-NLPs for a variety of in vivo applications

Time-dependent <i>in vivo</i> NiNLP biodistribution upon i.p. and i.n. administration.

<p>NiNLPs were administered by A) i.p. or B) i.n. routes and were assessed over 72 or 96 hours, respectively. Organ fluorescence was determined <i>ex vivo</i> and normalized to total organ weight. The normalized fluorescent intensity was quantitatively measured as a function of time. Data represent the average normalized fluorescence from groups of two animals, with standard error bars.</p

Stability of DMPC∶NLPs in 20% serum assessed by SEC over 24 hrs at 25°C.

<p>The indicated peaks correspond to intact NLP (t_R 9.5 min) and free apoE422k scaffold protein (t_R 12.2 min). Absorbance of AF647-labeled apoE422k absorbance was monitored at 600 nm.</p

Effect of repeated NiNLP administration on mouse body weights.

<p>Weights of A) male and B) female mice receiving daily NiNLP injections i.n. (30 µl) or i.p. (100 µl) for 14 consecutive days. Control mice received equal volumes of PBS i.p. over the same 14-day time course. Data represent averaged weights from groups of three animals, with standard deviation error bars.</p

Assessment of NiNLP immunogenicity.

<p>Groups of 10 female BALB/c mice were inoculated either i.n. or i.p. with NiNLP. As a positive control, a group of mice was injected with a known immunogenic recombinant subunit antigen (LcrV) co-administered with adjuvant (CpG). Serum IgG antibody titers against the scaffold protein, apoE422k (NiNLP-ip and NiNLP-in), or LcrV (LcrV+CpG-ip and LcrV+CpG-in) were assessed 4 weeks post-immunization. Each data point represents the titer value of an individual mouse.</p

Effect of repeated NiNLP administration on mouse organ weights.

<p>Weights of A) liver, B) kidney, C) lung, and D) spleen obtained from mice that received 25 µg of NiNLP i.n. (30 µl) or i.p. (100 µl) daily for 14 consecutive days. Control animals received an equal volume of PBS i.p.(100 µl) daily for 14 days. Normalized organ weights are represented as (organ weight, g)/(body weight, g). Data represent averaged organ weights from groups of three animals, with standard deviation error bars.</p

Stability of NLPs as a function of lipid content, temperature, time, and serum concentration.

<p>Integrated NLP peak area of the SEC chromatograms for A) DOPC∶NLPs incubated at 25°C and B) DMPC∶NLPs incubated at 25°C. C) t_1/2 of the DOPC∶NLPs (blue line) and DMPC∶NLPs (red line) incubated at 25°C. Integrated NLP peak area of the SEC chromatograms for D) DOPC∶NLPs incubated at 37°C and E) DMPC∶NLPs incubated at 37°C. F) t_1/2 of the DOPC∶NLPs (blue line) and DMPC∶NLPs (red line) incubated at 37°C. AF647-labeled apoE422k absorbance was monitored at 600 nm.</p

Histological analysis of the effect of repeated NiNLP administration on the microstructure of the liver.

<p>H&E stained sections of livers obtained from mice that had received 25 µg of NiNLP i.n. (30 µl) or i.p. (100 µl) daily for 14 consecutive days at 10×, 20× and 40× magnifications.</p

Conjugation of cODN and PF to the NLP platform.

<p>A) SEC analysis of the cODN∶NLP constructs at indicated cODN∶NLP molar ratios, monitored at 280 nm. The increase in absorbance at 280 nm and peak shift indicate successful incorporation of cODN. B) UV-Vis absorption spectra of SEC-purified cODN∶NLP (blue line) and apoE422k concentration matched NLP lacking the cODN (black line). The dashed lines represent the absorbance at 260 nm and 280 nm. C) Analysis of cODN incorporation into the NLP. The x-axis represents the cODN∶NLP ratio used during the NLP assembly reaction and the y-axis is the measured amount of cODN ultimately incorporated into the particle. D) SEC chromatograms of the PF∶NLP constructs at increasing PF-to-NLP ratios. E) UV-vis spectra of PF∶NLPs (blue line) and apoE422k concentration matched NLPs lacking the PF (black line). The dashed lines represent the absorbance at 280 nm and 368 nm. F) Analysis of PF incorporation efficiency into the NLP, represented as a function of the PF-to-NLP assembly ratio (x-axis) vs. measured PF-to-NLP ratio after purification (y-axis).</p