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

Characterization of Folic Acid and Poly(amidoamine) Dendrimer Interactions with Folate Binding Protein: A Force-Pulling Study

Atomic force microscopy force-pulling experiments have been used to measure the binding forces between folic acid (FA) conjugated poly­(amidoamine) (PAMAM) dendrimers and folate binding protein (FBP). The generation 5 (G5) PAMAM conjugates contained an average of 2.7, 4.7, and 7.2 FA per dendrimer. The most probable rupture force was measured to be 83, 201, and 189 pN for G5-FA_2.7, G5-FA_4.7, and G5-FA_7.2, respectively. Folic acid blocking experiments for G5-FA_7.2 reduced the frequency of successful binding events and increased the magnitude of the average rupture force to 274 pN. The force data are interpreted as arising from a network of van der Waals and electrostatic interactions that form between FBP and G5 PAMAM dendrimer, resulting in a binding strength far greater than that expected for an interaction between FA and FBP alone

Gel electrophoresis indicates NLP complex formation with YopB and/or YopD.

<p>Purified expressed YopB and/or YopD co-expressed with Δ49ApoA1 in the presence of lipid. <b>A)</b> Denatured 4–12% Bis-Tris PAGE gel, MES-SDS buffer, Native Tris-glycine buffer, detection by SyproRuby fluorescence total protein stain. <b>B)</b> Native 4–12% Tris-glycine PAGE gel, Native Tris-glycine buffer, detection by SyproRuby fluorescence total protein stain. The lanes are represented as follows with the predicted molecular weight based on native gel size; <b>1)</b> YopB with Δ49ApoA1, 237 kDa complex; <b>2)</b> YopB/D with Δ49ApoA1, 448 kDa complex; <b>3)</b> YopD with Δ49ApoA1, 397 kDa complex; <b>4)</b> Δ49ApoA1 only (empty-NLP), 393 kDa complex; <b>M)</b> Mass standard (kDa) SeeBlue Plus 2 (Invitrogen); <b>NM)</b> Protein mass standard (kDa) NativeMark (Invitrogen). <b>Grey arrow</b> YopB; <b>Black arrow</b> YopD; <b>White arrow</b> Δ49ApoA1.</p

Size distribution for co-expressed YopD- and/or YopB/D-NLPs as determined by FCS.

<p>Size distribution for co-expressed YopD- and/or YopB/D-NLPs as determined by FCS.</p

Anti-YopD western blot confirms YopD complex formation with NLPs.

<p>Purified expressed YopB and/or YopD co-expressed with Δ49ApoA1 in the presence of lipid. <b>A)</b> Native 4–20% Tris-glycine PAGE gel, Native Tris-glycine buffer, detection by SyproRuby fluorescence total protein stain. <b>B)</b> Anti-YopD western blot of a native 4–20% Tris-glycine PAGE gel, Native Tris-glycine buffer, detection by fluorescence of rhodamine conjugated secondary antibody. The lanes are represented as follows: <b>1)</b> YopB with Δ49ApoA1; <b>2)</b> YopB/D with Δ49ApoA1; <b>3)</b> YopD with Δ49ApoA1; <b>4)</b> Δ49ApoA1 only (empty-NLP); <b>NM</b>) Protein mass standard (kDa) NativeMark (Invitrogen).</p

Expression and Association of the <i>Yersinia pestis</i> Translocon Proteins, YopB and YopD, Are Facilitated by Nanolipoprotein Particles

<div><p><i>Yersinia pestis</i> enters host cells and evades host defenses, in part, through interactions between <i>Yersinia pestis</i> proteins and host membranes. One such interaction is through the type III secretion system, which uses a highly conserved and ordered complex for <i>Yersinia pestis</i> outer membrane effector protein translocation called the injectisome. The portion of the injectisome that interacts directly with host cell membranes is referred to as the translocon. The translocon is believed to form a pore allowing effector molecules to enter host cells. To facilitate mechanistic studies of the translocon, we have developed a cell-free approach for expressing translocon pore proteins as a complex supported in a bilayer membrane mimetic nano-scaffold known as a nanolipoprotein particle (NLP) Initial results show cell-free expression of <i>Yersinia pestis</i> outer membrane proteins YopB and YopD was enhanced in the presence of liposomes. However, these complexes tended to aggregate and precipitate. With the addition of co-expressed (NLP) forming components, the YopB and/or YopD complex was rendered soluble, increasing the yield of protein for biophysical studies. Biophysical methods such as Atomic Force Microscopy and Fluorescence Correlation Spectroscopy were used to confirm that the soluble YopB/D complex was associated with NLPs. An interaction between the YopB/D complex and NLP was validated by immunoprecipitation. The YopB/D translocon complex embedded in a NLP provides a platform for protein interaction studies between pathogen and host proteins. These studies will help elucidate the poorly understood mechanism which enables this pathogen to inject effector proteins into host cells, thus evading host defenses.</p></div

Atomic Force Microscopy (AFM) of empty- and Yop-NLPs.

<p><b>Left panels</b>, Atomic force microscopy (AFM) image of Empty- and Yop-NLPs prepared by co-expression. 100 nm scale bar. <b>Center panels</b>, AFM height distribution plots. <b>Right panels</b>, NLP cartoons with and without Yop insertion. <b>A)</b> empty-NLPs. <b>B)</b> YopB-NLPs. <b>Black Line</b>: empty-NLPs representing 70% of the total; <b>Green Line</b>: co-expressed YopB-NLPs representing 30% of total. <b>C)</b> YopB/D-NLPs. <b>Black Line</b>: empty-NLPs representing 61% of the total; <b>Green Line</b>: co-expressed YopB-NLPs representing 39% of the total. <b>D)</b> YopD-NLPs. <b>Black Line</b> represents all particles; YopD-NLPs were indistinguishable from empty-NLPs.</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

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

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