Extent of MHC Clustering Regulates Selectivity and Effectiveness of T Cell Responses.

MHC proteins that present peptide ligands for recognition by TCR form nanoscale clusters on the cell membrane of APCs. How the extent of MHC clustering controls productive TCR engagement and TCR-mediated signaling has not been systematically studied. To evaluate the role of MHC clustering, we exploited nanoscale discoidal membrane mimetics (nanolipoprotein particles) to capture and present peptide-MHC (pMHC) ligands at various densities. We examined the binding of these model membrane clusters to the surface of live human CD8+ T cells and the subsequent triggering of intracellular signaling. The data demonstrate that the proximity of pMHC ligands, high association rate of CD8-MHC interactions, and relatively long lifetime of cognate TCR-pMHC complexes emerge as essential parameters, explaining the significance of MHC clustering. Rapid rebinding of CD8 to MHC suggests a dual role of CD8 in facilitating the T cells' hunt for a rare foreign pMHC ligand and the induction of rapid T cell response. Thus, our findings provide a new understanding of how MHC clustering influences multivalent interactions of pMHC ligands with CD8 and TCR on live T cells that regulate Ag recognition, kinetics of intracellular signaling, and the selectivity and efficiency of T cell responses.

Cell-free production of a functional oligomeric form of a Chlamydia major outer-membrane protein (MOMP) for vaccine development.

Chlamydia is a prevalent sexually transmitted disease that infects more than 100 million people worldwide. Although most individuals infected with Chlamydia trachomatis are initially asymptomatic, symptoms can arise if left undiagnosed. Long-term infection can result in debilitating conditions such as pelvic inflammatory disease, infertility, and blindness. Chlamydia infection, therefore, constitutes a significant public health threat, underscoring the need for a Chlamydia-specific vaccine. Chlamydia strains express a major outer-membrane protein (MOMP) that has been shown to be an effective vaccine antigen. However, approaches to produce a functional recombinant MOMP protein for vaccine development are limited by poor solubility, low yield, and protein misfolding. Here, we used an Escherichia coli-based cell-free system to express a MOMP protein from the mouse-specific species Chlamydia muridarum (MoPn-MOMP or mMOMP). The codon-optimized mMOMP gene was co-translated with Δ49apolipoprotein A1 (Δ49ApoA1), a truncated version of mouse ApoA1 in which the N-terminal 49 amino acids were removed. This co-translation process produced mMOMP supported within a telodendrimer nanolipoprotein particle (mMOMP-tNLP). The cell-free expressed mMOMP-tNLPs contain mMOMP multimers similar to the native MOMP protein. This cell-free process produced on average 1.5 mg of purified, water-soluble mMOMP-tNLP complex in a 1-ml cell-free reaction. The mMOMP-tNLP particle also accommodated the co-localization of CpG oligodeoxynucleotide 1826, a single-stranded synthetic DNA adjuvant, eliciting an enhanced humoral immune response in vaccinated mice. Using our mMOMP-tNLP formulation, we demonstrate a unique approach to solubilizing and administering membrane-bound proteins for future vaccine development. This method can be applied to other previously difficult-to-obtain antigens while maintaining full functionality and immunogenicity.

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.

Imaging cerebroside-rich domains for phase and shape characterization in binary and ternary mixtures.

The objective of this paper is to review phase behavior and shape characterization of cerebroside-rich domains in binary and ternary lipid bilayers, as obtained by microscopy techniques. These lipid mixtures provide a format to examine molecular (e.g. headgroup, tail unsaturation, and tail hydroxylation) and thermodynamic (e.g. temperature and mole percentages) factors that determine phase behavior, molecular partitioning, crystal/atomic scale structure, and microstructure/shape (particularly of phase-separated domains). Microscopy can provide excellent spatial (often with high resolution) characterization of cerebroside-rich domains (and their surroundings) to identify, describe or infer with high certainty these characteristics. In the introduction to this review we review briefly the molecular structure, phase behavior, and intermolecular interactions of cerebrosides, in comparison to ceramides and sphingomyelins and in some binary and biological systems. The bulk of the review is then devoted to microscopy investigations of cerebroside-rich domain microstructure and shape dynamics in binary and ternary (one component is cholesterol) systems. Quantitative and/or high-resolution microscopy techniques have been used to interrogate cerebroside-rich domains such as freeze-fracture electron microscopy, atomic force microscopy, imaging elipsometry, two-photon fluorescence microscopy, and LAURDAN generalized polarization in addition to the laboratory workhorse technique of epifluorescence microscopy that allows a quick often qualitative assessment of microstructure and dynamics. We particularly focus on the information these microscopy investigations have revealed with respect to phase behavior, cholesterol partitioning, domain shape, and determinants of domain shape.

AFM for structure and dynamics of biomembranes.

We review structure and dynamic measurements of biomembranes by atomic force microscopy (AFM). We focus mainly on studies involving supported lipid bilayers (SLBs), particularly formation by vesicle rupture on flat and corrugated surfaces, nucleation and growth of domains in phase-separated systems, anesthetic-lipid interactions, and protein/peptide interactions in multicomponent systems. We show that carefully designed experiments along with real-time AFM imaging with superior lateral and z resolution (0.1 nm) have revealed quantitative details of the mechanisms and factors controlling vesicle rupture, domain shape and size, phase transformations, and some model biological interactions. The AFM tip can also be used as a mechanical transducer and incorporated in electrochemical measurements of membrane components; therefore, we touch on these important applications in both model and cell membranes.

Atomic force microscopy differentiates discrete size distributions between membrane protein containing and empty nanolipoprotein particles.

To better understand the incorporation of membrane proteins into discoidal nanolipoprotein particles (NLPs) we have used atomic force microscopy (AFM) to image and analyze NLPs assembled in the presence of bacteriorhodopsin (bR), lipoprotein E4 n-terminal 22k fragment scaffold and DMPC lipid. The self-assembly process produced two distinct NLP populations: those containing inserted bR (bR-NLPs) and those that did not (empty-NLPs). The bR-NLPs were distinguishable from empty-NLPs by an average increase in height of 1.0 nm as measured by AFM. Streptavidin binding to biotinylated bR confirmed that the original 1.0 nm height increase corresponds to br-NLP incorporation. AFM and ion mobility spectrometry (IMS) measurements suggest that NLP size did not vary around a single mean but instead there were several subpopulations, which were separated by discrete diameters. Interestingly, when bR was present during assembly the diameter distribution was shifted to larger particles and the larger particles had a greater likelihood of containing bR than smaller particles, suggesting that membrane proteins alter the mechanism of NLP assembly.

Kinetic analysis of his-tagged protein binding to nickel-chelating nanolipoprotein particles.

Nanolipoprotein particles (NLPs) are discoidal self-assembling membrane mimetics that have been primarily used as a platform for the solubilization and stabilization of membrane proteins. Nickel-chelating nanolipoprotein particles (NiNLPs) containing nickel-chelating lipids (Ni-lipid) for the targeted immobilization of His-tagged proteins hold promise as carriers of hydrophilic biological molecules for a range of applications. The effect of protein loading (i.e., the number of proteins bound per NiNLP) and Ni-lipid content on the time scales and kinetics of binding are important to various applications such as vaccine development, diagnostic imaging, and drug delivery. We have immobilized hexa-His-tagged LsrB, a Yersinia pestis transport protein, onto NiNLPs to examine the effect of protein binding stoichiometry and Ni-lipid content on the time scales and kinetics of protein binding by surface plasmon resonance (SPR). Data indicate that the dissociation half-time increases with Ni-lipid content up to a molar concentration of 35% and decreases as the number of bound protein per NiNLP increases. These findings indicate that the kinetics of protein binding are highly dependent on both the number of bound protein per NiNLP and Ni-lipid content.

Conjugation to nickel-chelating nanolipoprotein particles increases the potency and efficacy of subunit vaccines to prevent West Nile encephalitis.

Subunit antigens are attractive candidates for vaccine development, as they are safe, cost-effective, and rapidly produced. Nevertheless, subunit antigens often need to be adjuvanted and/or formulated to produce products with acceptable potency and efficacy. Here, we describe a simple method for improving the potency and efficacy of a recombinant subunit antigen by its immobilization on nickel-chelating nanolipoprotein particles (NiNLPs). NiNLPs are membrane mimetic nanoparticles that provide a delivery and presentation platform amenable to binding any recombinant subunit immunogens featuring a polyhistidine tag. A His-tagged, soluble truncated form of the West Nile virus (WNV) envelope protein (trE-His) was immobilized on NiNLPs. Single inoculations of the NiNLP-trE-His produced superior anti-WNV immune responses and provided significantly improved protection against a live WNV challenge compared to mice inoculated with trE-His alone. These results have broad implications in vaccine development and optimization, as NiNLP technology is well-suited to many types of vaccines, providing a universal platform for enhancing the potency and efficacy of recombinant subunit immunogens.

Cell-free co-expression of functional membrane proteins and apolipoprotein, forming soluble nanolipoprotein particles.

Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cell-free extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein Delta1-49 apolipoprotein A-I fragment (Delta49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR --> M transition. Importantly the functional bR was solubilized in discoidal bR.NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with Delta49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies.

Characterization of Bacillus anthracis Spore Proteins Using a Nanoscaffold Vaccine Platform.

Subunit vaccines are theoretically safe and easy to manufacture but require effective adjuvants and delivery systems to yield protective immunity, particularly at critical mucosal sites such as the lung. We investigated nanolipoprotein particles (NLPs) containing the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) as a platform for intranasal vaccination against Bacillus anthracis. Modified lipids enabled attachment of disparate spore and toxin protein antigens. Intranasal vaccination of mice with B. anthracis antigen-MPLA-NLP constructs induced robust IgG and IgA responses in serum and in bronchoalveolar and nasal lavage. Typically, a single dose sufficed to induce sustained antibody titers over time. When multiple immunizations were required for sustained titers, specific antibodies were detected earlier in the boost schedule with MPLA-NLP-mediated delivery than with free MPLA. Administering combinations of constructs induced responses to multiple antigens, indicating potential for a multivalent vaccine preparation. No off-target responses to the NLP scaffold protein were detected. In summary, the NLP platform enhances humoral and mucosal responses to intranasal immunization, indicating promise for NLPs as a flexible, robust vaccine platform against B. anthracis and potentially other inhalational pathogens.

Hydrogen production by a hyperthermophilic membrane-bound hydrogenase in water-soluble nanolipoprotein particles.

Hydrogenases constitute a promising class of enzymes for ex vivo hydrogen production. Implementation of such applications is currently hindered by oxygen sensitivity and, in the case of membrane-bound hydrogenases (MBHs), poor water solubility. Nanolipoprotein particles (NLPs) formed from apolipoproteins and phospholipids offer a novel means of incorporating MBHs into a well-defined water-soluble matrix that maintains the enzymatic activity and is amenable to incorporation into more complex architectures. We report the synthesis, hydrogen-evolving activity, and physical characterization of the first MBH-NLP assembly. This may ultimately lead to the development of biomimetic hydrogen-production devices.

Immobilization of His-tagged proteins on nickel-chelating nanolipoprotein particles.

Nanolipoprotein particles (NLPs) are nanometer-sized, discoidal particles that self-assemble from purified apolipoprotein and phospholipid. Their size and facile functionalization suggest potential application of NLPs as platforms for the presentation and delivery of recombinant proteins. To this end, we investigated incorporation of nickel-chelating lipids into NLPs (NiNLPs) and subsequent sequestration of polyhistidine (His)-tagged proteins. From initial lipid screens for NLP formation, the two phospholipids DMPC and DOPC were identified as suitable bulk lipids for incorporation of the nickel-chelating lipid DOGS-NTA-Ni into NLPs, and NiNLPs were successfully formed with varying amounts of DOGS-NTA-Ni. NiNLPs consisting of 10% DOGS-NTA-Ni with 90% bulk lipid (either DMPC or DOPC) were thoroughly characterized by size exclusion chromatography (SEC), non-denaturing gradient gel electrophoresis (NDGGE), and atomic force microscopy (AFM). Three different His-tagged proteins were sequestered on NiNLPs in a nickel-dependent manner, and the amount of immobilized protein was contingent on the size and composition of the NiNLP.

Cell-free expression for nanolipoprotein particles: building a high-throughput membrane protein solubility platform.

Membrane-associated proteins and protein complexes account for approximately a third or more of the proteins in the cell (1, 2). These complexes mediate essential cellular processes; including signal transduc-tion, transport, recognition, bioenergetics and cell-cell communication. In general, membrane proteins are challenging to study because of their insolubility and tendency to aggregate when removed from their protein lipid bilayer environment. This chapter is focused on describing a novel method for producing and solubilizing membrane proteins that can be easily adapted to high-throughput expression screening. This process is based on cell-free transcription and translation technology coupled with nanolipoprotein par ticles (NLPs), which are lipid bilayers confined within a ring of amphipathic protein of defined diameter. The NLPs act as a platform for inserting, solubilizing and characterizing functional membrane proteins. NLP component proteins (apolipoproteins), as well as membrane proteins can be produced by either traditional cell-based or as discussed here, cell-free expression methodologies.

Characterization and purification of polydisperse reconstituted lipoproteins and nanolipoprotein particles.

Heterogeneity is a fact that plagues the characterization and application of many self-assembled biological constructs. The importance of obtaining particle homogeneity in biological assemblies is a critical goal, as bulk analysis tools often require identical species for reliable interpretation of the results-indeed, important tools of analysis such as x-ray diffraction typically require over 90% purity for effectiveness. This issue bears particular importance in the case of lipoproteins. Lipid-binding proteins known as apolipoproteins can self assemble with liposomes to form reconstituted high density lipoproteins (rHDLs) or nanolipoprotein particles (NLPs) when used for biotechnology applications such as the solubilization of membrane proteins. Typically, the apolipoprotein and phospholipids reactants are self assembled and even with careful assembly protocols the product often contains heterogeneous particles. In fact, size polydispersity in rHDLs and NLPs published in the literature are frequently observed, which may confound the accurate use of analytical methods. In this article, we demonstrate a procedure for producing a pure, monodisperse NLP subpopulation from a polydisperse self-assembly using size exclusion chromatography (SEC) coupled with high resolution particle imaging by atomic force microscopy (AFM). In addition, NLPs have been shown to self assemble both in the presence and absence of detergents such as cholate, yet the effects of cholate on NLP polydispersity and separation has not been systematically examined. Therefore, we examined the separation properties of NLPs assembled in both the absence and presence of cholate using SEC and native gel electrophoresis. From this analysis, NLPs prepared with and without cholate showed particles with well defined diameters spanning a similar size range. However, cholate was shown to have a dramatic affect on NLP separation by SEC and native gel electrophoresis. Furthermore, under conditions where different sized NLPs were not sufficiently separated or purified by SEC, AFM was used to deconvolute the elution pattern of different sized NLPs. From this analysis we were able to purify an NLP subpopulation to 90% size homogeneity by taking extremely fine elutions from the SEC. With this purity, we generate high quality NLP crystals that were over 100 microm in size with little precipitate, which could not be obtained utilizing the traditional size exclusion techniques. This purification procedure and the methods for validation are broadly applicable to other lipoprotein particles.

Domain nucleation rates and interfacial line tensions in supported bilayers of ternary mixtures containing galactosylceramide.

Domains within the plane of the plasma membrane, referred to as membrane rafts, have been a topic of considerable interest in the field of membrane biophysics. Although model membrane systems have been used extensively to study lipid phase behavior as it relates to the existence of rafts, very little work has focused on either the initial stage of lipid domain nucleation, or the relevant physical parameters such as temperature and interfacial line tension which control nucleation. In this work, we utilize a method in which the kinetic process of lipid domain nucleation is imaged by atomic force microscopy and modeled using classical theory of nucleation to map interfacial line tension in ternary lipid mixtures. These mixtures consist of a fluid phase lipid component (1,2-dilauroyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, or 1,2-dioleoyl-sn-glycero-3-phosphocholine), a solid phase component (galactosylceramide), and cholesterol. Interfacial line tension measurements of galactosylceramide-rich domains track with our previously measured area/perimeter ratios and height mismatches measured here. Line tension also follows known trends in cholesterol interactions and partitioning, as we observed previously with area/perimeter ratios. Our line tension measurements are discussed in combination with recent line tension measurements to address line tension regulation by cholesterol and the dynamic nature of membrane rafts.

Direct visualization of phase transition dynamics in binary supported phospholipid bilayers using imaging ellipsometry.

Via imaging ellipsometry, we study the phase transition dynamics induced by selective gelation of one component in a binary supported phopholipid bilayer. We find the modulation of two attendant morphological features: emergence of extended defect chains due to a net change in the molecular areas and fractal-like domains suggesting weak line tension. A time-lapse analysis of the ellipsometric images reveals the cluster size of 4-20 molecules undergoing gelation indicating weak cooperativity. These results demonstrate the use of ellipsometry for in situ, label-free, non-contact, and large-area imaging of dynamics in interfacial films.

Lipid composition dictates serum stability of reconstituted high-density lipoproteins: implications for in vivo applications.

Nanolipoprotein particles (NLPs) are reconstituted high-density lipoproteins, consisting of a phospholipid bilayer stabilized by an apolipoprotein scaffold protein. This class of nanoparticle has been a vital tool in the study of membrane proteins, and in recent years has been increasingly used for in vivo applications. Previous work demonstrated that the composition of the lipid bilayer component affects the stability of these particles in serum solutions. In the current study, NLPs assembled with phosphatidylcholine lipids featuring different acyl chain structures were systematically tested to understand the effect that lipid composition has on NLP stability in both neat serum and cell culture media supplemented with 10% serum by volume. The time at which 50% of the particles dissociate, as well as the fraction of the initial population that remains resistant to dissociation, were correlated to key parameters obtained from all-atom simulations of the corresponding lipid bilayers. A significant correlation was observed between the compressibility modulus of the lipid bilayer and particle stability in these complex biological milieu. These results can be used as a reference to tune the stability of these versatile biological nanoparticles for in vitro and in vivo applications.

SOST/Sclerostin Improves Posttraumatic Osteoarthritis and Inhibits MMP2/3 Expression After Injury.

Patients with anterior cruciate ligament (ACL) rupture are two times as likely to develop posttraumatic osteoarthritis (PTOA). Annually, there are ∼900,000 knee injuries in the United States, which account for ∼12% of all osteoarthritis (OA) cases. PTOA leads to reduced physical activity, deconditioning of the musculoskeletal system, and in severe cases requires joint replacement to restore function. Therefore, treatments that would prevent cartilage degradation post-injury would provide attractive alternatives to surgery. Sclerostin (Sost), a Wnt antagonist and a potent negative regulator of bone formation, has recently been implicated in regulating chondrocyte function in OA. To determine whether elevated levels of Sost play a protective role in PTOA, we examined the progression of OA using a noninvasive tibial compression overload model in SOST transgenic (SOSTTG ) and knockout (Sost-/- ) mice. Here we report that SOSTTG mice develop moderate OA and display significantly less advanced PTOA phenotype at 16 weeks post-injury compared with wild-type (WT) controls and Sost-/- . In addition, SOSTTG built ∼50% and ∼65% less osteophyte volume than WT and Sost-/- , respectively. Quantification of metalloproteinase (MMP) activity showed that SOSTTG had ∼2-fold less MMP activation than WT or Sost-/- , and this was supported by a significant reduction in MMP2/3 protein levels, suggesting that elevated levels of SOST inhibit the activity of proteolytic enzymes known to degrade articular cartilage matrix. Furthermore, intra-articular administration of recombinant Sost protein, immediately post-injury, also significantly decreased MMP activity levels relative to PBS-treated controls, and Sost activation in response to injury was TNFα and NF-κB dependent. These results provide in vivo evidence that sclerostin functions as a protective molecule immediately after joint injury to prevent cartilage degradation. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

Lipid domains in supported lipid bilayer for atomic force microscopy.

Phase-separated supported lipid bilayers have been widely used to study the phase behavior of multicomponent lipid mixtures. One of the primary advantages of using supported lipid bilayers is that the two-dimensional platform of this model membrane system readily allows lipid-phase separation to be characterized by high-resolution imaging techniques such as atomic force microscopy (AFM). In addition, when supported lipid bilayers have been functionalized with a specific ligand, protein-membrane interactions can also be imaged and characterized through AFM. It has been recently demonstrated that when the technique of vesicle fusion is used to prepare supported lipid bilayers, the thermal history of the vesicles before deposition and the supported lipid bilayers after formation will have significant effects on the final phase-separated domain structures. In this chapter, three methods of vesicle preparations as well as three deposition conditions will be presented. Also, the techniques and strategies of using AFM to image multicomponent phase-separated supported lipid bilayers and protein binding will be discussed.

Enhancement of antigen-specific CD4+ and CD8+ T cell responses using a self-assembled biologic nanolipoprotein particle vaccine.

To address the need for vaccine platforms that induce robust cell-mediated immunity, we investigated the potential of utilizing self-assembling biologic nanolipoprotein particles (NLPs) as an antigen and adjuvant delivery system to induce antigen-specific murine T cell responses. We utilized OT-I and OT-II TCR-transgenic mice to investigate the effects of NLP-mediated delivery of the model antigen ovalbumin (OVA) on T cell activation. Delivery of OVA with the TLR4 agonist monophosphoryl lipid A (MPLA) in the context of NLPs significantly enhanced the activation of both CD4+ and CD8+ T cells in vitro compared to co-administration of free OVA and MPLA. Upon intranasal immunization of mice harboring TCR-transgenic cells, NLPs enhanced the adjuvant effects of MPLA and the in vivo delivery of OVA, leading to significantly increased expansion of CD4+ and CD8+ T cells in lung-draining lymph nodes. Therefore, NLPs are a promising vaccine platform for inducing T cell responses following intranasal administration.