We previously reported a protective antibody response in mice immunized with synthetic microparticle vaccines made using layer-by-layer fabrication (LbL-MP) and containing the conserved T1BT* epitopes from the P. falciparum circumsporozoite protein. To further optimize the vaccine candidate, a benchtop tangential flow filtration method (LbL-by-TFF) was developed and utilized to produce vaccine candidates that differed in the status of base layer crosslinking, inclusion of a TLR2 ligand in the antigenic peptide, and substitution of serine or alanine for an unpaired cysteine residue in the T* epitope. Studies in mice revealed consistent superiority of the Pam3Cys-modified candidates and a modest benefit of base layer crosslinking, as evidenced by higher and more persistent antibody titers (up to 18 months post-immunization), a qualitative improvement of T-cell responses toward a Th1 phenotype, and greater protection from live parasite challenges compared to the unmodified prototype candidate. Immunogenicity was also tested in a non-human primate model, the rhesus macaque. Base layer-crosslinked LbL-MP loaded with T1BT* peptide with or without covalently linked Pam3Cys elicited T1B-specific antibody responses and T1BT*-specific T-cell responses dominated by IFNγ secretion with lower levels of IL-5 secretion. The Pam3Cys-modified construct was more potent, generating antibody responses that neutralized wild-type P. falciparum in an in vitro hepatocyte invasion assay. IgG purified from individual macaques immunized with Pam3Cys.T1BT* LbL-MP protected naïve mice from challenges with transgenic P. berghei sporozoites that expressed the full-length PfCS protein, with 50–88% of passively immunized mice parasite-free for ≥15 days. Substitution of serine for an unpaired cysteine in the T* region of the T1BT* subunit did not adversely impact immune potency in the mouse while simplifying the manufacture of the antigenic peptide. In a Good Laboratory Practices compliant rabbit toxicology study, the base layer-crosslinked, Pam3Cys-modified, serine-substituted candidate was shown to be safe and immunogenic, eliciting parasite-neutralizing antibody responses and establishing the dose/route/regimen for a clinical evaluation of this novel synthetic microparticle pre-erythrocytic malaria vaccine candidate.

Layer-by-layer microparticle (LbL-MP) fabrication was used to produce synthetic vaccines presenting a fusion peptide containing RSV G protein CX3C chemokine motif and a CD8 epitope of the RSV matrix protein 2 (GM2) with or without a covalently linked TLR2 agonist (Pam3.GM2). Immunization of BALB/c mice with either GM2 or Pam3.GM2 LbL-MP in the absence of adjuvant elicited G-specific antibody responses and M2-specific CD8+ T-cell responses. Following challenge with RSV, mice immunized with the GM2 LbL-MP vaccine developed a Th2-biased immune response in the lungs with elevated levels of IL-4, IL-5, IL-13, and eotaxin in the bronchoalveolar lavage (BAL) fluid and a pulmonary influx of eosinophils. By comparison, mice immunized with the Pam3.GM2 LbL-MP vaccine had considerably lower to non-detectable levels of the Th2 cytokines and chemokines and very low numbers of eosinophils in the BAL fluid post-RSV challenge. In addition, mice immunized with the Pam3.GM2 LbL-MP also had higher levels of RSV G-specific IgG2a and IgG2b in the post-challenge BAL fluid compared to those immunized with the GM2 LbL-MP vaccine. While both candidates protected mice from infection following challenge, as evidenced by the reduction or elimination of RSV plaques, the inclusion of the TLR2 agonist yielded a more potent antibody response, greater protection, and a clear shift away from Th2/eosinophil responses. Since the failure of formalin-inactivated RSV (FI-RSV) vaccines tested in the 1960s has been hypothesized to be partly due to the ablation of host TLR engagement by the vaccine and inappropriate Th2 responses upon subsequent viral infection, these findings stress the importance of appropriate engagement of the innate immune response during initial exposure to RSV G CX3C.

Synthetic vaccines made by layer-by-layer fabrication of microparticles (LbL-MP represent a novel method of delivering peptide antigens to elicit protective immune responses in the absence of complex adjuvant and without concomitant inflammation. We incorporated LbL-MP into a microneedle patch that delivers the vaccine directly to the immunologically rich environment of the skin in a minimally-invasive manner. BALB/c mice immunized with microneedle patches loaded with LbL-MP bearing two conserved epitopes of respiratory syncytial virus (RSV) generated humoral and cellular immune responses and were protected from virus challenge. The vaccine contained a conserved CX3C chemokine mimic epitope from the RSV attachment (G) protein that contributes to Th2-skewed inflammatory responses. Patch-immunized mice developed RSV-G-specific antibody titers comparable to the titers induced by direct injection of the vaccine into the footpad. The mice developed T-cell responses, including effector CTL, against a CD8 epitope of the RSV-M2 protein included in the vaccine. While footpad injection elicited both Th2 (IL-5) and Th1 (IFNγ) responses, microneedle patch immunization elicited a dominant Th1 response with very low Th2 response. A similar pattern of Th1>Th2 was seen in C57BL/6 mice immunized with a patch loaded with LbL-MP containing epitopes of the P. falciparum circumsporozoite protein, suggesting that the Th1 dominance was not antigen- or strain-specific, but rather an outcome of LbL-MP microneedle patch immunization. These studies demonstrate that LbL-MP retain their integrity and potency when delivered via microneedle patch, and favor Th1 immune responses that may be more effective against acute infection.

Respiratory syncytial virus (RSV) is the single most important cause of serious lower respiratory tract infections in young children; however no effective treatment or vaccine is currently available. Previous studies have shown that therapeutic treatment with a monoclonal antibody (clone 131-2G) specific to the RSV G glycoprotein CX3C motif, mediates virus clearance and decreases leukocyte trafficking to the lungs of RSV-infected mice. In this study, we show that vaccination with layer-by-layer nanoparticles (LbL-NP) carrying the G protein CX3C motif induces blocking antibodies that prevent the interaction of the RSV G protein with the fractalkine receptor (CX3CR1) and protect mice against RSV replication and disease pathogenesis. Peptides with mutations in the CX3C motif induced antibodies with diminished capacity to block G protein-CX3CR1 binding. Passive transfer of these anti-G protein antibodies to mice infected with RSV improved virus clearance and decreased immune cell trafficking to the lungs. These data suggest that vaccination with LbL-NP loaded with the CX3C motif of the RSV G protein can prevent manifestations of RSV disease by preventing the interaction between the G protein and CX3CR1 and recruitment of immune cells to the airways.

We have previously shown that synthetic LbL-MP vaccines containing the conserved T1B repeat and T* epitopes from the P. falciparum circumsporozoite (PfCS) protein elicit a protective antibody response in mice. Immunogenicity and efficacy of these LbL-MP were tested in a non-human primate model, the rhesus macaque. LbL-MP were loaded with T1BT* peptide with or without covalently-linked Pam3Cys, a TLR2 ligand. Both constructs elicited T1B-specific antibody responses and T1BT*-specific T-cell responses dominated by IFNγ secretion with little or no IL-5 secretion. The Pam3Cys-modified construct was more potent, generating antibody responses that neutralized wild-type P. falciparum in an in vitro hepatocyte invasion assay. In addition, IgG was purified from the macaque immune sera and passively administered to naïve mice that were then challenged with 5,000 transgenic P. berghei sporozoites that expressed the full-length PfCS protein. IgG from individual macaques immunized with Pam3Cys.T1BT* LbL-MP protected mice from challenge, with 50-88% of mice parasite-free for ≥ 15 days. These results demonstrate that synthetic LbL-MP vaccines bearing the conserved T1BT* epitopes of PfCS can elicit protective antibody responses against P. falciparum in a non-human primate model.

BALB/c mice immunized with nanoparticle vaccines comprising RSV G protein CX3C polypeptides in absence of adjuvant had a neutralizing antibody response associated with reduced lung titers following RSV challenge. These mice also had an increased level of RSV G protein-specific IL-4 and IFNγ secreting cells in the lung, and an increased level of RSV M2-specific IL-4 and IFNγ CD8 T cells. There was a significant increase in M2-specific CD8 T cells that trafficked to the lung following RSV challenge despite the mice being vaccinated with a G protein epitope. Pulmonary cell analysis revealed no significant increase in neutrophils (Ly6Ghi Ly6Cint CD125loSiglecF-) or eosinophils (Ly6Gint Ly6Chi CD125hi SiglecF+) in vaccinated mice before or after RSV challenge. The results show that RSV G protein nanoparticle vaccination is safe and effective, induces a neutralizing protective antibody response, increased RSV G protein- and M2-specific T cell responses, and is not associated with pulmonary disease pathogenesis.

Nanoparticle vaccines were produced using layer-by-layer fabrication and incorporating respiratory syncytial virus (RSV) G protein polypeptides comprising the CX3C chemokine motif. BALB/c mice immunized with G protein nanoparticle vaccines produced a neutralizing antibody response that inhibited RSV replication in the lungs following RSV challenge. ELISPOT analysis showed that G nanoparticle vaccinated mice had increased levels of RSV G protein-specific IL-4 and IFN-γ secreting cells compared to controls following RSV challenge. Remarkably, RSV challenge of G protein nanoparticle vaccinated mice resulted in increased RSV M2-specific IL-4 and IFN-γ secreting T cells, and increased M2-specific H-2Kd-tetramer positive CD8+ T cells in the lungs compared to controls. Cell type analysis showed vaccination was not associated with increased pulmonary eosinophilia following RSV challenge. These results demonstrate that vaccination of mice with the RSV G protein nanoparticle vaccines induces a potent neutralizing antibody response, increased G protein- and M2- specific T cell responses, and a reduction in RSV disease pathogenesis.

Epitopes of the circumsporozoite (CS) protein of Plasmodium falciparum, the most pathogenic species of the malaria parasite, have been shown to elicit protective immunity in experimental animals and human volunteers. The mechanisms of immunity include parasite-neutralizing antibodies that can inhibit parasite motility in the skin at the site of infection and in the bloodstream during transit to the hepatocyte host cell and also block interaction with host cell receptors on hepatocytes. In addition, specific CD4+ and CD8+ cellular mechanisms target the intracellular hepatic forms, thus preventing release of erythrocytic stage parasites from the infected hepatocyte and the ensuing blood stage cycle responsible for clinical disease. An innovative method for producing particle vaccines, layer-by-layer (LbL) fabrication of polypeptide films on solid CaCO3 cores, was used to produce synthetic malaria vaccines containing a tri-epitope CS peptide T1BT comprising the antibody epitope of the CS repeat region (B) and two T-cell epitopes, the highly conserved T1 epitope and the universal epitope T. Mice immunized with microparticles loaded with T1BT peptide developed parasite-neutralizing antibodies and malaria-specific T-cell responses including cytotoxic effector T-cells. Protection from liver stage infection following challenge with live sporozoites from infected mosquitoes correlated with neutralizing antibody levels. Although some immunized mice with low or undetectable neutralizing antibodies were also protected, depletion of T-cells prior to challenge resulted in the majority of mice remaining resistant to challenge. In addition, mice immunized with microparticles bearing only T-cell epitopes were not protected, demonstrating that cellular immunity alone was not sufficient for protective immunity. Although the microparticles without adjuvant were immunogenic and protective, a simple modification with the lipopeptide TLR2 agonist Pam3Cys increased the potency and efficacy of the LbL vaccine candidate. This study demonstrates the potential of LbL particles as promising malaria vaccine candidates using the T1BT epitopes from the P. falciparum CS protein.

Abstract Microparticle vaccines containing the conserved T1B repeat and T* epitopes from the P. falciparum CS protein were synthesized via layer-by-layer (LbL) fabrication on a solid core; microcapsules were prepared by dissolution of the particle core. Mice immunized with microparticles or microcapsules yielded T1B-specific antibody responses that neutralized Plasmodium parasite in vitro. The mice also developed T1BT*-specific cellular responses, including Th1, Th2, and CD4+ cytotoxic effector cells. When challenged with PfPb, a recombinant P. bergheii expressing the T1B repeats from P. falciparum, 70-80% of the mice immunized with microparticle, and 40-50% immunized with microcapsule, showed >90% reduction in liver parasite burden compared to naïve challenged mice. A minority of mice with reduced parasite burden did not have potent PfPb-neutralizing antibody activity. Since depletion of either CD4+ or CD8+ cells prior to challenge did not ablate protection, the vaccine-induced protection appears to be primarily associated with antibody responses but may also involve other mechanisms not yet defined. An examination of cytokine and chemokine gene expression in the liver post-challenge did not reveal any evidence of inflammatory responses following LbL vaccination and PfPb challenge. These results demonstrate that synthetic LbL vaccines bearing the conserved T1BT* epitopes can elicit protective immunity against Plasmodium without triggering unwanted inflammatory responses.

Abstract Nanoparticle vaccines synthesized via layer-by-layer (LbL) fabrication were loaded with designed peptides representing epitopes of the attachment (G) and matrix (M2) proteins of respiratory syncytial virus. The CX3C chemokine mimic epitope of RSV-G has been proposed to contribute to inflammatory responses post-challenge while CD8+ T-cell responses against RSV M2 have been shown to limit the severity of infection and inflammatory pathology. Both monovalent designs (G or M2) and multivalent designs (G+M2) were tested. Mice immunized with RSV-G nanoparticles produced antibody responses that recognized the CX3C epitope only in its folded conformation and not in a linearized state. The same sera also bound native RSV-G protein, inhibited binding of RSV-G protein to the CX3CR1 chemokine receptor, and inhibited migration of human leukocytes toward RSV-G protein. Mice immunized with RSV M2 nanoparticles generated CD8+ T-cell responses and in vitro CTL activity against M2-labeled target cells. The multivalent vaccines containing both G and M2 elicited higher antibody responses to RSV-G and, surprisingly, more potent cellular responses against RSV-M2. These novel nanoparticle vaccines are currently being tested for the induction of neutralizing antibody responses and protection from viral challenge. If successful, the LbL nanoparticle fabrication strategy will provide an innovative approach to formulating safe and effective subunit vaccines for respiratory pathogens including RSV.

Nanoparticle vaccines induce potent immune responses in the absence of conventional adjuvant due to the recognition by immune cells of the particle structures, which mimic natural pathogens such as viruses and bacteria. Nanoparticle vaccines were fabricated by constructing artificial biofilms using layer-by-layer (LbL) deposition of oppositely charged polypeptides and target designed peptides on CaCO3 cores. LbL nanoparticles were efficiently internalized by dendritic cells in vitro by a mechanism that was at least partially phagocytic, and induced DC maturation without triggering secretion of inflammatory cytokines. LbL nanoparticle delivery of designed peptides to DC resulted in potent cross-presentation to CD8+ T-cells and more efficient presentation to CD4+ T-cells compared to presentation of soluble peptide. A single immunization of mice with LbL nanoparticles containing designed peptide induced vigorous T-cell responses characterized by a balanced effector (IFNγ) and Th2 (IL-4) ELISPOT profile and in vivo CTL activity. Mice immunized with LbL nanoparticles bearing ovalbumin-derived designed peptides were protected from challenge with Listeria monocytogenes ectopically expressing ovalbumin, confirming the relevance of the CTL/effector T-cell responses. LbL nanoparticles also elicited antibody responses to the target epitope but not to the matrix components of the nanoparticle, avoiding the vector or carrier affect that hampers utility of other vaccine platforms. The potency and efficacy of LbL nanoparticles administered in aqueous suspension without adjuvant or other formulation additive, and the absence of immune responses to the matrix components, suggest that this strategy may be useful in producing novel vaccines against multiple diseases.

Abstract Nanoparticle vaccines were synthesized using layer-by-layer (LbL) deposition of oppositely-charged polypeptides to build up an artificial film on CaCO3 nanocores. Designed peptides (DP) containing antigenic epitopes of interest were incorporated into the layers of the film, yielding nanoparticles that delivered the DP payload to dendritic cells via phagocytosis. LbL nanoparticles induced DC maturation without triggering secretion of inflammatory cytokines including TNFα and IL-6. LbL nanoparticle delivery of DP to DC resulted in potent cross-presentation to CD8+ T-cells and more efficient presentation to CD4+ T-cells compared to presentation of soluble peptide. Mice immunized with a single dose of LbL nanoparticles without adjuvant mounted vigorous T-cell responses detectable by IFNγ and IL-4 ELISPOT and in vivo CTL assay. Mice immunized with nanoparticles containing ovalbumin-derived DP were protected from challenge with Listeria monocytogenes ectopically expressing ovalbumin. Similar to the in vitro DC maturation results, immunization with LbL nanoparticles did not induce inflammatory symptoms locally (injection site reaction) or systemically (serum cytokine spike). The potency and efficacy of polypeptide LbL nanoparticles administered in aqueous suspension without adjuvant or other formulation additive, and the absence of overt inflammatory responses, suggest that this strategy may be useful in producing novel vaccines against multiple diseases.

The CETP inhibitor, torcetrapib, was prematurely terminated from phase 3 clinical trials due to an increase in cardiovascular and noncardiovascular mortality. Because nearly half of the latter deaths involved patients with infection, we have tested torcetrapib and other CETPIs to see if they interfere with lipopolysaccharide binding protein (LBP) or bactericidal/permeability increasing protein (BPI). No effect of these potent CETPIs on LPS binding to either protein was detected. Purified CETP itself bound weakly to LPS with a Kd ≥ 25 uM compared with 0.8 and 0.5 nM for LBP and BPI, respectively, and this binding was not blocked by torcetrapib. In whole blood, LPS induced tumor necrosis factor-α normally in the presence of torcetrapib. Furthermore, LPS had no effect on CETP activity. We conclude that the sepsis-related mortality of the ILLUMINATE trial was unlikely due to a direct effect of torcetrapib on LBP or BPI function, nor to inhibition of an interaction of CETP with LPS. Instead, we speculate that the negative outcome seen for patients with infections might be related to the changes in plasma lipoprotein composition and metabolism, or alternatively to the known off-target effects of torcetrapib, such as aldosterone elevation, which may have aggravated the effects of sepsis.

Proprotein convertase subtilisin-kexin type 9 (PCSK9) binds to the low-density lipoprotein receptor (LDLR) on target cells and lowers the level of receptor by impeding its recycling. PCSK9 is self-processed to a complex of its prodomain and catalytic domain like a typical protein convertase, but it does not develop normal proteolytic activity. Instead, its propeptide remains complexed with the catalytic domain, and the C-terminal Gln152 of the prodomain occupies the active site like a substrate for peptide synthesis. To probe its latent catalytic activity, PCSK9 and its complex with the soluble LDLR extracellular domain were separately transferred into H218O, and time point samples were analyzed by peptide mapping with mass spectrometry to measure the rate and extent of incorporation of 18O into the Gln152 carboxylate. In free wild-type or D374Y mutant PCSK9, the t1/2 for exchange of 18O for both oxygens was near 5 min. This slow process progressed to completion, with the distribution of oxygen isotopes in the Gln152 carboxylate finally matching that in solvent. In contrast, exchange reached its final state in <30 s in LDLR-complexed D374Y mutant PCSK9, but approximately 40% of the molecules gave data indicating the presence of only one 18O atom in Gln152. With support from further experiments, this was attributed to hydrolysis of acylenzyme in H216O during preparations for digestion and indicated that PCSK9 complexed with LDLR contains approximately 40% intramolecular acylenzyme at equilibrium. The synthetic EGF-A domain of LDLR induced similar effects as the full-length receptor. The data suggest the existence of distinct conformational states in free and receptor-bound PCSK9.

Novel fluorescent derivatives of serotonin have been synthesized and used as tracers for the development of a 5-HT2C fluorescence polarization assay. Serotonin analogs that feature a fluorescent probe attached through an ether linkage at the tryptamine 5-position have high affinity for the 5-HT2C receptor, and affinity is dependent on both linker length and pendent dye. These variables have been optimized to generate Cy3B derivative 5a, which has 10-fold higher 5-HT2C affinity relative to serotonin (Kd=0.23 nM). In receptor activation experiments, 5a acts as a full agonist of 5-HT2C. Upon binding to 5-HT2C cell membranes, 5a shows a robust increase in fluorescence polarization (FP) signal. In an FP binding assay using 5a as a tracer ligand, Ki values for known 5-HT2C agonists and antagonists showed excellent agreement with Ki values from radioligand binding (r2=0.93). The FP ligand assay is suitable for high-throughput drug screening applications with respect to speed of analysis, displaceable signal, precision, and sensitivity to various reagents. A 384-well-based high-throughput assay that is rapid, economical, and predictive of test compounds' ability to bind to the 5-HT2C receptor has been compiled and validated.

Cholesteryl ester transfer protein (CETP) transfers neutral lipids between different types of plasma lipoprotein. Inhibitors of CETP elevate the fraction of plasma cholesterol associated with high-density lipoproteins and are being developed as new agents for the prevention and treatment of cardiovascular disease. The molecular basis of their function is not yet fully understood. To aid in the study of inhibitor interactions with CETP, a torcetrapib-related compound was coupled to different biotin-terminated spacer groups, and the binding of CETP to the streptavidin-bound conjugates was monitored on agarose beads and in a surface plasmon resonance biosensor. CETP binding was poor with a 2.0 nm spacer arm, but efficient with polyethyleneglycol spacers of 3.5 or 4.6 nm. The conjugate based on a 4.6 nm spacer was used for further biosensor experiments. Soluble inhibitor blocked the binding of CETP to the immobilized drug, as did preincubation with a disulfide-containing covalent inhibitor. To provide a first estimate of the binding site for torcetrapib-like inhibitors, CETP was modified with a disulfide-containing agent that modifies Cys-13 of CETP. Mass spectrometry of the modified protein indicated that a single half-molecule of the disulfide was covalently bound to CETP, and peptide mapping after digestion with pepsin confirmed previous reports based on mutagenesis that Cys-13 was the site of modification. Modified CETP was unable to bind to the biosensor-mounted torcetrapib analog, indicating that the binding site on CETP for torcetrapib is in the lipid-binding pocket near the N-terminus of the protein. The crystal structure of CETP shows that the sulfhydryl group of Cys-13 resides at the bottom of this pocket.

Novel fluorescent derivatives of dofetilide (1) have been synthesized. Analogues that feature a fluorescent probe attached through an aliphatic spacer to the central tertiary nitrogen of 1 have high affinity for the hERG channel, and affinity is dependent on both linker length and pendent dye. These variables have been optimized to generate Cy3B derivative 10e, which has hERG channel affinity equivalent to that of dofetilide. When bound to cell membranes expressing the hERG channel, 10e shows a robust increase in fluorescence polarization (FP) signal. In a FP binding assay using 10e as tracer ligand, Ki values for several known hERG channel blockers were measured and excellent agreement with the literature Ki values was observed over an affinity range of 2 nM to 3 muM. 10e blocks hERG channel current in electrophysiological patch clamp experiments, and computational docking experiments predict that the dofetilide core of 10e binds hERG channel in a conformation similar to that previously predicted for 1. These analogues enable high-throughput hERG channel binding assays that are rapid, economical, and predictive of test compounds' potential for prolonged QT liabilities.