A novel allosteric inhibitor of macrophage migration inhibitory factor (MIF).

Macrophage migration inhibitory factor (MIF) is a catalytic cytokine and an upstream mediator of the inflammatory pathway. MIF has broad regulatory properties, dysregulation of which has been implicated in the pathology of multiple immunological diseases. Inhibition of MIF activity with small molecules has proven beneficial in a number of disease models. Known small molecule MIF inhibitors typically bind in the tautomerase site of the MIF trimer, often covalently modifying the catalytic proline. Allosteric MIF inhibitors, particularly those that associate with the protein by noncovalent interactions, could reveal novel ways to block MIF activity for therapeutic benefit and serve as chemical probes to elucidate the structural basis for the diverse regulatory properties of MIF. In this study, we report the identification and functional characterization of a novel allosteric MIF inhibitor. Identified from a high throughput screening effort, this sulfonated azo compound termed p425 strongly inhibited the ability of MIF to tautomerize 4-hydroxyphenyl pyruvate. Furthermore, p425 blocked the interaction of MIF with its receptor, CD74, and interfered with the pro-inflammatory activities of the cytokine. Structural studies revealed a unique mode of binding for p425, with a single molecule of the inhibitor occupying the interface of two MIF trimers. The inhibitor binds MIF mainly on the protein surface through hydrophobic interactions that are stabilized by hydrogen bonding with four highly specific residues from three different monomers. The mode of p425 binding reveals a unique way to block the activity of the cytokine for potential therapeutic benefit in MIF-associated diseases.

Immunogenicity, Efficacy, and Safety of a Novel Synthetic Microparticle Pre-Erythrocytic Malaria Vaccine in Multiple Host Species.

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.

Microparticle RSV Vaccines Presenting the G Protein CX3C Chemokine Motif in the Context of TLR Signaling Induce Protective Th1 Immune Responses and Prevent Pulmonary Eosinophilia Post-Challenge.

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.

Layer-By-Layer Nanoparticle Vaccines Carrying the G Protein CX3C Motif Protect against RSV Infection and Disease.

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.

Nanoparticle vaccines encompassing the respiratory syncytial virus (RSV) G protein CX3C chemokine motif induce robust immunity protecting from challenge and disease.

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.

Plasmodium falciparum synthetic LbL microparticle vaccine elicits protective neutralizing antibody and parasite-specific cellular immune responses.

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.

Synthetic nanoparticle vaccines produced by layer-by-layer assembly of artificial biofilms induce potent protective T-cell and antibody responses in vivo.

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 CaCO(3) 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.