Biology subjects

LaVake, C. J.

Publications and source records attributed to LaVake, C. J..

3 recordsLinked to original sources

bioRxiv2024

Immunodominant extracellular loops of Treponema pallidum FadL outer membrane proteins elicit antibodies with opsonic and growth-inhibitory activities

The global resurgence of syphilis has created a potent stimulus for vaccine development. To identify potentially protective antibodies (Abs) against Treponema pallidum (TPA), we used Pyrococcus furiosus thioredoxin (PfTrx) to display extracellular loops (ECLs) from three TPA outer membrane protein families (outer membrane factors for efflux pumps, eight-stranded {beta}-barrels, and FadLs) to assess their reactivity with immune rabbit serum (IRS). Five ECLs from the FadL orthologs TP0856, TP0858 and TP0865 were immunodominant. Rabbits and mice immunized with these five PfTrx constructs produced ECL-specific Abs that promoted opsonophagocytosis of TPA by rabbit peritoneal and murine bone marrow-derived macrophages at levels comparable to IRS and mouse syphilitic serum. ECL-specific rabbit and mouse Abs also impaired viability, motility, and cellular attachment of spirochetes during in vitro cultivation. The results support the use of ECL-based vaccines and suggest that ECL-specific Abs promote spirochete clearance via Fc receptor-independent as well as Fc receptor-dependent mechanisms. Author SummaryThe resurgence of syphilis emphasizes the critical need for vaccine development against Treponema pallidum (TPA). Research utilizing immune rabbit serum (IRS) suggests that an effective syphilis vaccine should induce "functional" antibodies (Abs) capable of enhancing the opsonophagocytosis of treponemes by activated macrophages. Structural models of TPA outer membrane proteins (OMPs), specifically the extracellular loops (ECLs), guided the identification of potential vaccine candidates. Antigenic analysis with IRS of individual ECLs from three TPA OMP families scaffolded onto Pyrococcus furiosus thioredoxin (PfTrx) revealed five FadL antigenic ECLs. Immunization with immunodominant ECL antigens elicited robust ECL-specific Abs, demonstrating functional opsonic activity in the opsonophagocytosis assays. Furthermore, these Abs effectively inhibited the growth inhibition of in vitro-cultivated TPA in both rabbit and mouse models. Our findings underscore the value of antigenic analysis in identifying promising TPA OMP ECL vaccine targets and highlight the multifaceted protective capacity of ECL Abs against TPA. This approach also extends to identifying potential OMP vaccinogens in other bacterial pathogens, offering valuable insights for broader vaccine development strategies.

immunology↗

bioRxiv2023

Use of Epivolve phage display to generate a monoclonal antibody with opsonic activity directed against a subdominant epitope on extracellular loop 4 of Treponema pallidum BamA (TP0326)

Syphilis, a sexually transmitted infection caused by the spirochete Treponema pallidum (Tp), is resurging globally. Opsonic antibodies (Abs) targeting surface-exposed epitopes of the spirochetes outer membrane proteins (OMPs) are believed to promote macrophage-mediated clearance of the bacterium during infection and are presumed to be key to vaccine development. Tps repertoire of outer membrane proteins includes BamA ({beta}-barrel assembly machinery subunit A/TP0326), the central component of the molecular machine that inserts newly exported OMP precursors into the OM lipid bilayer. BamA is a bipartite protein consisting of an 18-stranded {beta}-barrel with nine extracellular loops (ECLs) and five periplasmic POTRA (polypeptide transport-associated) domains. Antisera directed against BamA ECL4 promote internalization of Tp by rabbit peritoneal macrophages. Herein, we employed a novel two-stage, phage display strategy, termed "Epivolve" (for epitope evolution), to generate five site-directed murine monoclonal Abs (mAbs) targeting a centrally located peptide (S2) of BamA ECL4. Each of the five mAbs demonstrated reactivity by immunoblotting and ELISA to nanogram amounts of BamA ECL4 displayed by a Pyrococcus furiosus thioredoxin (PfTrx) scaffold (PfTrxBamA/ECL4). One mAb containing a unique amino acid sequence in both light and heavy chains showed activity in an opsonophagocytosis assay employing murine bone marrow-derived macrophages. Mice and rabbits hyperimmunized with PfTrxBamA/ECL4 produced opsonic antisera that strongly recognized the ECL presented in a heterologous scaffold and overlapping ECL4 peptides including S2. In contrast, Abs generated during Tp infection of mice and rabbits poorly recognized the peptides, indicating that S2 contains a subdominant epitope. Epivolve, which circumvents the natural immune response, can be utilized for the generation of mAbs that target subdominant opsonic epitopes in ECLs of Tp OMPs.

immunology↗

bioRxiv2019

Macrophage mediated recognition and clearance of Borrelia burgdorferi elicits MyD88-dependent and - independent phagosomal signals that contribute to phagocytosis and inflammation

Lyme disease is a tick-borne illness caused by the spirochete Borrelia burgdorferi (Bb). It is believed that the robust inflammatory response induced by the hosts innate immune system is responsible for the clinical manifestations associated with Bb infection. The macrophage plays a central role in the immune response to many bacterial infections and is thought to play a central role in activation of the innate immune response to Bb. Previous studies have shown that following phagocytosis of spirochetes by macrophages, phagosome maturation results in degradation of Bb and liberation of bacterial lipoproteins and nucleic acids, which are recognized by TLR2 and TLR8, respectively, and elicit MyD88-mediated phagosome signaling cascades. Bone marrow-derived macrophages (BMDMs) from MyD88-/- mice show significantly reduced spirochete uptake and inflammatory cytokine production when incubated with Bb ex vivo. Paradoxically, additional studies revealed that Bb-infected MyD88-/- mice exhibit inflammation in joint and heart tissues. To determine the contribution of MyD88 to macrophage-mediated spirochete clearance, we compared wildtype (WT) and MyD88-/- mice using a murine model of Lyme disease. MyD88-/- mice showed increased Bb burdens in hearts 28 days post infection, while H&E staining and immunohistochemistry showed significantly increased inflammation and greater macrophage infiltrate in the hearts of MyD88-/- mice. This suggests that Bb triggers MyD88-independent inflammatory pathways in macrophages to facilitate cell recruitment to tissues. Upon stimulation with Bb ex vivo, WT and MyD88-/- BMDMs exhibit significant differences in bacteria uptake, suggesting that MyD88 signaling mediates cytoskeleton remodeling and the formation of membrane protrusions to enhance bacteria phagocytosis. A comprehensive transcriptome comparison in Bb-infected WT and MyD88-/- BMDMs identified a large cohort of MyD88-dependent genes that are differentially expressed in response to Bb, including genes involved in actin and cytoskeleton organization (Daam1, Fmnl1). We also identified a cohort of differentially-expressed MyD88-independent chemokines (Cxcl2, Ccl9) known to recruit macrophages. We identified master regulators and generated networks which model potential signaling pathways that mediate both phagocytosis and the inflammatory response. These data provide strong evidence that MyD88-dependent and -independent phagosomal signaling cascades in macrophages play significant roles in the ability of these cells to phagocytose Bb and mediate infection.\n\nAUTHOR SUMMARYMacrophages play prominent roles in bacteria recognition and clearance, including Borrelia burgdorferi (Bb), the Lyme disease spirochete. To elucidate mechanisms by which MyD88/TLR signaling enhances clearance of Bb by macrophages, we studied Bb-infected wildtype (WT) and MyD88-/- mice and Bb-stimulated bone marrow-derived macrophages (BMDMs). Bb-infected MyD88-/- mice show increased bacterial burdens, macrophage infiltration and altered gene expression in inflamed heart tissue. MyD88-/- BMDMs exhibit impaired uptake of spirochetes but comparable maturation of phagosomes following internalization of spirochetes. RNA-sequencing of infected WT and MyD88-/- BMDMs identified a large cohort of differentially expressed MyD88-dependent genes involved in re-organization of actin and cytoskeleton during phagocytosis along with several MyD88-independent chemokines involved in inflammatory cell recruitment. We computationally generated networks which identified several MyD88-independent master regulators (Cxcl2 and Vcam1) and MyD88-dependent intermediate proteins (Rhoq and Cyfip1) that are known to mediate inflammation and phagocytosis respectively. These results provide mechanistic insights into MyD88-mediated phagosomal signaling enhancing Bb uptake and clearance.

immunology↗