Characterization of prostanoids response to Bordetella pertussis antigen BscF and Tdap in LPS-challenged monocytes.

Prostanoids are potent inflammatory mediators that play a regulatory role in the innate immune activation of the adaptive immune response to determine the duration of protection against infection. We aim to quantify the modulation of prostanoids profiles in lipopolysaccharide (LPS)-stimulated THP-1 cells treated with the novel pertussis antigen BscF. We compared the effect with pertussis antigens present in the current Tdap vaccine to understand the immunomodulatory effect that might contribute to the diminished Tdap vaccine effectiveness. The inflammatory challenge with LPS induced a robust elevation of most prostanoid family members compared to the control treatment. Treatment with BscF and Tdap significantly reduced the LPS-stimulated elevation of prostaglandins (PGs) D2, E2, and F2α, as well as thromboxane (TX) A2 levels. An opposite trend was observed for PGI2, as both antigens accelerated the LPS-stimulated upregulation. Further, we quantified cyclooxygenases (COXs) that catalyze the biosynthesis of prostanoids and found that both antigens significantly reduced LPS-stimulated COX-1 and COX-2, demonstrating that the waning of acellular pertussis vaccines' protective immunity may be due to other downstream enzymes not related to COXs. Our present study validates the potential role of BscF as an adjuvant, resulting in the next-generation pertussis vaccine discovery.

Modulation of Inflammatory Signaling Molecules in Bordetella pertussis Antigen-Challenged Human Monocytes in Presence of Adrenergic Agonists.

BscF is a type III secretion system (T3SS) needle protein from Bordetella pertussis and has previously been shown to induce a sufficient Th1 and Th17 response in human monocytes and mice as a prerequisite for long-lasting protective immunity against pertussis infection. In our current study, we aim to compare the modulation of inflammatory signaling molecules as a direct measure of the immune response to the B. pertussis antigens BscF and Tdap in the presence or absence of the adrenergic receptor agonists phenylephrine (PE) or isoproterenol (ISO) to observe differences that may contribute to the diminished protective immunity of the current acellular pertussis (aP) vaccine, Tdap. Stimulation of human monocyte THP-1 cells with LPS, BscF, and Tdap induced a robust elevation of CCL20, CXCL10, PGE2, and PGF2α among most chemokine and prostanoid members when compared with the control treatment. Treatment with the adrenergic agonist PE or ISO significantly enhanced the BscF- and Tdap-stimulated modulation of CCL20 and CXCL10 but not PGE2 and PGF2α, suggesting that adrenergic modulation of pertussis antigen responses might be a new therapeutic strategy to improve the longevity of pertussis immunity. Stimulation of THP-1 cells with BscF alone initiated significant expression of CXCL10 and PGF2α but not when Tdap was used, suggesting that BscF might be an important pertussis antigen for next-generation pertussis vaccines or when combined with the current aP vaccine. Our data offer opportunities for designing new therapeutic approaches against pertussis infection.

Dengue virus specific IgY provides protection following lethal dengue virus challenge and is neutralizing in the absence of inducing antibody dependent enhancement.

Dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are severe disease manifestations that can occur following sequential infection with different dengue virus serotypes (DENV1-4). At present, there are no licensed therapies to treat DENV-induced disease. DHF and DSS are thought to be mediated by serotype cross-reactive antibodies that facilitate antibody-dependent enhancement (ADE) by binding to viral antigens and then Fcγ receptors (FcγR) on target myeloid cells. Using genetically engineered DENV-specific antibodies, it has been shown that the interaction between the Fc portion of serotype cross-reactive antibodies and FcγR is required to induce ADE. Additionally, it was demonstrated that these antibodies were as neutralizing as their non-modified variants, were incapable of inducing ADE, and were therapeutic following a lethal, antibody-enhanced infection. Therefore, we hypothesized that avian IgY, which do not interact with mammalian FcγR, would provide a novel therapy for DENV-induced disease. We demonstrate here that goose-derived anti-DENV2 IgY neutralized DENV2 and did not induce ADE in vitro. Anti-DENV2 IgY was also protective in vivo when administered 24 hours following a lethal DENV2 infection. We were also able to demonstrate via epitope mapping that both full-length and alternatively spliced anti-DENV2 IgY recognized different epitopes, including epitopes that have not been previously identified. These observations provide evidence for the potential therapeutic applications of goose-derived anti-DENV2 IgY.

A Method for Characterizing the Type III Secretion System's Contribution to Pathogenesis: Homologous Recombination to Generate Yersinia pestis Type III Secretion System Mutants.

The type III (T3S) secretion system of many gram-negative bacteria is a surface-exposed protein secretion apparatus used to directly inject bacterial effector molecules into eukaryotic cells. These effector molecules contribute to bacterial pathogenesis in many ways, and have been shown to be crucial for infectivity. Here, we describe a protocol for using homologous recombination to generate T3S system mutants to assess the role of different T3S system proteins in bacterial pathogenesis.

Expression and Purification of N-Terminally His-Tagged Recombinant Type III Secretion Proteins.

The ability to express and purify recombinant needle proteins from the Type III Secretion System (T3SS) of many gram-negative bacteria has allowed us to develop novel experimental approaches, both in vitro and in vivo, to identify unique roles for T3SS in bacterial pathogenesis. In addition, these purified needle proteins have shown to be promising immunotherapies acting as both protective antigens and adjuvants, presumably due to their immune activating properties. Here, we describe the expression and purification of recombinant T3SS needle proteins.

Mouse Immunization with Purified Needle Proteins from Type III Secretion Systems and the Characterization of the Immune Response to These Proteins.

Many Gram-negative pathogens utilize a type III secretion (T3S) system to directly deliver effector molecules into host eukaryotic cells to manipulate cellular processes. These surface-exposed syringe-like structures are highly conserved, necessary for pathogenesis, and hence are therapeutic targets against a number of Gram-negative pathogens. Here we describe a protocol for using purified needle proteins to immunize mice, and subsequently, ways to characterize the immune response to immunization.