Avid binding by B cells to the Plasmodium circumsporozoite protein repeat suppresses responses to protective subdominant epitopes

Antibodies targeting the NANP/NVDP repeat domain of the Plasmodium falciparum circumsporozoite protein (CSP ) can protect against malaria. However, it has also been suggested that the CSP is a decoy that prevents the immune system from mounting responses against other domains of CSP. Here, we show that, following parasite immunization, B cell responses to the CSP are immunodominant over responses to other CSP domains despite the presence of similar numbers of naive B cells able to bind these regions. We find that this immunodominance is driven by avid binding of the CSP to cognate B cells that are able to expand at the expense of B cells with other specificities. We further show that mice immunized with repeat-truncated CSP molecules develop responses to subdominant epitopes and are protected against malaria. These data demonstrate that the CSP functions as a decoy, but truncated CSP molecules may be an approach for malaria vaccination.We thank Rajagopal Murugan and Hedda Wardemann (Deutsches Krebsforschungszentrum, Heidelberg, Germany) for providing the C-terminal antibodies 1710 and 3919 and Azza Idris, Joe Francica, and Robert Seder (Vaccine Research Center, National Institutes of Health, Bethesda, MD) for the provision of mAb15 and CSPCterm and CSPNterm peptides. We thank Michael Devoy, Harpreet Vohra, and Catherine Gillespie of the Imaging and Cytometry Facility at the John Curtin School of Medical Research for assistance with flow cytometry and multi-photon microscopy. We also thank Theresa Neeman of the ANU statistical consulting unit for assistance with statistical analysis of the data. This work was funded by the Bill and Melinda Gates Foundation (OPP1151018) and the National Health and Medical Research Council (GNT1158404). D.C. is supported by Deutsche Forschungsgemeinschaft International Research Training Group 2290 and is the recipient of PhD scholarship from the Australian National Universit

TLR-3 Stimulation Skews M2 Macrophages to M1 Through IFN-αβ Signaling and Restricts Tumor Progression

During tumor progression, macrophages shift their protective M1-phenotype to pro-tumorigenic M2-subtype. Therefore, conversion of M2 to M1 phenotype may be a potential therapeutic intervention. TLRs are important pathogen recognition receptors expressed by cells of the immune system. Recently, a crucial role of TLR-3 has been suggested in cancer. Consequently, in the current study, we defined the role of TLR-3 in the reversion of M2-macrophages to M1. We analyzed the role of TLR-3 stimulation for skewing M2-macrophages to M1 at mRNA and protein level through qRT-PCR, flow cytometry, western blotting, and ELISA. The effectiveness of TLR-3L stimulation to revert M2-macrophages to M1 was evaluated in the murine tumor model. To determine the role of IFN-αβ signaling in vitro and in vivo, we used Ifnar1−/− macrophages and anti-IFN-αβ antibodies, respectively. We observed upregulation of M1-specific markers MHC-II and costimulatory molecules like CD86, CD80, and CD40 on M2-macrophages upon TLR-3 stimulation. In contrast, reduced expression of M2-indicators CD206, Tim-3, and pro-inflammatory cytokines was noticed. The administration of TLR-3L in the murine tumor reverted the M2-macrophages to M1-phenotype and regressed the tumor growth. The mechanism deciphered for macrophage reversion and controlling the tumor growth is dependent on IFN-αβ signaling pathway. The results indicate that the signaling through TLR-3 is important in protection against tumors by skewing M2-macrophages to protective M1-subtype

Morbid Sequences Suggest Molecular Mimicry between Microbial Peptides and Self-Antigens: A Possibility of Inciting Autoimmunity

Understanding etiology of autoimmune diseases has been a great challenge for designing drugs and vaccines. The pathophysiology of many autoimmune diseases may be attributed to molecular mimicry provoked by microbes. Molecular mimicry hypothesizes that a sequence homology between foreign and self-peptides leads to cross-activation of autoreactive T cells. Different microbial proteins are implicated in various autoimmune diseases, including multiple sclerosis, human type 1 diabetes, primary biliary cirrhosis and rheumatoid arthritis. It may be imperative to identify the microbial epitopes that initiate the activation of autoreactive T cells. Consequently, in the present study, we employed immunoinformatics tools to delineate homologous antigenic regions between microbes and human proteins at not only the sequence level but at the structural level too. Interestingly, many cross-reactive MHC class II binding epitopes were detected from an array of microbes. Further, these peptides possess a potential to skew immune response toward Th1-like patterns. The present study divulges many microbial target proteins, their putative MHC-binding epitopes, and predicted structures to establish the fact that both sequence and structure are two important aspects for understanding the relationship between molecular mimicry and autoimmune diseases. Such findings may enable us in designing potential immunotherapies to tolerize autoreactive T cells

Bolstering Immunity through Pattern Recognition Receptors: A Unique Approach to Control Tuberculosis

The global control of tuberculosis (TB) presents a continuous health challenge to mankind. Despite having effective drugs, TB still has a devastating impact on human health. Contributing reasons include the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb), the AIDS-pandemic, and the absence of effective vaccines against the disease. Indeed, alternative and effective methods of TB treatment and control are urgently needed. One such approach may be to more effectively engage the immune system; particularly the frontline pattern recognition receptor (PRR) systems of the host, which sense pathogen-associated molecular patterns (PAMPs) of Mtb. It is well known that 95% of individuals infected with Mtb in latent form remain healthy throughout their life. Therefore, we propose that clues can be found to control the remainder by successfully manipulating the innate immune mechanisms, particularly of nasal and mucosal cavities. This article highlights the importance of signaling through PRRs in restricting Mtb entry and subsequently preventing its infection. Furthermore, we discuss whether this unique therapy employing PRRs in combination with drugs can help in reducing the dose and duration of current TB regimen

Antibody Feedback Limits the Expansion of B Cell Responses to Malaria Vaccination but Drives Diversification of the Humoral Response

Generating sufficient antibody to block infection is a key challenge for vaccines against malaria. Here, we show that antibody titers to a key target, the repeat region of the Plasmodium falciparum circumsporozoite protein (PfCSP), plateaued after two immunizations in a clinical trial of the radiation-attenuated sporozoite vaccine. To understand the mechanisms limiting vaccine responsiveness, we developed immunoglobulin (Ig)-knockin mice with elevated numbers of PfCSP-binding B cells. We determined that recall responses were inhibited by antibody feedback, potentially via epitope masking of the immunodominant PfCSP repeat region. Importantly, the amount of antibody that prevents boosting is below the amount of antibody required for protection. Finally, while antibody feedback limited responses to the PfCSP repeat region in vaccinated volunteers, potentially protective subdominant responses to PfCSP C-terminal regions expanded with subsequent boosts. These data suggest that antibody feedback drives the diversification of immune responses and that vaccination for malaria will require targeting multiple antigens.Production and characterization of PfSPZ Vaccine were supported in part by National Insitute of Allergy and Infectious Diseases Small Business Innovation Research grants 5R44AI055229-11 (to S.L.H.), 5R44AI058499-08 (to S.L.H.), and 5R44AI058375-08 (to S.L.H.)

Presentation_1_TLR-3 Stimulation Skews M2 Macrophages to M1 Through IFN-αβ Signaling and Restricts Tumor Progression.PDF

<p>During tumor progression, macrophages shift their protective M1-phenotype to pro-tumorigenic M2-subtype. Therefore, conversion of M2 to M1 phenotype may be a potential therapeutic intervention. TLRs are important pathogen recognition receptors expressed by cells of the immune system. Recently, a crucial role of TLR-3 has been suggested in cancer. Consequently, in the current study, we defined the role of TLR-3 in the reversion of M2-macrophages to M1. We analyzed the role of TLR-3 stimulation for skewing M2-macrophages to M1 at mRNA and protein level through qRT-PCR, flow cytometry, western blotting, and ELISA. The effectiveness of TLR-3L stimulation to revert M2-macrophages to M1 was evaluated in the murine tumor model. To determine the role of IFN-αβ signaling in vitro and in vivo, we used Ifnar1^−/− macrophages and anti-IFN-αβ antibodies, respectively. We observed upregulation of M1-specific markers MHC-II and costimulatory molecules like CD86, CD80, and CD40 on M2-macrophages upon TLR-3 stimulation. In contrast, reduced expression of M2-indicators CD206, Tim-3, and pro-inflammatory cytokines was noticed. The administration of TLR-3L in the murine tumor reverted the M2-macrophages to M1-phenotype and regressed the tumor growth. The mechanism deciphered for macrophage reversion and controlling the tumor growth is dependent on IFN-αβ signaling pathway. The results indicate that the signaling through TLR-3 is important in protection against tumors by skewing M2-macrophages to protective M1-subtype.</p