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Anthrax protective antigen (PA), the receptor-binding component of anthrax toxin, elicits toxin-neutralizing antibodies which provide protection against anthrax disease. PA binds to two mammalian receptors, capillary morphogenesis protein-2 (CMG2) and tumor endothelial marker-8 (TEM8). We previously observed that binding of PA to its receptors plays a role in eliciting a strong toxin-neutralizing antibody response. In this study, we examined the roles that individual receptors play in mediating the toxin-neutralizing antibody response. Mice immunized with PA that binds preferentially to CMG2 elicited a toxin-neutralizing antibody response similar to that elicited by wild-type PA, whereas the antibody response elicited by PA that binds preferentially to TEM8 was significantly lower. Also, the toxin-neutralizing antibody response elicited by wild-type PA in CMG2-null mice was found to be significantly lower than that induced in CMG2-sufficient mice, further supporting a predominant role for the CMG2 receptor in mediating a protective antibody response to PA.

Development of recombinant protective antigen (rPA)-based anthrax vaccines has been hindered by a lack of stability of the vaccines associated with spontaneous deamidation of asparagine (Asn) residues of the rPA antigen during storage. In this study, we explored the role that two deamidation-prone Asn residues located directly adjacent to the receptor binding site of PA, Asn713 and Asn719, play in the stability of rPA-based anthrax vaccines. We modified these residues to glutamine (Gln) and generated rPA(N713Q/N719Q), since Gln would not be expected to deamidate on a time scale relevant to vaccine storage. While wild-type rPA vaccine formulated with aluminum hydroxide lost immunogenicity upon storage, as measured by induction of toxin-neutralizing antibodies in mice, the rPA(N713Q/N719Q) vaccine did not exhibit a significant loss in immunogenicity. This finding suggests that modification of Asn713 and Asn719 of rPA to deamidation-resistant amino acids may improve the stability of rPA-based anthrax vaccines.

•GC1109 is a novel recombinant protective antigen anthrax vaccine.•We evaluate the immunogenicity and safety of GC1109 in healthy adult volunteers.•Participants were randomized to three doses of GC1109 groups or placebo group.•GC1109 was immunogenic after three doses of intramuscular administration.•Although vaccine-related adverse events were frequent, most of them were mild. The demand on effective and safe anthrax vaccine is increasing as a part of the preparedness for possible bioterrorism in the future. We performed a randomized, single-blind, placebo controlled phase II clinical study to evaluate the immunogenicity and safety of a novel recombinant protective antigen (rPA) anthrax vaccine, GC1109, in healthy adult volunteers. Participants were randomized to experiment groups (0.3 mL, 0.5 mL, and 1.0 mL of GC1109) or placebo group (normal saline 0.5 mL) in 2:2:2:1 ratio. They received respective vaccines intramuscularly at 0, 4 and 8 weeks. Immunogenicity was evaluated by seroconversion rate and geometric mean titer (GMT) of lethal toxin neutralizing assay (TNA) and anti-PA IgG by ELISA. Safety was assessed by laboratory tests, and solicited and unsolicited adverse events on diary cards. 30, 29, 30 participants were randomized to 0.3, 0.5, and 1.0 mL of GC1109 groups, respectively, while 15 to placebo group. 92 participants received all three doses. In per-protocol analysis, TNA GMTs at week 12 were 296.5, 285.2, and 433.2 in the three groups, respectively. Seroconversion rates measured by ELISA were 100% at week 12 in the three groups. Local and systemic vaccine-related adverse events were frequent; however, most of them were mild, and no serious events were observed. A new rPA anthrax vaccine GC1109 was immunogenic after three doses of intramuscular administration, and was well-tolerated.

In this study, we have investigated the immunogenicity and protective efficacy of a niosomal formulation encapsulating protective antigen (PA) and PA domain 4 (D4) of . Nonionic surfactant-based vesicles (NISV) + PA and NISV + D4 were prepared from span-60 and cholesterol by reverse-phase evaporation method and were evaluated for in vitro characteristics and immunological studies. Particle characterization using transmission electron microscopy and atomic force microscopy analysis showed that the niosomal formulation was spherical in shape. The entrapment efficiency values were calculated to be 58.5% and 44.75% for PA and D4, respectively. Confocal microscopy and flow cytometry studies showed an enhanced uptake of antigen in THP1 macrophages by niosome as compared to antigen only. An in vitro release assay showed a burst release of antigen from niosome within 24 hours followed by a gradual release for 144 hours. Immunological studies showed that both PA- and D4-encapsulated niosome elicited a robust IgG titer. Antibody isotyping and cytokine profile showed that NISV + PA and NISV + D4 enhanced both Th1 and Th2 responses in mice, suggesting a mixed Th1/Th2 response. Both NISV + PA and NISV + D4 elicited high levels of anti-inflammatory cytokine interleukin-10 with low levels of pro-inflammatory cytokine tumor necrosis factor-α, suggesting the anti-inflammatory property of niosome. Both the niosomal formulations were also able to confer protection against BA infection as compared to only PA and D4. PA and D4 encapsulated NISV formulation could modulate both the Th1 and Th2 adaptive immune system and was found to be a better prophylactic against anthrax.

Background Disease caused by Bacillus anthracis is often accompanied by high mortality primarily due to toxin-mediated injury. In the early disease course, anthrax toxins are secreted; thus, antibiotic use is limited to the early stage. In this regard, antibodies against the toxin component, protective antigen (PA), play an important role in protecting against anthrax. Therefore, we developed PA21, a fully human anti-PA immunoglobulin G monoclonal antibody. Methods Combining human Fab was screened from a phage library with human heavy constant regions. Enzyme-linked immune sorbent assay, Western blot analysis and immunoprecipitation test evaluated the binding ability of PA21. Moreover, the affinity and neutralizing activity of the antibody was detected in vitro while the protective effectiveness in 60 rats was also examined in vivo. Results The Fischer 344 rats challenged with the lethal toxin can be protected by PA21 at a concentration of 0.067 mg/kg. All six rats remained alive although PA21 was injected 24 h before the toxin challenge. PA21 did not influence the binding of PA to cell receptors and that of a lethal factor to PA. Conclusion The PA21 monoclonal antibody against PA can be used for emergency prophylaxis and anthrax treatment.

► Recombinant proteins Com1, Mip and GroEL were cloned and used to stimulate BMDCs. ► Mice receiving Com1 or Mip-pulsed DCs showed lower bacterial loads than control. ► Mice receiving Com1 or Mip-pulsed DCs exhibited greater IFN-γ-positive cells. The recombinant membrane-associated proteins of Coxiella burnetii, Com1, Mip and GroEL, were used in vitro to stimulate BALB/c mouse bone marrow-derived dendritic cells (BMDCs). The antigen-activated BMDCs were transferred into naïve BALB/c mice. Seven days after challenge of C. burnetii, the bacterial loads of mice receiving BMDCs activated with Com1 or Mip, but not GroEL, were significantly lower than that of mice receiving BMDCs pulsed with TrxA (Esherichia coli thioredoxin) in a quantitative polymerase chain reaction assay. After in vitro interaction with cognate antigen-pulsed BMDCs, the percentages of CD69-positive cells and TNF-α-positive cells in CD4+ and CD8+ T cells isolated from the spleens of mice receiving Com1-, Mip-, or GroEL-pulsed BMDCs were significantly higher than that of mice receiving mock-pulsed BMDCs in flow cytometric analysis. The percentages of IFN-γ-positive cells in CD4+ and CD8+ T cells from mice receiving Com1- or Mip-pulsed BMDCs were significantly greater than that of mice receiving GroEL-pulsed BMDCs. However, the percentage of IL-4-positive cells in CD4+ T cells of mice receiving GroEL-pulsed BMDCs was obviously higher than that of mice receiving Com1- or Mip-pulsed BMDCs. Our results demonstrate that Com1 and Mip are protective antigens and strongly indicate that they favor to induce IFN-γ-producing Th1 and Tc1 cells, whereas the non-protective antigen GroEL is biased to induce a Th2 response. Therefore, Com1 and Mip are key antigens to induce a protective immune response against C. burnetii infection.

Tuberculosis (TB) is the leading cause of death of any single infectious agent, having led to 1.4 million deaths in 2019 alone. Moreover, an estimated one-quarter of the global population is latently infected with Mycobacterium tuberculosis (MTB), presenting a huge pool of potential future disease. Nonetheless, the only currently licensed TB vaccine fails to prevent the activation of latent TB infections (LTBI). These facts together illustrate the desperate need for a more effective TB vaccine strategy that can prevent both primary infection and the activation of LTBI. In this study, we employed a machine learning-based reverse vaccinology approach to predict the likelihood that each protein within the proteome of MTB laboratory reference strain H37Rv would be a protective antigen (PAg). The proteins predicted most likely to be a PAg were assessed for their belonging to a protein family of previously established PAgs, the relevance of their biological processes to MTB virulence and latency, and finally the immunogenic potential that they may provide in terms of the number of promiscuous epitopes within each. This study led to the identification of 16 proteins with the greatest vaccine potential for further in vitro and in vivo studies. It also demonstrates the value of computational methods in vaccine development.

Vector-borne diseases, mainly transmitted by ticks, have a significant impact on the productivity and health of the animals in the developing countries. The quest for vaccine candidates to control these arthropod vectors remains pivotal and only rationale approach in controlling these diseases. Several candidate antigens have been screened over the years of which 4D8 tick protective antigen have shown promising results in controlling ixodid tick infestations. The 4D8 tick protective antigen is a structural and functional ortholog of insect subolesin/akirin and an evolutionary conserved protein, associated with multiple essential cellular processes in tick biology. Variable efficacy of recombinant subolesin/4D8 vaccine in field trials propels the approach towards synthetic peptide vaccine through reverse vaccinology. Advancement in reverse vaccinology has opened a new paradigm in the search of a perfect candidate antigen. The present study focused on full length amplification, cloning and sequencing of the 4D8 gene from Hyalomma antolicum. Combination of bioinformatics analysis and computational modeling tools identified linear as well as conformational B-cell and MHC class II epitopes conserved in many ixodid ticks which may be useful to design an universal synthetic peptide vaccine against multiple tick species. Western blot analysis using rabbit antisera against recombinant 4D8 protein has also confirmed the immunogenic potential of the candidate.

Immune surveillance against pathogens and tumours in the central nervous system is thought to be limited owing to the lack of lymphatic drainage. However, the characterization of the meningeal lymphatic network has shed light on previously unappreciated ways that an immune response can be elicited to antigens that are expressed in the brain 1 – 3 . Despite progress in our understanding of the development and structure of the meningeal lymphatic system, the contribution of this network in evoking a protective antigen-specific immune response in the brain remains unclear. Here, using a mouse model of glioblastoma, we show that the meningeal lymphatic vasculature can be manipulated to mount better immune responses against brain tumours. The immunity that is mediated by CD8 T cells to the glioblastoma antigen is very limited when the tumour is confined to the central nervous system, resulting in uncontrolled tumour growth. However, ectopic expression of vascular endothelial growth factor C (VEGF-C) promotes enhanced priming of CD8 T cells in the draining deep cervical lymph nodes, migration of CD8 T cells into the tumour, rapid clearance of the glioblastoma and a long-lasting antitumour memory response. Furthermore, transfection of an mRNA construct that expresses VEGF-C works synergistically with checkpoint blockade therapy to eradicate existing glioblastoma. These results reveal the capacity of VEGF-C to promote immune surveillance of tumours, and suggest a new therapeutic approach to treat brain tumours. In a mouse model of glioblastoma, treatment with VEGF-C increases lymphatic drainage in the central nervous system and improves the immune response, suggesting that modulating meningeal lymphatics could enhance checkpoint inhibitor therapy.

The precise assembly and engineering of molecular machines capable of handling biomolecules play crucial roles in most single-molecule methods. In this work we use components from all three domains of life to fabricate an integrated multiprotein complex that controls the unfolding and threading of individual proteins across a nanopore. This 900 kDa multicomponent device was made in two steps. First, we designed a stable and low-noise β-barrel nanopore sensor by linking the transmembrane region of bacterial protective antigen to a mammalian proteasome activator. An archaeal 20S proteasome was then built into the artificial nanopore to control the unfolding and linearized transport of proteins across the nanopore. This multicomponent molecular machine opens the door to two approaches in single-molecule protein analysis, in which selected substrate proteins are unfolded, fed to into the proteasomal chamber and then addressed either as fragmented peptides or intact polypeptides.An integrated multiprotein nanopore has been fabricated using components from all three domains of life. This molecular machine opens the door to two approaches in single-molecule protein analysis, in which selected substrate proteins are unfolded, fed to into the proteasomal chamber and then processed either as fragmented peptides or intact polypeptides.