AAV-mediated delivery of an anti-BACE1 VHH alleviates pathology in an Alzheimer's disease model.

Single domain antibodies (VHHs) are potentially disruptive therapeutics, with important biological value for treatment of several diseases, including neurological disorders. However, VHHs have not been widely used in the central nervous system (CNS), largely because of their restricted blood-brain barrier (BBB) penetration. Here, we propose a gene transfer strategy based on BBB-crossing adeno-associated virus (AAV)-based vectors to deliver VHH directly into the CNS. As a proof-of-concept, we explored the potential of AAV-delivered VHH to inhibit BACE1, a well-characterized target in Alzheimer's disease. First, we generated a panel of VHHs targeting BACE1, one of which, VHH-B9, shows high selectivity for BACE1 and efficacy in lowering BACE1 activity in vitro. We further demonstrate that a single systemic dose of AAV-VHH-B9 produces positive long-term (12 months plus) effects on amyloid load, neuroinflammation, synaptic function, and cognitive performance, in the AppNL-G-F Alzheimer's mouse model. These results constitute a novel therapeutic approach for neurodegenerative diseases, which is applicable to a range of CNS disease targets.

Passive immunotherapy with a novel antibody against 3pE-modified Aß demonstrates potential for enhanced efficacy and favorable safety in combination with BACE inhibitor treatment in plaque-depositing mice.

The imbalance between production and clearance of amyloid ß (Aß) peptides and their resulting accumulation in the brain is an early and crucial step in the pathogenesis of Alzheimer's disease (AD). Therefore, Aß is strongly positioned as a promising and extensively validated therapeutic target for AD. Investigational disease-modifying approaches aiming at reducing cerebral Aß concentrations include prevention of de novo production of Aß through inhibition of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1), and clearance of Aß deposits via passive Aß immunotherapy. We have developed a novel, high affinity antibody against Aß peptides bearing a pyroglutamate residue at amino acid position 3 (3pE), an Aß species abundantly present in plaque deposits in AD brains. Here, we describe the preclinical characterization of this antibody, and demonstrate a significant reduction in amyloid burden in the absence of microhemorrhages in different mouse models with established plaque deposition. Moreover, we combined antibody treatment with chronic BACE1 inhibitor treatment and demonstrate significant clearance of pre-existing amyloid deposits in transgenic mouse brain, without induction of microhemorrhages and other histopathological findings. Together, these data confirm significant potential for the 3pE-specific antibody to be developed as a passive immunotherapy approach that balances efficacy and safety. Moreover, our studies suggest further enhanced treatment efficacy and favorable safety after combination of the 3pE-specific antibody with BACE1 inhibitor treatment.

Vaccination with Secreted Aspartyl Proteinase 2 Protein from Candida parapsilosis Can Enhance Survival of Mice during C. tropicalis-Mediated Systemic Candidiasis.

The rising incidence of non-albicans Candida species globally, along with the emergence of drug resistance, is a cause for concern. This study investigated the protective efficacy of secreted aspartyl proteinase 2 (Sap2) in systemic C. tropicalis infection. Vaccination with recombinant Sap2 (rSap2) protein from C. parapsilosis enhanced survival of mice compared to rSap2 vaccinations from C. albicans (P = 0.02), C. tropicalis (P = 0.06), and sham immunization (P = 0.04). Compared to sham-immunized mice, the fungal CFU number was significantly reduced in organs of Sap2-parapsilosis-immunized mice. Histopathologically, increased neutrophilic recruitment was observed in Sap2-parapsilosis- and Sap2-tropicalis-immunized mice. Among different rSap2 proteins, Sap2-parapsilosis vaccination induced increased titers of Sap2-specific Ig, IgG, and IgM antibodies, which could bind whole fungus. Between different groups, sera from Sap2-parapsilosis-vaccinated mice exhibited increased C. tropicalis biofilm inhibition ability in vitro and enhanced neutrophil-mediated fungal killing. Passive transfer of anti-Sap2-parapsilosis immune serum in naive mice significantly reduced fungal burdens compared to those in mice receiving anti-sham immune serum. Higher numbers of plasma cells and Candida-binding B cells in Sap2-vaccinated mice suggest a role of B cells during early stages of Sap2-mediated immune response. Additionally, increased levels of Th1/Th2/Th17 cytokines observed in Sap2-parapsilosis-vaccinated mice indicate immunomodulatory properties of Sap2. Epitope analysis performed using identified B-cell epitopes provides a basis to understand differences in immunogenicity observed among Sap2-antigens and can aid the development of a multivalent or multiepitope anti-Candida vaccine(s). In summary, our results suggest that Sap2-parapsilosis vaccination can improve mouse survival during C. tropicalis infection by inducing both humoral and cellular immunity, and higher titers of Sap2-induced antibodies are beneficial during systemic candidiasis.

Correlation between Antibodies to Bacterial Lipopolysaccharides and Barrier Proteins in Sera Positive for ASCA and ANCA.

Individuals with intestinal barrier dysfunction are more prone to autoimmunity. Lipopolysaccharides (LPS) from gut bacteria have been shown to play a role in systemic inflammation, leading to the opening of the gut and blood-brain barrier (BBB). This study aims to measure antibodies against LPS and barrier proteins in samples positive for anti-Saccharomyces cerevisiae antibodies (ASCA) and anti-neutrophil cytoplasmic antibodies (ANCA) and compare them with these same antibodies in controls to determine whether a correlation between LPS and barrier proteins could be found. We obtained 94 ASCA- and 94 ANCA-positive blood samples, as well as 188 blood samples from healthy controls. Samples were assessed for antibodies to LPS, zonulin+occludin, S100B, and aquaporin-4 (AQP4). Results show significant elevation in antibodies in about 30% of ASCA- and ANCA-positive sera and demonstrate positive linear relationships between these antibodies. The findings suggest that individuals positive for ASCA and ANCA have increased odds of developing intestinal and BBB permeability compared to healthy subjects. The levels of LPS antibodies in both ASCA- and ANCA-positive and negative specimens showed from low and moderate to high correlation with antibodies to barrier proteins. This study shows that LPS, by damaging the gut and BBBs, contribute to the extra-intestinal manifestation of IBD. We conclude that IBD patients should be screened for LPS antibodies in an effort to detect or prevent possible barrier damage at the earliest stage possible to abrogate disease symptoms in IBS and associated disorders.

Optimal conditions for the storage of German cockroach extract.

Allergen extracts are commonly utilized for diagnosis and immunotherapy; however, the stability of protease­rich extracts is important for a precise diagnosis and treatment efficacy. The present study determines the optimal conditions for the storage of German cockroach allergen extract. Cockroach extracts were reconstituted in four buffers: normal saline (NS), 50% glycerol in NS, 0.3% phenol in NS, or 0.3% phenol and 50% glycerol in NS. The extracts in different buffers were stored either at room temperature (18­26˚C, RT) or refrigerated (2­8˚C). Subsequently, the protein concentration and allergen content (Bla g 1 and Bla g 2) in the extracts were examined for the course of one year. Extract potency was estimated by inhibition ELISA. At least 90.5% protein, 94.4% Bla g 1, 65.2% Bla g 2, and 91.4% potency remained after one year when 50% glycerol NS was added to the extract with refrigeration. However, less than 13.7% protein, 17.1% Bla g 1, 0% Bla g 2 and 32.5% potency were maintained after one year when 50% glycerol NS was not added to the extract and was maintained at RT. The addition of 0.3% phenol NS did not show significant effects on extract stability. The addition of 50% glycerol NS and refrigerated storage temperature were found to be important factors for increasing the shelf life of protease­rich cockroach extract.

Functional selection of protease inhibitory antibodies.

Critical for diverse biological processes, proteases represent one of the largest families of pharmaceutical targets. To inhibit pathogenic proteases with desired selectivity, monoclonal antibodies (mAbs) hold great promise as research tools and therapeutic agents. However, identification of mAbs with inhibitory functions is challenging because current antibody discovery methods rely on binding rather than inhibition. This study developed a highly efficient selection method for protease inhibitory mAbs by coexpressing 3 recombinant proteins in the periplasmic space of Escherichia coli-an antibody clone, a protease of interest, and a ß-lactamase modified by insertion of a protease cleavable peptide sequence. During functional selection, inhibitory antibodies prevent the protease from cleaving the modified ß-lactamase, thereby allowing the cell to survive in the presence of ampicillin. Using this method to select from synthetic human antibody libraries, we isolated panels of mAbs inhibiting 5 targets of 4 main protease classes: matrix metalloproteinases (MMP-14, a predominant target in metastasis; MMP-9, in neuropathic pain), ß-secretase 1 (BACE-1, an aspartic protease in Alzheimer's disease), cathepsin B (a cysteine protease in cancer), and Alp2 (a serine protease in aspergillosis). Notably, 37 of 41 identified binders were inhibitory. Isolated mAb inhibitors exhibited nanomolar potency, exclusive selectivity, excellent proteolytic stability, and desired biological functions. Particularly, anti-Alp2 Fab A4A1 had a binding affinity of 11 nM and inhibition potency of 14 nM, anti-BACE1 IgG B2B2 reduced amyloid beta (Aß40) production by 80% in cellular assays, and IgG L13 inhibited MMP-9 but not MMP-2/-12/-14 and significantly relieved neuropathic pain development in mice.

Candida vaginitis: virulence, host response and vaccine prospects.

Vulvovaginal candidiasis is a common mucosal infection affecting a large proportion of women with some of them affected by recurrent often intractable forms of the disease. Thus, there is an increasing interest in understanding the pathogenesis of this disease. The aim of our work was to characterize, in animal models of vaginal candidiasis, the components of the host-fungus interaction at the mucosal level.The evidence of an immune response in the vaginal compartment was very encouraging to identify the proper targets for new strategies for vaccination or immunotherapy of vaginal candidiasis. Aspartyl-proteinase (Sap2), which is an important immunodominant antigens and virulence factors of C.albicans acting in mucosal infections, was assembled with virosomes and a vaccine PEV7 was obtained. The results obtained in the mouse model and in the clinical trial conducted by Pevion on women have evidenced that the vaccine PEV7, intravaginally administered, has an encouraging therapeutic potential for the treatment of recurrent vulvovaginal candidiasis. This opens the way to a modality for anti-Candida protection at mucosal level.

A novel blood-feeding detoxification pathway in Nippostrongylus brasiliensis L3 reveals a potential checkpoint for arresting hookworm development.

As part of on-going efforts to control hookworm infection, the "human hookworm vaccine initiative" has recognised blood feeding as a feasible therapeutic target for inducing immunity against hookworm infection. To this end, molecular approaches have been used to identify candidate targets, such as Necator americanus (Na) haemoglobinase aspartic protease-1 (APR-1), with immunogenicity profiled in canine and hamster models. We sought to accelerate the immune analysis of these identified therapeutic targets by developing an appropriate mouse model. Here we demonstrate that Nippostrongylus brasiliensis (Nb), a phylogenetically distant strongylid nematode of rodents, begins blood feeding early in its development and that immunisation with Na-APR-1 can block its growth and completion of its life cycle. Furthermore, we identify a new haem detoxification pathway in Nb required for blood feeding that can be blocked by drugs of the quinolone family, reducing both infection burden and the associated anaemia in rodents. Collectively, our findings show that haem metabolism has potential as a checkpoint for interrupting hookworm development in early stages of the hookworm life cycle and that the Nippostrongylus brasiliensis rodent model is relevant for identifying novel therapeutic targets against human hookworm.

Molecular Pathways for Immune Recognition of Preproinsulin Signal Peptide in Type 1 Diabetes.

The signal peptide region of preproinsulin (PPI) contains epitopes targeted by HLA-A-restricted (HLA-A0201, A2402) cytotoxic T cells as part of the pathogenesis of ß-cell destruction in type 1 diabetes. We extended the discovery of the PPI epitope to disease-associated HLA-B*1801 and HLA-B*3906 (risk) and HLA-A*1101 and HLA-B*3801 (protective) alleles, revealing that four of six alleles present epitopes derived from the signal peptide region. During cotranslational translocation of PPI, its signal peptide is cleaved and retained within the endoplasmic reticulum (ER) membrane, implying it is processed for immune recognition outside of the canonical proteasome-directed pathway. Using in vitro translocation assays with specific inhibitors and gene knockout in PPI-expressing target cells, we show that PPI signal peptide antigen processing requires signal peptide peptidase (SPP). The intramembrane protease SPP generates cytoplasm-proximal epitopes, which are transporter associated with antigen processing (TAP), ER-luminal epitopes, which are TAP independent, each presented by different HLA class I molecules and N-terminal trimmed by ER aminopeptidase 1 for optimal presentation. In vivo, TAP expression is significantly upregulated and correlated with HLA class I hyperexpression in insulin-containing islets of patients with type 1 diabetes. Thus, PPI signal peptide epitopes are processed by SPP and loaded for HLA-guided immune recognition via pathways that are enhanced during disease pathogenesis.

ICAM1+ neutrophils promote chronic inflammation via ASPRV1 in B cell-dependent autoimmune encephalomyelitis.

Neutrophils contribute to demyelinating autoimmune diseases, yet their phenotype and functions have been elusive to date. Here, we demonstrate that ICAM1 surface expression distinguishes extra- from intravascular neutrophils in the mouse CNS during experimental autoimmune encephalomyelitis (EAE). Transcriptomic analysis of these 2 subpopulations indicated that neutrophils, once extravasated, acquire macrophage-like properties, including the potential for immunostimulation and MHC class II-mediated antigen presentation. In corroboration, super-resolution (3D stimulated emission-depletion [STED]) microscopy revealed neutrophils forming synapses with T and B cells in situ. Further, neutrophils specifically express the aspartic retroviral-like protease ASPRV1, which increases in the CNS during EAE and severe cases of multiple sclerosis. Without ASPRV1, mice immunized with a new B cell-dependent myelin antigen (but not with the traditional myelin oligodendrocyte glycoprotein peptide) develop a chronic phase of EAE that is less severe and even completely fades in many individuals. Therefore, ICAM1+ macrophage-like neutrophils can play both shared and nonredundant roles in autoimmune demyelination, among them perpetuating inflammation via ASPRV1.

Widespread brain distribution and activity following i.c.v. infusion of anti-ß-secretase (BACE1) in nonhuman primates.

BACKGROUND AND PURPOSE: The potential for therapeutic antibody treatment of neurological diseases is limited by poor penetration across the blood-brain barrier. I.c.v. delivery is a promising route to the brain; however, it is unclear how efficiently antibodies delivered i.c.v. penetrate the cerebrospinal spinal fluid (CSF)-brain barrier and distribute throughout the brain parenchyma. EXPERIMENTAL APPROACH: We evaluated the pharmacokinetics and pharmacodynamics of an inhibitory monoclonal antibody against ß-secretase 1 (anti-BACE1) following continuous infusion into the left lateral ventricle of healthy adult cynomolgus monkeys. KEY RESULTS: Animals infused with anti-BACE1 i.c.v. showed a robust and sustained reduction (~70%) of CSF amyloid-ß (Aß) peptides. Antibody distribution was near uniform across the brain parenchyma, ranging from 20 to 40 nM, resulting in a ~50% reduction of Aß in the cortical parenchyma. In contrast, animals administered anti-BACE1 i.v. showed no significant change in CSF or cortical Aß levels and had a low (~0.6 nM) antibody concentration in the brain. CONCLUSION AND IMPLICATIONS: I.c.v. administration of anti-BACE1 resulted in enhanced BACE1 target engagement and inhibition, with a corresponding dramatic reduction in CNS Aß concentrations, due to enhanced brain exposure to antibody.

Immune response in bovine neosporosis: Protection or contribution to the pathogenesis of abortion.

Neospora caninum is a protozoan parasite with a preference for cattle and dogs as hosts. When N. caninum infection occurs in cattle it induces abortion, bovine neosporosis being a main cause of abortion worldwide. In dairy cattle, the economic burden of neosporosis-associated abortion is so great that it might results in closure of a farm. However, not all infected cows abort and it is not yet understood why this occurs. At present there is no effective treatment or vaccine. This review provides insights on how immune response against the parasite determines protection or contribution to abortion. Aspects on markers of risk of abortion are also discussed. Humoral immune responses are not protective against N. caninum but seropositivity and antibody level can be good markers for a diagnosis of bovine neosporosis and its associated abortion risk. In addition, humoral mechanisms against N. caninum infection and abortion differ in pure-breed and cross-breed pregnant dairy and beef cattle. Concentrations of Pregnancy Associated glycoprotein -2 (PAG-2) can also be used to predict abortion. A partially protective immune response encompasses increased IFN-γ expression, which has to be counterbalanced by other cytokines such as IL-12 and IL-10, especially towards the end of pregnancy. Although IFN-γ is required to limit parasite proliferation a critical threshold of the IFN-γ response is also required to limit adverse effects on pregnancy. In clinical terms, it may be stated that IFN-γ production and cross-breed pregnancy can protect Neospora-infected dairy cows against abortion.

N-glycan in cockroach allergen regulates human basophil function.

INTRODUCTION: Cockroach allergen exposure elicits cockroach sensitization and poses an increased risk for asthma. However, the major components in cockroach allergen and the mechanisms underlying the induction of cockroach allergen-induced allergy and asthma remain largely elusive. We sought to examine the role of cockroach-associated glycan in regulating human basophil function. METHODS: N-linked glycans from naturally purified cockroach allergen Bla g 2 were characterized by MALDI-TOF mass spectrometry. Binding of cockroach allergen to serum IgE from cockroach allergic subjects was determined by solid-phase binding immunoassays. Role of cockroach associated glycan in histamine release and IL-4 production from human basophils was examined. Expression of C-type lectin receptors (CLRs) and their role in mediating glycan-uptake in the basophils was also investigated. RESULTS: MALDI-TOF mass spectrometric analysis of N-glycan from Bla g 2 showed complex hybrid-types of glycans that terminated with mannose, galactose, and/or N-acetyl glucosamine (GlcNAc). Deglycosylated Bla g 2 showed reduced binding to IgE and was less capable of inducing histamine release from human basophils. In contrast, N-glycan derived from Bla g 2 significantly inhibited histamine release and IL-4 production from basophils passively sensitized with serum from cockroach allergic subjects. An analysis of CLRs revealed the expression of DC-SIGN and DCIR, but not MRC1 and dectin-1, in human basophils. Neutralizing antibody to DCIR, but not DC-SIGN, significantly inhibited Bla g 2 uptake by human basophils. A dose-dependent bindings of cockroach allergen to DCIR was also observed. CONCLUSIONS: These observations indicate a previously unrecognized role for cockroach allergen-associated glycans in allergen-induced immune reactions, and DCIR may play a role in mediating the regulation of glycan on basophil function.

Perinatal Bacterial Exposure Contributes to IL-13 Aeroallergen Response.

There is a high prevalence of aeroallergen sensitivity in asthmatic populations, and seroreactivity to aeroallergens early in infancy is associated with increased risk of developing asthma later in life. In addition to allergen sensitivity, asthma development has been associated with differential microbial exposure and infection in early life. We have previously shown that cord blood mononuclear cells respond to common aeroallergens (i.e., house dust mite [Der f1] and cockroach [Bla g2]) as assayed by lymphoproliferation and cytokine (IL-13 and IFN-γ) production. We hypothesized that there is a relationship between perinatal microbial exposure and response to specific aeroallergens. To test this hypothesis, we isolated DNA from cord blood serum samples with known lymphoproliferative and cytokine responses to Bla g2 and Der f1. Bacterial 16S ribosomal DNA amplicon libraries were generated and analyzed using high throughput sequencing of cord blood serum samples. In our analysis, we identified major compositional differences, including diversity and abundance of specific taxa, between groups whose IL-13 response to Der f1 and Bla g2 differed. We demonstrate a strong association between the ratio of Acinetobacter to Proteobacteria and IL-13 production and the probability of IL-13 production after allergen exposure. IL-13 concentrations in serum were also significantly correlated with the diversity of bacterial DNA. Together, these results underscore the relationship between immune responses to allergens and bacterial exposure during perinatal development.

Pax8, Napsin A, and CD10 as Immunohistochemical Markers of Canine Renal Cell Carcinoma.

Pax8, napsin A, and CD10 are useful immunohistochemical markers of human renal cell carcinoma (RCC); however, their diagnostic utility in canine RCC is unclear. Forty formalin-fixed paraffin-embedded renal cell carcinomas from dogs (15 papillary, 12 solid, and 13 tubular) and 10 metastases were evaluated for expression of Pax8, napsin A, and CD10. Thirty-nine (98%), 24 (60%), and 19 (50%) tumors expressed Pax8 (nuclear labeling), napsin A (cytoplasmic labeling), and CD10 (cytoplasmic and membranous labeling), respectively. Pax8 was expressed in 92% of solid, 100% of papillary, and 100% of tubular tumors. Napsin A was expressed in 58% of solid, 60% of papillary, and 62% of tubular RCC. CD10 was expressed in 33% of solid, 47% of papillary, and 62% of tubular RCC. Pax8 was expressed in 80% of the metastatic tumors, napsin A in 60%, and CD10 in 50%. Additionally, Pax8 immunoreactivity was stronger overall than that of napsin A or CD10. In summary, Pax8 is a more sensitive marker than napsin A or CD10 for primary and metastatic canine RCC; its nuclear and more intense reactivity also makes it easier to interpret. Tubular and papillary RCCs were more likely than solid RCC to express all 3 markers. These findings highlight the utility of Pax8 as an immunohistochemical marker in diagnosing all major subtypes of canine primary and metastatic renal cell carcinoma.

Dual Signal Amplification Electrochemical Biosensor for Monitoring the Activity and Inhibition of the Alzheimer's Related Protease ß-Secretase.

The protease BACE1 (the ß-site amyloid precursor protein cleaving enzyme 1) catalyzes the first step in the synthesis of ß-amyloids (Aß), peptides that accumulate in the brain in Alzheimer's disease (AD). Measurement of BACE1 activity is important for the development of BACE1 inhibitors to slow or stop AD. To measure BACE1 cleavage of the electrode-immobilized substrate peptide, we developed a redox-generating hydroxyapatite (HAP) probe which generates electrochemical current by reaction of the nanoparticle with molybdate (MoO42-). The probe combines alkaline phosphatase (ALP) for dual signal amplification and Aß antibody to bind the probe to the immobilized peptide substrate on the surface of the electrode. We measured the activity of BACE1 at concentrations ranging from 0.25 to 100 U/mL. The use of the dual-signal HAP-ALP probe increased the signal by an order of magnitude compared to HAP-only probe, enabling detection limits as low as 0.1 U/mL. To measure the inhibition of BACE1 activity, the BACE1 inhibitor OM99-2 was added to 25 U/mL of BACE1 in concentrations ranging from 5 to 150 nM. The observed detection limit of inhibition is 10 nM of OM99-2. These results demonstrate the capabilities of this novel biosensor to measure BACE1 activity and inhibitors of BACE1 activity. To the best of our knowledge this is the first report that reaction of HAP nanoparticles with molybdate can generate electrochemical current. This dual signal amplification strategy can be extended to other electrochemical assays and adapted for wide applications.

Novel nanoscale bacteriophage-based single-domain antibodies for the therapy of systemic infection caused by Candida albicans.

Candida albicans (C. albicans) is an important human commensal and opportunistic fungal pathogen. Secreted aspartyl proteinases (Saps) are a major virulence trait of C. albicans, and among these proteases Sap2 has the highest expression levels. It is possible that antibodies against Sap2 could provide an antifungal effect. In this study, two phages displaying anti-rSap2 single chain variable fragments (scFvs) were screened from human single fold scFv libraries, and their potential therapeutic roles were evaluated using a murine model infected by C. albicans. The in vivo efficacies were assessed by mortality rates, fungal burden and histological examination. Overall survival rates were significantly increased while the colony counts and infectious foci were significantly decreased after treatment with the scFv-phages relative to the control groups. In order to investigate the immune response provoked by scFv-phages, three kinds of cytokines (Th1, Th2 and Th17 types) were measured and a clear immune response was observed. These findings suggest that anti-rSap2 scFv-phages have potential in the therapy of systemic infection caused by C. albicans.

Immunoproteomic Analysis of Antibody Responses to Extracellular Proteins of Candida albicans Revealing the Importance of Glycosylation for Antigen Recognition.

During infection, the human pathogenic fungus Candida albicans undergoes a yeast-to-hypha transition, secretes numerous proteins for invasion of host tissues, and modulates the host's immune response. Little is known about the interplay of C. albicans secreted proteins and the host adaptive immune system. Here, we applied a combined 2D gel- and LC-MS/MS-based approach for the characterization of C. albicans extracellular proteins during the yeast-to-hypha transition, which led to a comprehensive C. albicans secretome map. The serological responses to C. albicans extracellular proteins were investigated by a 2D-immunoblotting approach combined with MS for protein identification. On the basis of the screening of sera from candidemia and three groups of noncandidemia patients, a core set of 19 immunodominant antibodies against secreted proteins of C. albicans was identified, seven of which represent potential diagnostic markers for candidemia (Xog1, Lip4, Asc1, Met6, Tsa1, Tpi1, and Prx1). Intriguingly, some secreted, strongly glycosylated protein antigens showed high cross-reactivity with sera from noncandidemia control groups. Enzymatic deglycosylation of proteins secreted from hyphae significantly impaired sera antibody recognition. Furthermore, deglycosylation of the recombinantly produced, secreted aspartyl protease Sap6 confirmed a significant contribution of glycan epitopes to the recognition of Sap6 by antibodies in patient's sera.

Characterization of a sensitive mouse Aß40 PD biomarker assay for Alzheimer's disease drug development in wild-type mice.

AIM: Transgenic mice that overexpress human amyloid precursor protein with Swedish or London (APPswe or APPlon) mutations have been widely used for preclinical Alzheimer's disease (AD) drug development. AD patients, however, rarely possess these mutations or overexpress APP. RESULTS: We developed a sensitive ELISA that specifically and accurately measures low levels of endogenous Aß40 in mouse plasma, brain and CSF. In wild-type mice treated with a bispecific anti-TfR/BACE1 antibody, significant Aß reductions were observed in the periphery and the brain. APPlon transgenic mice showed a slightly less reduction, whereas APPswe mice did not have any decrease. CONCLUSION: This sensitive and well-characterized mouse Aß40 assay enables the use of wild-type mice for preclinical PK/PD and efficacy studies of potential AD therapeutics.

Plasmodium falciparum ookinete expression of plasmepsin VII and plasmepsin X.

BACKGROUND: Plasmodium invasion of the mosquito midgut is a population bottleneck in the parasite lifecycle. Interference with molecular mechanisms by which the ookinete invades the mosquito midgut is one potential approach to developing malaria transmission-blocking strategies. Plasmodium aspartic proteases are one such class of potential targets: plasmepsin IV (known to be present in the asexual stage food vacuole) was previously shown to be involved in Plasmodium gallinaceum infection of the mosquito midgut, and plasmepsins VII and plasmepsin X (not known to be present in the asexual stage food vacuole) are upregulated in Plasmodium falciparum mosquito stages. These (and other) parasite-derived enzymes that play essential roles during ookinete midgut invasion are prime candidates for transmission-blocking vaccines. METHODS: Reverse transcriptase PCR (RT-PCR) was used to determine timing of P. falciparum plasmepsin VII (PfPM VII) and plasmepsin X (PfPM X) mRNA transcripts in parasite mosquito midgut stages. Protein expression was confirmed by western immunoblot and immunofluorescence assays (IFA) using anti-peptide monoclonal antibodies (mAbs) against immunogenic regions of PfPM VII and PfPM X. These antibodies were also used in standard membrane feeding assays (SMFA) to determine whether inhibition of these proteases would affect parasite transmission to mosquitoes. The Mann-Whitney U test was used to analyse mosquito transmission assay results. RESULTS: RT-PCR, western immunoblot and immunofluorescence assay confirmed expression of PfPM VII and PfPM X in mosquito stages. Whereas PfPM VII was expressed in zygotes and ookinetes, PfPM X was expressed in gametes, zygotes, and ookinetes. Antibodies against PfPM VII and PfPM X decreased P. falciparum invasion of the mosquito midgut when used at high concentrations, indicating that these proteases play a role in Plasmodium mosquito midgut invasion. Failure to generate genetic knockouts of these genes limited determination of the precise role of these proteases in parasite transmission but suggests that they are essential during the intraerythrocytic life cycle. CONCLUSIONS: PfPM VII and PfPM X are present in the mosquito-infective stages of P. falciparum. Standard membrane feeding assays demonstrate that antibodies against these proteins reduce the infectivity of P. falciparum for mosquitoes, suggesting their viability as transmission-blocking vaccine candidates. Further study of the role of these plasmepsins in P. falciparum biology is warranted.