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Immunogenicity in mice and non-human primates of the Group A Streptococcal J8 peptide vaccine candidate conjugated to CRM197. - Human vaccines & immunotherapeutics
Vaccine development for Group A streptococcal (GAS) infection has been extensively focused on the N-terminal hypervariable or the C-terminal conserved regions of the M protein, a major virulence factor of GAS. We evaluated the immunogenicity and functional activity of the conserved C-terminal peptide vaccine candidate, J8, conjugated to CRM197, in two mouse strains: C3H (H2(k)) and Balb/c (H2(d)), and in rhesus macaques. Mice were immunized with J8-CRM197 formulated with Amorphous Aluminum Hydroxyphosphate Sulfate Adjuvant (AAHSA), and non-human primates were immunized with J8-CRM197 formulated with AAHSA, ISCOMATRIX (TM) adjuvant, or AAHSA/ISCOMATRIX adjuvant. J8-CRM197 was immunogenic in mice from both H2(k) and H2(d) backgrounds, and the antibodies generated bound to the surface of four different GAS serotypes and had functional bacterial opsonic activity. Mice immunized with J8-CRM197/AAHSA demonstrated varying degrees of protection from lethal challenge. We also demonstrated that J8-CRM197 is immunogenic in non-human primates. Our data confirm the utility of J8 as a potential GAS vaccine candidate and demonstrate that CRM197 is an acceptable protein carrier for this peptide.
Sporozoite neutralizing antibodies elicited in mice and rhesus macaques immunized with a Plasmodium falciparum repeat peptide conjugated to meningococcal outer membrane protein complex. - Frontiers in cellular and infection microbiology
Antibodies that neutralize infectivity of malaria sporozoites target the central repeat region of the circumsporozoite (CS) protein, which in Plasmodium falciparum is comprised primarily of 30-40 tandem NANP tetramer repeats. We evaluated immunogenicity of an alum-adsorbed (NANP)(6) peptide conjugated to an outer membrane protein complex (OMPC) derived from Neisseria meningitidis, a carrier protein used in a licensed Haemophilus influenzae pediatric vaccine. Mice immunized with (NANP)(6)-OMPC adsorbed to Merck's alum adjuvant (MAA), with or without IscomatrixÂ® as co-adjuvant, developed high levels of anti-repeat peptide antibody that inhibited in vitro invasion of human hepatoma cells by transgenic P. berghei sporozoites that express P. falciparum CS repeats (PfPb). Inhibition of sporozoite invasion in vitro correlated with in vivo resistance to challenge by the bites of PfPb-infected mosquitoes. Challenged mice had >90% reduction of hepatic stage parasites as measured by real-time PCR, and either sterile immunity, i.e., no detectable blood stage parasites, or delayed prepatent periods which indicate neutralization of a majority, but not all, sporozoites. Rhesus macaques immunized with two doses of (NANP)(6)-OMPC/MAA formulated with IscomatrixÂ® developed anti-repeat antibodies that persisted for ~2 years. A third dose of (NANP)(6)-OMPC/MAA+ IscomatrixÂ® at that time elicited strong anamnestic antibody responses. Rhesus macaque immune sera obtained post second and third dose of vaccine displayed high levels of sporozoite neutralizing activity in vitro that correlated with presence of high anti-repeat antibody titers. These preclinical studies in mice of different MHC haplotypes and a non-human primate support use of CS peptide-OMPC conjugates as a highly immunogenic platform to evaluate CS protective epitopes. Potential pre-erythrocytic vaccines can be combined with sexual blood stage vaccines as a multi-antigen malaria vaccine to block invasion and transmission of Plasmodium parasites.
Efficacy of a capsule conjugate vaccine against inhalational anthrax in rabbits and monkeys. - Vaccine
Bacillus anthracis, the causative agent of anthrax, is recognized as one of the most serious bioterrorism threats. The current human vaccines are based on the protective antigen component of the anthrax toxins. Concern about possible vaccine resistant strains and reliance on a single antigen has prompted the search for additional immunogens. Bacterial capsules, as surface-expressed virulence factors, are well-established components of several licensed vaccines. In a previous study we showed that an anthrax vaccine consisting of the B. anthracis poly-Î³-D-glutamic acid capsule covalently conjugated to the outer membrane protein complex of Neisseria meningitidis serotype B protected mice against parenteral B. anthracis challenge. Here we tested this vaccine in rabbits and monkeys against an aerosol spore challenge. The vaccine induced anti-capsule antibody responses in both species, measured by ELISA and a macrophage opsono-adherence assay. While rabbits were not protected against a high aerosol challenge dose, significant protection was observed in monkeys receiving the capsule conjugate vaccine. The results confirm that the capsule is a protective immunogen against anthrax, being the first non-toxin antigen shown to be efficacious in monkeys and suggest that addition of capsule may broaden and enhance the protection afforded by protective antigen-based vaccines.Published by Elsevier Ltd.
High throughput monitoring of amyloid-Î²(42) assembly into soluble oligomers achieved by sensitive conformation state-dependent immunoassays. - Journal of Alzheimer's disease : JAD
Accumulation of small soluble assemblies of amyloid-Î² (AÎ²)(42) in the brain is thought to play a key role in the pathogenesis of Alzheimer's disease. As a result, there has been much interest in finding small molecules that inhibit the formation of synaptotoxic AÎ²(42) oligomers that necessitates sensitive methods for detecting the initial steps in the oligomerization of AÎ²(42). Modeling suggests that oligomerized AÎ²(42) adopts a conformation in which the C-terminus is embedded in the center, whereas the N-terminus is exposed at the periphery of the oligomer. Here we report that an inverse change in AÎ²(42) C-terminal and N-terminal epitope accessibility provides the basis of a sensitive method for assessing early steps in AÎ²(42) oligomerization. Using ELISA and AlphaLISA, we found that AÎ²(42) C-terminal immunoreactivity decreased in a time- and concentration-dependent manner under conditions favoring oligomerization. This reduction was accompanied by an increase in the N-terminal immunoreactivity, suggesting that assemblies with multiple exposed N-terminal epitopes were detected. Importantly the assay generates a robust window between monomers and oligomers at as low as 1 nM AÎ²(42). Using this assay, known oligomerization inhibitors produced a dose-dependent unmasking of the AÎ²(42) C-terminal epitope. After automation, the assay proved to be highly reproducible and effective for high throughput screening of small molecules that inhibit AÎ²(42) oligomerization.
Small molecule mimetics of an HIV-1 gp41 fusion intermediate as vaccine leads. - The Journal of biological chemistry
We describe here a novel platform technology for the discovery of small molecule mimetics of conformational epitopes on protein antigens. As a model system, we selected mimetics of a conserved hydrophobic pocket within the N-heptad repeat region of the HIV-1 envelope protein, gp41. The human monoclonal antibody, D5, binds to this target and exhibits broadly neutralizing activity against HIV-1. We exploited the antigen-binding property of D5 to select complementary small molecules using a high throughput screen of a diverse chemical collection. The resulting small molecule leads were rendered immunogenic by linking them to a carrier protein and were shown to elicit N-heptad repeat-binding antibodies in a fraction of immunized mice. Plasma from HIV-1-infected subjects shown previously to contain broadly neutralizing antibodies was found to contain antibodies capable of binding to haptens represented in the benzylpiperidine leads identified as a result of the high throughput screen, further validating these molecules as vaccine leads. Our results suggest a new paradigm for vaccine discovery using a medicinal chemistry approach to identify lead molecules that, when optimized, could become vaccine candidates for infectious diseases that have been refractory to conventional vaccine development.
Design of an HA2-based Escherichia coli expressed influenza immunogen that protects mice from pathogenic challenge. - Proceedings of the National Academy of Sciences of the United States of America
Influenza HA is the primary target of neutralizing antibodies during infection, and its sequence undergoes genetic drift and shift in response to immune pressure. The receptor binding HA1 subunit of HA shows much higher sequence variability relative to the metastable, fusion-active HA2 subunit, presumably because neutralizing antibodies are primarily targeted against the former in natural infection. We have designed an HA2-based immunogen using a protein minimization approach that incorporates designed mutations to destabilize the low pH conformation of HA2. The resulting construct (HA6) was expressed in Escherichia coli and refolded from inclusion bodies. Biophysical studies and mutational analysis of the protein indicate that it is folded into the desired neutral pH conformation competent to bind the broadly neutralizing HA2 directed monoclonal 12D1, not the low pH conformation observed in previous studies. HA6 was highly immunogenic in mice and the mice were protected against lethal challenge by the homologous A/HK/68 mouse-adapted virus. An HA6-like construct from another H3 strain (A/Phil/2/82) also protected mice against A/HK/68 challenge. Regions included in HA6 are highly conserved within a subtype and are fairly well conserved within a clade. Targeting the highly conserved HA2 subunit with a bacterially produced immunogen is a vaccine strategy that may aid in pandemic preparedness.
Vaccination with peptide mimetics of the gp41 prehairpin fusion intermediate yields neutralizing antisera against HIV-1 isolates. - Proceedings of the National Academy of Sciences of the United States of America
Eliciting a broadly neutralizing polyclonal antibody response against HIV-1 remains a major challenge. One approach to vaccine development is prevention of HIV-1 entry into cells by blocking the fusion of viral and cell membranes. More specifically, our goal is to elicit neutralizing antibodies that target a transient viral entry intermediate (the prehairpin intermediate) formed by the HIV-1 gp41 protein. Because this intermediate is transient, a stable mimetic is required to elicit an immune response. Previously, a series of engineered peptides was used to select a mAb (denoted D5) that binds to the surface of the gp41 prehairpin intermediate, as demonstrated by x-ray crystallographic studies. D5 inhibits the replication of HIV-1 clinical isolates, providing proof-of-principle for this vaccine approach. Here, we describe a series of peptide mimetics of the gp41 prehairpin intermediate designed to permit a systematic analysis of the immune response generated in animals. To improve the chances of detecting weak neutralizing polyclonal responses, two strategies were employed in the initial screening: use of a neutralization-hypersensitive virus and concentration of the IgG fraction from immunized animal sera. This allowed incremental improvements through iterative cycles of design, which led to vaccine candidates capable of generating a polyclonal antibody response, detectable in unfractionated sera, that neutralize tier 1 HIV-1 and simian HIV primary isolates in vitro. Our findings serve as a starting point for the design of more potent immunogens to elicit a broadly neutralizing response against the gp41 prehairpin intermediate.
Oligomers of beta-amyloid are sequestered into and seed new plaques in the brains of an AD mouse model. - Experimental neurology
Amyloid plaque deposition in the brain is a hallmark of Alzheimer's disease, but recent evidence indicates that the disease may be primarily caused by soluble amyloid-beta (1-42) (Abeta) oligomers or Abeta-derived diffusible ligands (ADDLs). ADDLs induce cognitive deficits in animal models and are thought to assemble in vitro by a mechanism apart from plaque formation. To investigate the in vivo relationship of ADDLs and plaques, biotin-labeled ADDLs (bADDLs) or amylin oligomers (bAMs) were injected into the hippocampus of hAPP overexpressing mice. The brains were collected 1 or 5 weeks after the last treatment and were processed for immunohistochemistry. Staining of tissue 1 week post-treatment showed bADDLs had diffused throughout the tissue and incorporated into plaques. Additionally, small deposits of thioflavin S-negative bADDLs were observed. At 5 weeks post-treatment, thioflavin S-positive material continued to accumulate around plaques containing bADDLs. Thioflavin S-positive material also accrued around bADDL deposits, implying that bADDLs were capable of seeding new plaques. In contrast, bAMs cleared from the brain and did not accumulate in plaques. Together, these data indicate that ADDLs are able to contribute to in vivo plaque formation in a peptide-specific manner.Copyright (c) 2009 Elsevier Inc. All rights reserved.
Staphylococcus aureus capsule type 8 antibodies provide inconsistent efficacy in murine models of staphylococcal infection. - Human vaccines
Staphylococcus aureus is a clinically important capsule-forming bacterium. The capsule polysaccharide (CPs) occurs as different chemical structures depending on the serotype of the organism, but one form, capsular polysaccharide type 8 (CPs8) found in clinical isolates, is largely unstudied. The potential of CPs8 as a vaccine target was evaluated using two approaches. The first approach used a conjugate vaccine, made by chemically linking purified CPs8 to the outer membrane protein complex of N. meningitidis serotype B (OMPC). In efficacy studies, the CPs8-OMPC conjugate vaccine was immunogenic in Balb/c mice, however the immune response gave no protection from death after a lethal intravenous (IV) challenge with S. aureus Becker. In the second approach, two monoclonal antibodies were produced against CPs8 (mAbs 8E8 and 1C10). These were found to have functional activity in an opsonophagocytic killing assay (OPA), and provided protection from a lethal challenge when bacteria were pre-opsonized ex vivo before intra-peritoneal (IP) challenge. However, mAb 8E8 was not efficacious in the lethal challenge model, in which antibodies were passively transferred to the peritoneum and the animals were infected via the tail vein 18-24 h later. Additionally, the monoclonal antibodies did not opsonize capsule-expressing S. aureus Becker obtained from in vivo growth conditions. These results indicated that functional capsule antibodies may not be sufficient for protection from S. aureus under all in vivo conditions.
Amino acid analysis of peptide loading ratios in conjugate vaccines: a comparison of direct electrochemical detection and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate pre-column derivatization methods. - Bioconjugate chemistry
Amino acid analysis using direct electrochemical detection was compared with precolumn fluorescent derivatization using 6-aminoquinolyl- N-hydroxysuccinimidyl carbamate (AQC) for evaluation of the degree of covalent coupling of peptides to a carrier-protein complex derived from the bacteria Neisseria meningitidis. AQC derivatization was found to give superior sensitivity compared to electrochemical detection, with less interference from sample components such as carbohydrates or buffer salts. Hydrolysis time and temperature were optimized for maximal recoveries of the marker amino acid 6-aminohexanoic acid (epsilon-Ahx) and the unique amino acids S-dicarboxyethyl cysteine (SDCEC) and S-carboxymethyl homocysteine (SCHMC), which are generated upon the hydrolysis of the covalent linkage between the peptide and the carrier protein. Quantitation of these amino acids enabled the determination of the ratio of peptide to protein in the conjugate samples.
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