Stabilized trimeric peptide immunogens of the complete HIV-1 gp41 N-heptad repeat and their use as HIV-1 vaccine candidates.

Efforts to develop an HIV-1 vaccine include those focusing on conserved structural elements as the target of broadly neutralizing monoclonal antibodies. MAb D5 binds to a highly conserved hydrophobic pocket on the gp41 N-heptad repeat (NHR) coiled coil and neutralizes through prevention of viral fusion and entry. Assessment of 17-mer and 36-mer NHR peptides presenting the D5 epitope in rodent immunogenicity studies showed that the longer peptide elicited higher titers of neutralizing antibodies, suggesting that neutralizing epitopes outside of the D5 pocket may exist. Although the magnitude and breadth of neutralization elicited by NHR-targeting antigens are lower than that observed for antibodies directed to other epitopes on the envelope glycoprotein complex, it has been shown that NHR-directed antibodies are potentiated in TZM-bl cells containing the FcγRI receptor. Herein, we report the design and evaluation of covalently stabilized trimeric 51-mer peptides encompassing the complete gp41 NHR. We demonstrate that these peptide trimers function as effective antiviral entry inhibitors and retain the ability to present the D5 epitope. We further demonstrate in rodent and nonhuman primate immunization studies that our 51-mer constructs elicit a broader repertoire of neutralizing antibody and improved cross-clade neutralization of primary HIV-1 isolates relative to 17-mer and 36-mer NHR peptides in A3R5 and FcγR1-enhanced TZM-bl assays. These results demonstrate that sensitive neutralization assays can be used for structural enhancement of moderately potent neutralizing epitopes. Finally, we present expanded trimeric peptide designs which include unique low-molecular-weight scaffolds that provide versatility in our immunogen presentation strategy.

Orthogonal Comparison of Analytical Methods by Theoretical Reconstruction from Bottom-up Assay Data.

In the never-ending endeavor to produce stable and efficacious protein therapeutics, biopharmaceutical companies often employ numerous analytical techniques to characterize and quantify a drug candidate's stability. Mass spectrometry, due to the information-rich data it produces, is commonly used in its numerous configurations to ascertain chemical and structural stability. At issue is the comparison of the various configurations utilized, that is, comparing bottom-up methods such as proteolytic digest followed by reversed phase LC-MS with intact LC-MS methods. Similar issues also arise when using capillary isoelectric focusing to see how charge variants change over time, that is, monitoring the progression of charge altering modifications like deamidation. To this end, site-specific degradations as quantified from bottom-up methods like peptide mapping can be used to build reconstructions of both theoretical intact mass spectra as well as theoretical electropherograms. The result can then be superimposed over the experimental data to qualitatively, and perhaps quantitatively, evaluate differences. In theory, if both experimental bottom-up data and intact data are accurate, the theoretical reconstruction produced from the bottom-up data should perfectly overlay with that of the experimental data. Valuable secondary information can also be ascertained from reconstructions, such as whether modifications are stochastic, as well as a detailed view of all possible combinations of modifications and their quantities used in the reconstruction. This comparison is also useful in determining unknown mass differences in deconvoluted intact protein spectra that may be a result of multiple modifications in combination. The comparison of data from alternate sources provides a holistic and more comprehensive view of the molecule under study.

Surfactant Impact on Interfacial Protein Aggregation and Utilization of Surface Tension to Predict Surfactant Requirements for Biological Formulations.

Biological drug products are formulated with excipients to maintain stability over the shelf life of the product. Surfactants are added to the drug product to stabilize air-water interfaces known to induce protein aggregation. Early formulation development is focused on maintaining protein conformation and colloidal stability over the course of the drug product shelf life but rarely considers stability through dose preparation and administration. Specifically, intravenous (IV) bag preparation exposes the therapeutic protein to a different solution environment concurrently diluting the stabilizing excipients that had been added to the drug product formulation. Mixing in IV bags can generate dynamic changes in the air-water interfacial area known to cause protein aggregation if not sufficiently protected. Therefore, understanding the surfactant requirements for drug product end-to-end stability in early formulation development provides critical information for a right-first-time approach to drug product formulation and robust clinical preparation. The goal of these studies was to understand if interfacial properties of proteins could predict surfactant formulation requirements for end-to-end stability. Specifically, the interfacial properties of five proteins were measured in 0.9% saline and 5% dextrose. Furthermore, shaking studies were conducted to identify the minimum surfactant concentration required to prevent subvisible and visible particle formulation in each diluent. The impact of surfactant type and concentration on particle generation and size was explored. A mathematical model was generated to predict the minimum surfactant concentration required to prevent interface-driven aggregation in each diluent based on the change in surface pressure upon exposure of the protein to the interface. The model was tested under typical IV-preparation conditions with experimental output closely matching the model prediction. By employing this model and better understanding the role of surfactants in interfacial stability, drug product development can generate robust end-to-end large molecule formulations across shelf life, dose preparation, and administration.

Spectroscopic analysis of chlamydial major outer membrane protein in support of structure elucidation.

Chlamydial major outer membrane protein (MOMP) is the major protein constituent of the bacterial pathogen Chlamydia trachomatis. Chlamydia trachomatis Serovars D-K are the leading cause of genital tract infections which can lead to infertility or ectopic pregnancies. A vaccine against Chlamydia is highly desirable but currently not available. MOMP accounts for ~ 60% of the chlamydial protein mass and is considered to be one of the lead vaccine candidates against C. trachomatis. We report on the spectroscopic analysis of C. trachomatis native MOMP Serovars D, E, F, and J as well as C. muridarum MOMP by size exclusion chromatography multi angle light scattering (SEC MALS), circular dichroism (CD) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). MOMP was purified from the native bacterium grown in either adherent HeLa cells or in different suspension cell lines. Our results confirm that MOMP forms homo-trimers in detergent micelles. The secondary structure composition of C. trachomatis MOMP was conserved across serovars, but different from composition of C. muridarum MOMP with a 13% (CD) to 18% (ATR-FTIR) reduction in ß-sheet conformation for C. trachomatis MOMP. When Serovar E MOMP was isolated from suspension cell lines the α-helix content increased by 7% (CD) to 13% (ATIR-FTIR). Maintenance of a native-like tertiary and quaternary structure in subunit vaccines is important for the generation of protective antibodies. This biophysical characterization of MOMP presented here serves, in the absence of functional assays, as a method for monitoring the structural integrity of MOMP.

Detection and Measurement of Methionine Oxidation in Proteins.

Methionine oxidation is a prevalent modification found in proteins both in biological settings and in the manufacturing of biotherapeutic molecules. In cells, the oxidation of specific methionine sites can modulate protein function or promote interactions that trigger signaling pathways. In biotherapeutic development, the formation of oxidative species could be detrimental to the efficacy or safety of the drug product. Thus, methionine oxidation is a critical quality attribute that needs to be monitored throughout development. Here we describe a method using LC/MS/MS to identify site-specific methionine modifications in proteins. Antibodies are stressed with hydrogen peroxide, and the level of Met oxidation is compared to that of reference molecules. The protocols presented here are not specific to methionine and can be used more generally to identify other PTM risk sites in molecules after various types of treatments. © 2017 by John Wiley & Sons, Inc.

Protection of rhesus macaques against inhalational anthrax with a Bacillus anthracis capsule conjugate vaccine.

The efficacy of currently licensed anthrax vaccines is largely attributable to a single Bacillus anthracis immunogen, protective antigen. To broaden protection against possible strains resistant to protective antigen-based vaccines, we previously developed a vaccine in which the anthrax polyglutamic acid capsule was covalently conjugated to the outer membrane protein complex of Neisseria meningitidis serotype B and demonstrated that two doses of 2.5µg of this vaccine conferred partial protection of rhesus macaques against inhalational anthrax . Here, we demonstrate complete protection of rhesus macaques against inhalational anthrax with a higher 50µg dose of the same capsule conjugate vaccine. These results indicate that B. anthracis capsule is a highly effective vaccine component that should be considered for incorporation in future generation anthrax vaccines.

Immunogenicity in mice and non-human primates of the Group A Streptococcal J8 peptide vaccine candidate conjugated to CRM197.

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.

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 Iscomatrix® 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 Iscomatrix® developed anti-repeat antibodies that persisted for ~2 years. A third dose of (NANP)(6)-OMPC/MAA+ Iscomatrix® 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.

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-γ-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.

High throughput monitoring of amyloid-ß(42) assembly into soluble oligomers achieved by sensitive conformation state-dependent immunoassays.

Accumulation of small soluble assemblies of amyloid-ß (Aß)(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 Aß(42) oligomers that necessitates sensitive methods for detecting the initial steps in the oligomerization of Aß(42). Modeling suggests that oligomerized Aß(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 Aß(42) C-terminal and N-terminal epitope accessibility provides the basis of a sensitive method for assessing early steps in Aß(42) oligomerization. Using ELISA and AlphaLISA, we found that Aß(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 Aß(42). Using this assay, known oligomerization inhibitors produced a dose-dependent unmasking of the Aß(42) C-terminal epitope. After automation, the assay proved to be highly reproducible and effective for high throughput screening of small molecules that inhibit Aß(42) oligomerization.

Small molecule mimetics of an HIV-1 gp41 fusion intermediate as vaccine leads.

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.

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.

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.

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.

Staphylococcus aureus capsule type 8 antibodies provide inconsistent efficacy in murine models of staphylococcal infection.

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.

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.

Solution state characterization of amyloid beta-derived diffusible ligands.

A growing body of evidence suggests that soluble oligomeric forms of the amyloid beta peptide known as amyloid-derived diffusible ligands (ADDLs) are the toxic species responsible for neurodegeneration associated with Alzheimer's disease. Accurate biophysical characterization of ADDL preparations is hampered by the peptide's strong tendency to self-associate and the effect of factors such as ionic strength, temperature, and pH on its behavior. In addition, amyloid peptides are known to interact with common laboratory excipients, specifically detergents, further complicating the results from standard analytical methods such as denaturing polyacrylamide gel electrophoresis. We have studied the solution behavior of various amyloid peptide preparations using analytical ultracentrifugation and size exclusion chromatography coupled with multiangle laser light scattering. Our results indicate that ADDL preparations exist in solution primarily as a binary mixture of a monomeric peptide and high-molecular mass oligomers. We relate our findings to previously described characterizations utilizing atomic force microscopy and electrophoretic methods and demonstrate that low-molecular mass oligomers identified by gel electrophoresis likely represent artifacts induced by the peptide's interaction with detergent, while atomic force microscopy results are likely skewed by differential binding of monomeric and oligomeric peptide species. Finally, we confirm that only the high-molecular mass oligomeric components of an ADDL preparation are capable of binding to subpopulations of primary hippocampal neurons in vitro.

A recombinant 63-kDa form of Bacillus anthracis protective antigen produced in the yeast Saccharomyces cerevisiae provides protection in rabbit and primate inhalational challenge models of anthrax infection.

Infection by Bacillus anthracis is preventable by prophylactic vaccination with several naturally derived and recombinant vaccine preparations. Existing data suggests that protection is mediated by antibodies directed against the protective antigen (PA) component of the anthrax toxin complex. PA is an 83-kDa protein cleaved in vivo to yield a biologically active 63-kDa protein. In an effort to evaluate the potential of yeast as an expression system for the production of recombinant PA, and to determine if the yeast-purified rPA63 can protect from a lethal inhalational challenge, the sequence of the 63-kDa form of PA was codon-optimized and expressed in the yeast Saccharomyces cerevisiae. Highly purified rPA63 isolated from Saccharomyces under denaturing conditions demonstrated reduced biological activity in a macrophage-killing assay compared to non-denatured rPA83 purified from Escherichia coli. Rabbits and non-human primates (NHP) immunized with rPA63 and later challenged with a lethal dose of B. anthracis spores were generally protected from infection. These results indicate that epitopes present in the 63-kDa from of PA can protect rabbits and non-human primates from a lethal spore challenge, and further suggest that a fully functional rPA63 is not required in order to provide these epitopes.

Immunogenicity and protective efficacy of Bacillus anthracis poly-gamma-D-glutamic acid capsule covalently coupled to a protein carrier using a novel triazine-based conjugation strategy.

The capsular polypeptide of Bacillus anthracis is composed of a unique polyglutamic acid polymer in which D-glutamate monomers are joined by gamma-peptidyl bonds. The capsule is poorly immunogenic, and efforts at exploiting the polymer for vaccine development have focused on increasing its inherent immunogenicity through chemical coupling to immune-stimulating protein carriers. The usual strategy has employed carbodiimide-based condensing reagents for activation of free alpha-carboxyl groups, despite reports that this chemistry may lead to chain scission. We have purified the high molecular mass capsule to >95% homogeneity and have demonstrated that the polymer contains >99% poly-gamma-D-glutamic acid. The predominant structure of the polymer as assessed by circular dichroism and multiangle laser light scattering was unordered at near-neutral pH. We investigated the effects of various activation chemistries, and we demonstrated that carbodiimide treatment under aqueous conditions results in significant cleavage of the gamma-peptidyl bond, whereas scission is significantly reduced in nonaqueous polar solvents, although undesired side chain modification was still observed. An activation chemistry was developed using the triazine-based reagent 4-(4,6-dimethoxy (1,3,5)triazin-2-yl)-4-methylmorpholinium chloride, which allowed for controlled and reproducible derivatization of alpha-carbonyls. In a two-pot reaction scheme, activated capsule was derivatized with a sulfhydryl-reactive heterobifunctional moiety and was subsequently coupled to thiolated carrier protein. This conjugate elicited very high capsule-specific immune titers in mice. More importantly, mice immunized with conjugated capsule exhibited good protection against lethal challenge from a virulent B. anthracis strain in two models of infection. We also showed, for the first time, that treatment of capsule with carbodiimide significantly reduced recognition by capsule-specific antisera concurrent with the reagent-induced reduction of polymer mass. The data suggested that for vaccine development, maintenance of the high mass of the polymer may be important.