The Effect of Physicochemical Modification on the Function of Antibodies Induced by Anti-Nicotine Vaccine in Mice.

Smoking remains one of the major causes of morbidity and mortality worldwide. One approach to assisting smoking cessation is via anti-nicotine vaccines, composed of nicotine-like haptens conjugated to a carrier protein plus adjuvant(s). We have previously shown that the carrier, hapten, linker, hapten load, degree of conjugate aggregation, and presence of adducts can each influence the function (nicotine-binding capacity) of the antibody (Ab) induced. Herein, we extend those findings and show that tertiary structure is also critical to the induction of functional immune responses and that this can be influenced by conjugation conditions. We evaluated immunogenicity in mice using six lots of NIC7-CRM, a conjugate of 5-aminoethoxy-nicotine (Hapten 7), and a single point (glycine 52 to glutamic acid) mutant nontoxic form of diphtheria toxin, cross-reactive material 197 (CRM197), which were synthesized under different reaction conditions resulting in conjugates with equivalent molecular characteristics (hapten load, aggregates, adducts), but a different tertiary structure. When tested in mice, better functional responses (reduced nicotine in the brain of immunized animals relative to non-immunized controls) were obtained with conjugates with a more closed structure than those with an open conformation. These studies highlight the need for a better understanding of the physicochemical properties of small molecule conjugate vaccines.

Immunogenicity of a peptide-based anti-IgE conjugate vaccine in non-human primates.

The anti-human immunoglobulin E (IgE) monoclonal antibody, omalizumab (Xolair®, Genentech, South San Fransisco, CA), is effective in the treatment of poorly controlled moderate to severe allergic asthma and chronic idiopathic urticaria. It acts by specifically binding to the constant domain (Cϵ3) of free human IgE in the blood and interstitial fluid. Although efficacious, use of omalizumab is limited due to restrictions on patient weight and pre-existing IgE levels, and frequent dosing (q2-4 weeks). A vaccine inducing anti-IgE antibodies has the potential for similar clinical benefits with less frequent dosing and relatively lower cost of goods. We developed a vaccine containing two IgE peptide-conjugates targeting the Cϵ3 domain of human IgE. As part of preclinical evaluation of the vaccine to optimize formulation and dose prior to initiating clinical studies, we evaluated the vaccine in non-human primates, and demonstrate the induction of anti-peptide antibodies that can bind to conformationally intact human IgE and are capable, at least in some animals, of substantial lowering circulating IgE levels.

The effect of preexisting anti-carrier immunity on subsequent responses to CRM197 or Qb-VLP conjugate vaccines.

CONTEXT: Certain antigens, such as haptens (small molecules), short peptides, and carbohydrates (e.g. bacterial polysaccharides) are non- or poorly immunogenic unless conjugated to a carrier molecule that provides a structural scaffold for antigen presentation as well as T cell help required for B-cell activation and maturation. However, the carriers themselves are immunogenic and resulting carrier-specific immune responses may impact the immunogenicity of other conjugate vaccines using the same carrier that are administered subsequently. OBJECTIVE: Herein, using two different carriers (cross-reactive material 197, CRM and Qb-VLP), we examined in mice the impact that preexisting anti-carrier antibodies (Ab) had on subsequent immune responses to conjugates with either the same or a different carrier. METHOD: For this purpose, we used two nicotine hapten conjugates (NIC7-CRM or NIC-Qb), two IgE peptide conjugates (Y-CRM or Y-Qb), and a pneumococcal polysaccharide conjugate (Prevnar 13(®)). RESULTS: Prior exposure to CRM or Qb-VLP significantly reduced subsequent responses to the conjugated antigen having the homologous carrier, with the exception of Prevnar 13® where anti-polysaccharide responses were similar to those in animals without preexisting anti-carrier Ab. CONCLUSION: Collectively, the data suggest that the relative sizes of the antigen and carrier, as well as the conjugation density for a given conjugate impact the extent of anti-carrier suppression. All animals developed anti-carrier responses with repeat vaccination and the differences in Ab titer between groups with and without preexisting anti-carrier responses became less apparent; however, anti-carrier effects were more durable for Ab function.

Support for the revocation of general safety test regulations in biologics license applications.

The United States Food and Drug Administration recently removed the requirement for a General Safety Test (GST) for biologics in the Code of Federal Regulations (21 CFR 610.11). The GST, as well as abnormal toxicity (European Pharmacopeia) and innocuity tests (World Health Organization), were designed to test for extraneous toxic contaminants on each product lot intended for human use. Tests require one-week observations for general health and weight following injection of specified volumes of product batches into guinea pigs and mice. At the volumes specified, dose-related toxicity may result when the product is pharmacologically active in rodents. With vaccines, required doses may be > 3 logs higher than intended human dose on a weight-adjusted basis and if an immune modulatory adjuvant is included, systemic immune hyperactivation may cause toxicity. Herein, using the CpG/alum adjuvant combination we evaluated the different test protocols and showed their unsuitability for this adjuvant combination.

Anti-IgE Qb-VLP Conjugate Vaccine Self-Adjuvants through Activation of TLR7.

Qb bacteriophage virus-like particles (Qb-VLP) are utilized as carriers to enhance immune responses to weakly or non-immunogenic antigens such as peptides and haptens. Qb-VLPs are formed through the self-assembly of multiple Qb capsid protein monomers, a process which traps a large amount of bacterial RNA in the core of the VLP. Bacterial RNA is known to activate the innate immune system via TLR 7 and 8 found within the endosomes of certain immune cells and has been shown to contribute to the immunogenicity of Qb-VLP vaccines. Herein, we evaluated an anti-IgE vaccine comprised of two IgE peptides (Y and P) conjugated to Qb-VLP (Qb-Y and Qb-P, respectively) for in vitro stimulation of human PBMCs and in vivo immunogenicity in mice. The in vitro secretion of IFN-α from human PBMCs exposed to Qb-Y is consistent with TLR7 activation. Immunization of mice with the IgE peptide Qb-VLP conjugates induced high titers of anti-IgE antibodies in wild-type mice, but significantly lower titers in TLR7 knockout mice, supporting the self-adjuvanting role of the RNA. Inclusion of alum and alum/CpG as adjuvants partially or completely compensated for the lack of TLR7 activation in TLR7-deficient mice. Our study demonstrates the key role that TLR7 plays in the immunogenicity of the IgE peptide Qb-VLP conjugate vaccine.

Co-crystal structure and inhibition of factor Xa by PD0313052 identifies structurally stabilized active site residues of factor Xa and prothrombinase.

The enzyme complex prothrombinase plays a pivotal role in fibrin clot development through the production of thrombin, making this enzyme complex an attractive target for therapeutic regulation. This study both functionally and structurally characterizes a potent, highly selective, active site directed inhibitor of human factor Xa and prothrombinase, PD0313052, and identifies structurally conserved residues in factor Xa and prothrombinase. Analyses of the association and dissociation of PD0313052 with human factor Xa identified a reversible, slow-onset mechanism of inhibition and a simple, single-step bimolecular association between factor Xa and PD0313052. This interaction was governed by association (k(on)) and dissociation (k(off)) rate constants of (1.0 +/- 0.1) x 10(7) M(-1) s(-1) and (1.9 +/- 0.5) x 10(-3) s(-1), respectively. The inhibition of human factor Xa by PD0313052 displayed significant tight-binding character described by a Ki* = 0.29 +/- 0.08 nM. Similar analyses of the inhibition of human prothrombinase by PD0313052 also identified a slow-onset mechanism with a Ki* = 0.17 +/- 0.03 nM and a k(on) and k(off) of (0.7 +/- 0.1) x 10(7) M(-1) s(-1) and (1.7 +/- 0.8) x 10(-3) s(-1), respectively. Crystals of factor Xa and PD0313052 demonstrated hydrogen bonding contacts within the S1-S4 pocket at residues Ser195, Asp189, Gly219, and Gly216, as well as interactions with aromatic residues within the S4 pocket. Overall, these data demonstrate that the inhibition of human factor Xa by PD0313052 occurs via a slow, tight-binding mechanism and indicate that active site residues of human factor Xa, including the catalytic Ser195, are effectively unaltered following assembly into prothrombinase.