Novel structural insights for a pair of monoclonal antibodies recognizing non-overlapping epitopes of the glucosyltransferase domain of Clostridium difficile toxin B.

Clostridium difficile toxins are the primary causative agents for hospital-acquired diarrhea and pseudomembranous colitis. Numerous monoclonal antibodies (mAbs) targeting different domains of Clostridium difficile toxin have been reported. Here we report the crystal structures of two mAbs, B1 and B2, in complex with the glycosyltransferase domain (GTD) of the Clostridium difficile toxin B (TcdB). B2 bound to the N-terminal 4 helix bundle of the GTD, a conserved membrane localization domain (MLD) found in the large clostridial glycosylating toxin family implicated in targeting plasma membrane. B1 bound to a distinct epitope at the hinge region between the MLD and the catalytic subdomain of the GTD. Functional studies revealed the potency of these mAbs in vitro and in vivo to be synergistic when given in combination.

Deciphering the domain specificity of C. difficile toxin neutralizing antibodies.

Clostridium difficile infection (CDI) is the principal cause of nosocomial diarrhea and pseudomembranous colitis associated with antibiotic therapy. The pathological effects of CDI are primarily attributed to toxins A (TcdA) and B (TcdB). Adequate toxin-specific antibody responses are associated with asymptomatic carriage, whereas insufficient humoral responses are associated with recurrent CDI. While the data supporting the importance of anti-toxin antibodies are substantial, clarity about the toxin domain specificity of these antibodies is more limited. To investigate this matter, combinations of human mAbs targeting multiple domains of TcdB were assessed using toxin neutralization assays. These data revealed that a combination of mAbs specific to all major toxin domains had improved neutralizing potency when compared to equivalent concentrations of a single mAb or a combination of mAbs against one or two domains. The function and toxin domain binding specificity of serum antibodies elicited by immunization of hamsters with a toxoid vaccine candidate was also assessed. Immunization with a toxoid vaccine candidate provoked toxin neutralizing antibodies specific to multiple domains of both TcdA and TcdB. When assessed in a toxin neutralization assay, polyclonal sera displayed greater activity against elevated concentrations of toxins than equivalent concentrations of individual mAbs. These data suggest a potential benefit of any antibody based therapeutic or prophylactic treatment that targets multiple toxin domains.

A multimechanistic antibody targeting the receptor binding site potently cross-protects against influenza B viruses.

Influenza B virus causes considerable disease burden worldwide annually, highlighting the limitations of current influenza vaccines and antiviral drugs. In recent years, broadly neutralizing antibodies (bnAbs) against hemagglutinin (HA) have emerged as a new approach for combating influenza. We describe the generation and characterization of a chimeric monoclonal antibody, C12G6, that cross-neutralizes representative viruses spanning the 76 years of influenza B antigenic evolution since 1940, including viruses belonging to the Yamagata, Victoria, and earlier lineages. Notably, C12G6 exhibits broad cross-lineage hemagglutination inhibition activity against influenza B viruses and has higher potency and breadth of neutralization when compared to four previously reported influenza B bnAbs. In vivo, C12G6 confers stronger cross-protection against Yamagata and Victoria lineages of influenza B viruses in mice and ferrets than other bnAbs or the anti-influenza drug oseltamivir and has an additive antiviral effect when administered in combination with oseltamivir. Epitope mapping indicated that C12G6 targets a conserved epitope that overlaps with the receptor binding site in the HA region of influenza B virus, indicating why it neutralizes virus so potently. Mechanistic analyses revealed that C12G6 inhibits influenza B viruses via multiple mechanisms, including preventing viral entry, egress, and HA-mediated membrane fusion and triggering antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity responses. C12G6 is therefore a promising candidate for the development of prophylactics or therapeutics against influenza B infection and may inform the design of a truly universal influenza vaccine.

Incorporation of RG1 epitope concatemers into a self-adjuvanting Flagellin-L2 vaccine broaden durable protection against cutaneous challenge with diverse human papillomavirus genotypes.

AIM: To achieve durable and broad protection against human papillomaviruses by vaccination with multimers of minor capsid antigen L2 using self-adjuvanting fusions with the toll-like receptor-5 (TLR5) ligand bacterial flagellin (Fla) instead of co-formulation with alum. METHODS: Fla fusions with L2 protective epitopes comprising residues 11-200, 11-88 and/or 17-38 of a single or multiple HPV types were produced in E. coli and their capacity to activate TLR5 signaling was assessed. Immunogenicity was evaluated serially following administration of 3 intramuscular doses of Fla-L2 multimer without exogenous adjuvant, followed by challenge 1, 3, 6 or 12months later, and efficacy compared to vaccination with human doses of L1 VLP vaccines (Gardasil and Cervarix) or L2 multimer formulated in alum. Serum antibody responses were assessed by peptide ELISA, in vitro neutralization assays and passive transfer to naïve rabbits in which End-Point Protection Titers (EPPT) were determined using serial dilutions of pooled immune sera collected 1, 3, 6 or 12months after completing active immunization. Efficacy was assessed by determining wart volume following concurrent challenge at different sites with HPV6/16/18/31/45/58 'quasivirions' containing cottontail rabbit papillomavirus (CRPV) genomes. RESULTS: Vaccination in the absence of exogenous adjuvant with Fla-HPV16 L2 11-200 fusion protein elicited durable protection against HPV16, but limited cross-protection against other HPV types. Peptide mapping data suggested the importance of the 17-38 aa region in conferring immunity. Indeed, addition of L2 residues 17-38 of HPV6/18/31/39/52 to a Fla-HPV16 L2 11-200 or 11-88 elicited broader protection via active or passive immunization, similar to that seen with vaccination with an alum-adjuvanted L2 multimer comprising the aa 11-88 peptides of five or eight genital HPV types. CONCLUSIONS: Vaccination with flagellin fused L2 multimers provided lasting (>1year) immunity without the need for an exogenous adjuvant. Inclusion of the L2 amino acid 17-38 region in such multi-HPV type fusions expanded the spectrum of protection.

Core bead chromatography for preparation of highly pure, infectious respiratory syncytial virus in the negative purification mode.

Respiratory syncytial virus (RSV) is an important human pathogen, and is the most frequent viral cause of severe respiratory disease in infants. In addition, it is increasingly being recognized as an important cause of respiratory disease in the elderly and immunocompromised. Although a passive prophylactic treatment does exist for high-risk neonates and children, the overall disease burden warrants the development of a safe and effective prophylactic vaccine for use in otherwise healthy newborns and children. RSV is known to be an extremely labile virus, prone to aggregation and loss of infectious titer during virus handling and preparation procedures. To date infective RSV virions have been prepared by methods which are not readily scalable, such as density gradient ultracentrifugation. In this study we describe a scalable, chromatography-based purification procedure for preparation of highly pure, infectious RSV. The purification scheme is based on core bead technology and hollow fiber tangential flow filtration (TFF) and results in a ∼60% recovery of infectious virus titer. This method can be used to prepare highly purified wild type or live-attenuated vaccine strain viruses with titers as high as 1×10(8) plaque forming units per mL. A live-attenuated RSV vaccine prepared by this method was found to be immunogenic and protective in vivo, and its purity was 50-200-fold greater with respect to host cell dsDNA and Vero host cell proteins, than the raw feed stream. The results presented here can be considered a starting point for downstream process development of a live-attenuated vaccine approach for prevention of disease by RSV.

Design and Characterization of a Computationally Optimized Broadly Reactive Hemagglutinin Vaccine for H1N1 Influenza Viruses.

UNLABELLED: One of the challenges of developing influenza A vaccines is the diversity of antigenically distinct isolates. Previously, a novel hemagglutinin (HA) for H5N1 influenza was derived from a methodology termed computationally optimized broadly reactive antigen (COBRA). This COBRA HA elicited a broad antibody response against H5N1 isolates from different clades. We now report the development and characterization of a COBRA-based vaccine for both seasonal and pandemic H1N1 influenza virus isolates. Nine prototype H1N1 COBRA HA proteins were developed and tested in mice using a virus-like particle (VLP) format for the elicitation of broadly reactive, functional antibody responses and protection against viral challenge. These candidates were designed to recognize H1N1 viruses isolated within the last 30 years. In addition, several COBRA candidates were designed based on sequences of H1N1 viruses spanning the past 100 years, including modern pandemic H1N1 isolates. Four of the 9 H1N1 COBRA HA proteins (X1, X3, X6, and P1) had the broadest hemagglutination inhibition (HAI) activity against a panel of 17 H1N1 viruses. These vaccines were used in cocktails or prime-boost combinations. The most effective regimens that both elicited the broadest HAI response and protected mice against a pandemic H1N1 challenge were vaccines that contained the P1 COBRA VLP and either the X3 or X6 COBRA VLP vaccine. These mice had little or no detectable viral replication, comparable to that observed with a matched licensed vaccine. This is the first report describing a COBRA-based HA vaccine strategy that elicits a universal, broadly reactive, protective response against seasonal and pandemic H1N1 isolates. IMPORTANCE: Universal influenza vaccine approaches have the potential to be paradigm shifting for the influenza vaccine field, with the goal of replacing the current standard of care with broadly cross-protective vaccines. We have used COBRA technology to develop an HA head-based strategy that elicits antibodies against many H1 strains that have undergone genetic drift and has potential as a "subtype universal" vaccine. Nine HA COBRA candidates were developed, and these vaccines were used alone, in cocktails or in prime-boost combinations. The most effective regimens elicited the broadest hemagglutination inhibition (HAI) response against a panel of H1N1 viruses isolated over the past 100 years. This is the first report describing a COBRA-based HA vaccine strategy that elicits a broadly reactive response against seasonal and pandemic H1N1 isolates.

A Combination of Three Fully Human Toxin A- and Toxin B-Specific Monoclonal Antibodies Protects against Challenge with Highly Virulent Epidemic Strains of Clostridium difficile in the Hamster Model.

Clostridium difficile infection (CDI) is the principal cause of nosocomial diarrhea and pseudomembranous colitis associated with antibiotic therapy. Recent increases in the number of outbreaks attributed to highly virulent antibiotic-resistant strains underscore the importance of identifying efficacious alternatives to antibiotics to control this infection. CDI is mediated by two large exotoxins, toxins A and B. Strong humoral toxin-specific immune responses are associated with recovery and a lack of disease recurrence, whereas insufficient humoral responses are associated with recurrent CDI. Multiple approaches targeting these toxins, including intravenous immunoglobulin, neutralizing polymers, active vaccines, and, most recently, monoclonal antibodies (MAbs), have been explored, with various degrees of success. In this study, we describe the characterization of the first MAbs isolated from healthy human donors using a high-throughput B-cell cloning strategy. The MAbs were selected based on their ability to inhibit the actions of toxins A and B in vitro and because of their in vivo efficacy in a hamster challenge model. A potent 2-MAb cocktail was identified and then further potentiated by the addition of a second anti-toxin B MAb. This 3-MAb combination protected animals against mortality and also reduced the severity and duration of diarrhea associated with challenge with highly virulent strains of C. difficile toxinotypes 0 and III. This highly efficacious cocktail consists of one MAb specific to the receptor binding domain of toxin A and two MAbs specific to nonoverlapping regions of the glucosyltransferase domain of toxin B. This MAb combination offers great potential as a nonantibiotic treatment for the prevention of recurrent CDI.

Preparation of pure, high titer, pseudoinfectious Flavivirus particles by hollow fiber tangential flow filtration and anion exchange chromatography.

Purification of enveloped viruses such as live flavivirus vaccine candidates poses a challenge as one must retain viral infectivity to preserve immunogenicity. Here we describe a laboratory-scale purification procedure for two replication defective (single-cycle) flavivirus variants for use in a pre-clinical setting. The two step purification scheme based on hollow fiber tangential flow filtration (TFF) followed by anion exchange chromatography using convective interaction media (CIM(®)) monoliths results in a ∼60% recovery of infectious virus titer and can be used to prepare nearly homogenous, highly purified vaccine viruses with titers as high as 1×10(9) focus forming units per mL. Flavivirus virions prepared by this method are 2 and 3 orders of magnitude more pure with respect to dsDNA and BHK host cell proteins, respectively, as compared to the raw feed stream.

Low doses of flagellin-L2 multimer vaccines protect against challenge with diverse papillomavirus genotypes.

Genetically modified bacterial flagellin (Fla), a Toll-like receptor-5 (TLR5) ligand, was evaluated as a fusion partner for human papillomavirus (HPV) L2-based immunogens in two animal challenge models; either cutaneous inoculation of rabbits with HPV 'quasivirions' containing cottontail rabbit papillomavirus (CRPV) genomes that induce warts, or intra-vaginal inoculation of mice with HPV 'pseudovirions' encapsidating a luciferase reporter plasmid and measurement of bioluminescence to determine infectivity. An Escherichia coli production system was developed for flagellin-L2 (Fla-L2) fusions containing either monomeric HPV-16 L2 a.a. 11(×11-200) or oligomeric L2 comprising a fusion of the a.a. 11-88 peptides of five (Fla∼5×11-88) or eight (Fla∼8×11-88) genital HPV types. Immunogenicity and bioactivity of Fla-L2 constructs were assessed using an in vitro neutralization and cell-based TLR-5 binding assay, respectively. Efficacy was evaluated following active immunization of rabbits or mice administered 3 intramuscular doses of Fla-L2 recombinants without exogenous adjuvant, followed by challenge. In addition, passive immunization studies of naïve rabbits with serial dilutions of pooled immune sera were used to determine End-Point Protection Titers (EPPT) for each formulation against a broader spectrum of HPV quasivirions. Efficacy was assessed for up to 10 weeks on the basis of wart volume induced following challenge and results compared to licensed L1-VLP vaccines (Gardasil and Cervarix). Following active immunization at doses as low as 1 µg, Fla-L2 fusions afforded complete protection against infection (mice) and disease (rabbits) following either homologous or heterologous HPV challenge. Passive immunization with anti-L2 immune sera discriminated between the different vaccine candidates under evaluation, demonstrated the protective role of antibody and suggested the superiority of this oligomeric L2-TLR5 agonist fusion approach compared to L1-based vaccines in its ability to cross-protect against non-vaccine HPV types.

High-purity preparation of HSV-2 vaccine candidate ACAM529 is immunogenic and efficacious in vivo.

Genital herpes is a sexually transmitted infection (STI) caused by herpes simplex virus 2 (HSV-2) and to a lesser extent herpes simplex virus 1 (HSV-1). Infection by HSV-2 is life-long and is associated with significant cost to healthcare systems and social stigma despite the highly prevalent nature of the disease. For instance, the proportion of HSV-2 seropositive to seronegative adults is approximately 1 in 5 in the US and greater than 4 in 5 in some areas of sub-Saharan Africa. The replication-defective vaccine strain virus dl5-29 was re-derived using cells appropriate for GMP manufacturing and renamed ACAM529. Immunization with dl5-29 was previously reported to be protective both in mice and in guinea pigs, however these studies were performed with vaccine that was purified using methods that cannot be scaled for manufacturing of clinical material. Here we describe methods which serve as a major step towards preparation of ACAM529 which may be suitable for testing in humans. ACAM529 can be harvested from infected cell culture of the trans-complementing cell line AV529 clone 19 (AV529-19) without mechanical cell disruption. ACAM529 may then be purified with respect to host cell DNA and proteins by a novel purification scheme, which includes a combination of endonuclease treatment, depth filtration, anion-exchange chromatography and ultrafiltration/diafiltration (UF/DF). The resultant virus retains infectivity and is ∼ 200-fold more pure with respect to host cell DNA and proteins than is ACAM529 purified by ultracentrifugation. Additionally, we describe a side-by-side comparison of chromatography-purified ACAM529 with sucrose cushion-purified ACAM529, which shows that both preparations are equally immunogenic and protective when tested in vivo.

Immunogenicity and efficacy of intramuscular replication-defective and subunit vaccines against herpes simplex virus type 2 in the mouse genital model.

Herpes simplex virus type 2 (HSV-2) is a sexually transmitted virus that is highly prevalent worldwide, causing a range of symptoms that result in significant healthcare costs and human suffering. ACAM529 is a replication-defective vaccine candidate prepared by growing the previously described dl5-29 on a cell line appropriate for GMP manufacturing. This vaccine, when administered subcutaneously, was previously shown to protect mice from a lethal vaginal HSV-2 challenge and to afford better protection than adjuvanted glycoprotein D (gD) in guinea pigs. Here we show that ACAM529 given via the intramuscular route affords significantly greater immunogenicity and protection in comparison with subcutaneous administration in the mouse vaginal HSV-2 challenge model. Further, we describe a side-by-side comparison of intramuscular ACAM529 with a gD vaccine across a range of challenge virus doses. While differences in protection against death are not significant, ACAM529 protects significantly better against mucosal infection, reducing peak challenge virus shedding at the highest challenge dose by over 500-fold versus 5-fold for gD. Over 27% (11/40) of ACAM529-immunized animals were protected from viral shedding while 2.5% (1/40) were protected by the gD vaccine. Similarly, 35% (7/20) of mice vaccinated with ACAM529 were protected from infection of their dorsal root ganglia while none of the gD-vaccinated mice were protected. These results indicate that measuring infection of the vaginal mucosa and of dorsal root ganglia over a range of challenge doses is more sensitive than evaluating survival at a single challenge dose as a means of directly comparing vaccine efficacy in the mouse vaginal challenge model. The data also support further investigation of ACAM529 for prophylaxis in human subjects.