Selection and Characterization of Murine Monoclonal Antibodies to Staphylococcus aureus Iron-Regulated Surface Determinant B with Functional Activity In Vitro and In Vivo▿ †

In an effort to characterize important epitopes of Staphylococcus aureus iron-regulated surface determinant B (IsdB), murine IsdB-specific monoclonal antibodies (MAbs) were isolated and characterized. A panel of 12 MAbs was isolated. All 12 MAbs recognized IsdB in enzyme-linked immunosorbent assays and Western blots; 10 recognized native IsdB expressed by S. aureus. The antigen epitope binding of eight of the MAbs was examined further. Three methods were used to assess binding diversity: MAb binding to IsdB muteins, pairwise binding to recombinant IsdB, and pairwise binding to IsdB-expressing bacteria. Data from these analyses indicated that MAbs could be grouped based on distinct or nonoverlapping epitope recognition. Also, MAb binding to recombinant IsdB required a significant portion of intact antigen, implying conformational epitope recognition. Four MAbs with nonoverlapping epitopes were evaluated for in vitro opsonophagocytic killing (OPK) activity and efficacy in murine challenge models. These were isotype switched from immunoglobulin G1 (IgG1) to IgG2b to potentially enhance activity; however, this isotype switch did not appear to enhance functional activity. MAb 2H2 exhibited OPK activity (≥50% killing in the in vitro OPK assay) and was protective in two lethal challenge models and a sublethal indwelling catheter model. MAb 13C7 did not exhibit OPK (<50% killing in the in vitro assay) and was protective in one lethal challenge model. Neither MAb 13G11 nor MAb 1G3 exhibited OPK activity in vitro or was active in a lethal challenge model. The data suggest that several nonoverlapping epitopes are recognized by the IsdB-specific MAbs, but not all of these epitopes induce protective antibodies

Development of a Novel Vaccine Containing Binary Toxin for the Prevention of Clostridium difficile Disease with Enhanced Efficacy against NAP1 Strains.

Clostridium difficile infections (CDI) are a leading cause of nosocomial diarrhea in the developed world. The main virulence factors of the bacterium are the large clostridial toxins (LCTs), TcdA and TcdB, which are largely responsible for the symptoms of the disease. Recent outbreaks of CDI have been associated with the emergence of hypervirulent strains, such as NAP1/BI/027, many strains of which also produce a third toxin, binary toxin (CDTa and CDTb). These hypervirulent strains have been associated with increased morbidity and higher mortality. Here we present pre-clinical data describing a novel tetravalent vaccine composed of attenuated forms of TcdA, TcdB and binary toxin components CDTa and CDTb. We demonstrate, using the Syrian golden hamster model of CDI, that the inclusion of binary toxin components CDTa and CDTb significantly improves the efficacy of the vaccine against challenge with NAP1 strains in comparison to vaccines containing only TcdA and TcdB antigens, while providing comparable efficacy against challenge with the prototypic, non-epidemic strain VPI10463. This combination vaccine elicits high neutralizing antibody titers against TcdA, TcdB and binary toxin in both hamsters and rhesus macaques. Finally we present data that binary toxin alone can act as a virulence factor in animal models. Taken together, these data strongly support the inclusion of binary toxin in a vaccine against CDI to provide enhanced protection from epidemic strains of C. difficile

Tetravalent vaccine generates high neutralizing antibody titers in rhesus macaques.

<p>Rhesus macaques (n = 5) were given 3 immunizations with the tetravalent vaccine formulated with either ISCOMATRIX™ or ISCOMATRIX^™ plus AAHS on d0, d7, and d30. Control animals were immunized on the same days with a formalin inactivated toxoid vaccine adjuvanted with Rehydragel. Serum samples were collected on days 0, 7, 21 and 45. Individual (Day 45) or pooled (days 0, 7 and 21) serum samples were preincubated with active toxin prior to being added to Vero cells. ED50 values indicate the serum dilution at which the area of the Vero cell monolayer is reduced by 50%. Strong neutralizing antibody titers were generated against (A) TcdA, (B) TcdB, and (C) binary toxin. Day 45 titers in these graphs represent the mean value of the individual neutralizing titers. Neutralizing antibody titers to D) TcdA, E) TcdB, and F) binary toxin were measured from individual rhesus macaque serum samples on day 45. Statistical analysis was performed using One-way ANOVA and Tukey multiple comparisons test. * = p<0.05, ns = no significant difference.</p

Purified vaccine components.

<p>Three micrograms 5mTcdA (lane A, 308kD), 5mTcdB (lane B, 269kD), proCDTb (lane C, 94kD) and 4mCDTa (lane D, 48kD) were loaded on a 8–16% Tris-Glycine (Invitrogen, Carlsbad, CA). GelCode Blue Stain Reagent (Thermo Fisher Scientific, Waltham, MA) was then used to stain the gel.</p

Potency of recombinant and native toxins in a cytotoxicity assay and mouse toxicity model.

<p>Potency of recombinant and native toxins in a cytotoxicity assay and mouse toxicity model.</p

Recombinant toxins and vaccines used in this study.

<p>Recombinant toxins and vaccines used in this study.</p

Efficacy and immunogenicity of bivalent vaccine in hamsters.

<p>Kaplan-Meier graphs show survival curves for hamsters that were immunized intramuscularly four times with the bivalent vaccine containing 10μg 5mTxdA and 10μg 5mTxdB adjuvanted with ISCOMATRIX^™ and AAHS or adjuvant alone. Following the final immunization, hamsters were bled prior to challenge with either (A) VPI10463 (707 cfu, p<0.0001) or (B) BI17 (470 cfu, p>0.05) spores. Statistical differences in survival curves were calculated using Mantel-Cox test. (C) Pooled serum samples (day 0, 21, 42, 63 and 77) from the BI17 challenged hamsters were tested in an <i>in vitro</i> assay against Vero cells to measure neutralizing antibody activity against TcdA and TcdB. The ED50 was calculated as the serum dose that reduced cytotoxicity by 50%. Similar titers were observed from hamsters in the VPI10463 challenge. (D) Individual serum samples from day 77 from the VPI10463 and BI17 challenged hamsters were also tested for neutralizing antibody activity against TcdA and TcdB. Comparison of neutralizing antibody titers was performed using unpaired, two tailed t-test. * = p<0.05, *** = p<0.001, ns = no significant difference.</p

A Novel Staphylococcus aureus Vaccine: Iron Surface Determinant B Induces Rapid Antibody Responses in Rhesus Macaques and Specific Increased Survival in a Murine S. aureus Sepsis Model

Staphylococcus aureus is a major cause of nosocomial infections worldwide, and the rate of resistance to clinically relevant antibiotics, such as methicillin, is increasing; furthermore, there has been an increase in the number of methicillin-resistant S. aureus community-acquired infections. Effective treatment and prevention strategies are urgently needed. We investigated the potential of the S. aureus surface protein iron surface determinant B (IsdB) as a prophylactic vaccine against S. aureus infection. IsdB is an iron-sequestering protein that is conserved in diverse S. aureus clinical isolates, both methicillin resistant and methicillin sensitive, and it is expressed on the surface of all isolates tested. The vaccine was highly immunogenic in mice when it was formulated with amorphous aluminum hydroxyphosphate sulfate adjuvant, and the resulting antibody responses were associated with reproducible and significant protection in animal models of infection. The specificity of the protective immune responses in mice was demonstrated by using an S. aureus strain deficient for IsdB and HarA, a protein with a high level of identity to IsdB. We also demonstrated that IsdB is highly immunogenic in rhesus macaques, inducing a more-than-fivefold increase in antibody titers after a single immunization. Based on the data presented here, IsdB has excellent prospects for use as a vaccine against S. aureus disease in humans