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.Staphylococcus aureus is a gram-positive bacterium that is notable for the frequency and severity of infections that it causes in hospitalized patients. These infections range from localized skin infections to bacteremia and septic shock. In the past 20 years there has been a dramatic increase in the incidence of nosocomial staphylococcal infections; this increase parallels the increased use of intravascular devices and invasive procedures. S. aureus has been identified as one of the three most frequent nosocomial pathogens and is responsible for approximately 25% of the 2 million nosocomial infections reported in the United States each year (38, 39). A second trend has been the increase in the incidence of methicillin-resistant S. aureus, largely due to selective antibiotic pressure. Resistant strains were initially identified in tertiary care hospitals but have been increasingly reported among infections in the community (25, 30). Resistance to methicillin is often accompanied by resistance to other antibiotics; a CDC survey showed that the proportion of methicillin-resistant isolates which were susceptible only to vancomycin rose from 22.8% to 56. 2% from 1987 to 1997 (18). More recently, S. aureus strains with intermediate susceptibility or resistance to vancomycin have been reported (11,24,36). Infections caused by multidrug-resistant S. aureus limit therapeutic options, and they may be associated with higher mortality and higher costs than infections caused by susceptible staphylococci. There is clearly a need for new treatment and prevention strategies.In an immunological survey of S. aureus su...

Optimization increased both the analytical sensitivity and the clinical specificity of the assay to more effectively discriminate the low-titer antibody response of HPV-infected persons from noninfected individuals. The characteristics of the assay that were optimized included monoclonal antibody (MAb) specificity, scaling up the conjugation of virus-like particles (VLPs) to microspheres, VLP concentration, MAb concentration, sample matrix, sample dilution, incubation time, heat inactivation of sample sera, and detergent effects on assay buffer. The assay was automated by use of a TECAN Genesis Workstation, thus improving assay throughput, reproducibility, and operator safety. Following optimization, the assay was validated using several distinct serum panels from individuals determined to be at low and high risk for HPV infection. The validated assay was then used to determine the clinical serostatus cutoff. This high-throughput assay has proven useful for performing epidemiology studies and evaluating the efficacy of prophylactic HPV vaccines.Cervical cancer is the second most common cancer in women worldwide. Every year, 450,000 women are diagnosed with cervical cancer and 220,000 succumb to this disease (27). Current approaches to cervical cancer control involve lifelong screening using the Papanicolau (Pap) test (13). The goal of screening is to detect precancerous lesions so that they can be removed prior to the development of cancer. Despite widespread Pap testing, there were an estimated 10,520 new cases of cervical cancer and nearly 4,000 cervical cancer-related deaths in the United States in 2004 (1). The national health care burden of current screening systems combined with direct costs of treating precancerous and cancerous lesions is in excess of 3.5 billion U.S. dollars per annum (7).Infection with human papillomavirus (HPV) is the first and obligate step in the development of cervical cancer (3, 4). Infection of the cervical epithelium with HPV results in expression of the E6 and E7 proteins, which have been shown to be potent oncogenes. More than 35 different HPV types are capable of infecting the human genital tract (2, 4, 28). Of these, four types cause the majority of the HPV-related cervical pathology. HPV 16 and 18 together account for 74.6% of all cervical cancers (23), whereas HPV6 and -11 cause a significant fraction of precancerous lesions which rarely develop into cervical cancer but morphologically are indistinguishable from lesions from more dangerous HPV types (37). HVP 6 and 11 are responsible for approximately 90% of all genital wart cases (37).The HPV LI capsid protein, when expressed recombinantly, assembles into empty viral capsids or "virus like particles" (VLPs) (12,15,16,29). Several prophylactic vaccines based on HPV LI VLPs are currently in phases II and III clinical development (14,17,36). The VLPs in the vaccine present the immune system with the conformational, neutralizing epitopes found on the natural virus and prime the immune system to generate antibodies that neutra...

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Human papillomavirus (HPV) types 6, 11, 16 and 18 L1 virus-like particles (VLPs) have been used to generate the prophylactic quadrivalent vaccine, Gardasil ® . There is a high degree of L1 homology between HPV types and it is likely that there is a substantial degree of surface exposed viral epitope similarity. An investigation of vaccine-induced antibody binding and neutralization was undertaken focusing on A7 species members, HPV 18 and 45. Polyclonal sera from Gardasil ® recipients and from HPV 18 L1 VLP recipients were evaluated. Vaccine-induced antibodies were found to cross-neutralize HPV 45 pseudovirions (PsV) in vitro. Examination of a panel of monoclonal antibodies made against L1 VLPs revealed the presence of conformational, neutralizing epitopes on the surface of VLPs that may be shared between HPV 18 and HPV 45. These data demonstrate that Gardasil(r) immunization induces antibodies capable of neutralizing HPV 18 PsV and HPV 45 PsV in vitro.

BackgroundHuman papillomavirus (HPV) vaccines based on major capsid protein L1 are licensed in over 100 countries to prevent HPV infections. The yeast-derived recombinant quadrivalent HPV L1 vaccine, GARDASIL(R), has played an important role in reducing cancer and genital warts since its introduction in 2006. The L1 proteins self-assemble into virus-like particles (VLPs).ResultsVLPs were subjected to post-purification disassembly and reassembly (D/R) treatment during bioprocessing to improve VLP immunoreactivity and stability. The post-D/R HPV16 VLPs and their complex with H16.V5 neutralizing antibody Fab fragments were visualized by cryo electron microscopy, showing VLPs densely decorated with antibody. Along with structural improvements, post-D/R VLPs showed markedly higher antigenicity to conformational and neutralizing monoclonal antibodies (mAbs) H16.V5, H16.E70 and H263.A2, whereas binding to mAbs recognizing linear epitopes (H16.J4, H16.O7, and H16.H5) was greatly reduced.Strikingly, post-D/R VLPs showed no detectable binding to H16.H5, indicating that the H16.H5 epitope is not accessible in fully assembled VLPs. An atomic homology model of the entireHPV16 VLP was generated based on previously determined high-resolution structures of bovine papillomavirus and HPV16 L1 pentameric capsomeres.ConclusionsD/R treatment of HPV16 L1 VLPs produces more homogeneous VLPs with more virion-like antibody reactivity. These effects can be attributed to a combination of more complete and regular assembly of the VLPs, better folding of L1, reduced non-specific disulfide-mediated aggregation and increased stability of the VLPs. Markedly different antigenicity of HPV16 VLPs was observed upon D/R treatment with a panel of monoclonal antibodies targeting neutralization sensitive epitopes. Multiple epitope-specific assays with a panel of mAbs with different properties and epitopes are required to gain a better understanding of the immunochemical properties of VLPs and to correlate the observed changes at the molecular level. Mapping of known antibody epitopes to the homology model explains the changes in antibody reactivity upon D/R. In particular, the H16.H5 epitope is partially occluded by intercapsomeric interactions involving the L1 C-terminal arm. The homology model allows a more precise mapping of antibody epitopes. This work provides a better understanding of VLPs in current vaccines and could guide the design of improved vaccines or therapeutics.

An in vitro relative potency (IVRP) assay has been developed as an alternative to the mouse potency assay used to release Merck's human papillomavirus (HPV) vaccine, Gardasil ® , for early phase clinical trials. The mouse potency assay is a classical, in vivo assay, which requires 4-6 weeks to complete and exhibits variability on the order of 40% relative standard deviation (RSD). The IVRP assay is a sandwich-type immunoassay that is used to measure relative antigenicity of the vaccine product. The IVRP assay can be completed in three days, has a variability of approximately 10% RSD and does not require the sacrifice of live animals. Because antigen detection is achieved using H16.V5, a neutralizing monoclonal antibody, which binds to a clinically-relevant epitope, the relative antigenicity measured by the IVRP assay is believed to be a good predictor of in vivo potency.In this study, the relationship between immunogenicity, as measured by the mouse potency assay and antigenicity as measured by the IVRP assay, is demonstrated. Freshly manufactured and aged samples produced using two different manufacturing processes were tested using both methods. The results demonstrate that there is an inverse correlation between the IVRP and mouse potency assays. Additionally, clinical results indicate IVRP is predictive of human immunogenicity. Thus, antigenicity, as defined by the H16.V5 epitope, can be used as a surrogate for immunogenicity and the IVRP assay is suitable for use as the sole potency test for Gardasil samples.