Visualization of immune pathways that enhance the neutralizing antibody response to vaccines after primary immunization.

We examined the relationship between the association of a vaccine antigen with immune cells in secondary lymphoid organs shortly after immunization and the resulting neutralizing antibody response induced by that antigen using three antigenic forms of anthrax protective antigen (PA) that induce qualitatively different antibody responses. The three PA forms used were wild-type PA, which binds to anthrax toxin receptors and elicits a robust antibody response that includes both neutralizing and non-neutralizing antibodies; a receptor-binding-deficient (RBD) mutant form of PA, which does not bind cellular receptors and elicits only barely detectable antibody responses; and an engineered chimeric form of PA, which binds cholera toxin receptors and elicits a robust total antibody response but a poor neutralizing antibody response. We found that both wild-type PA and the PA chimera associated with immune cells in secondary lymphoid organs after immunization, but the RBD mutant PA exhibited minimal association, revealing a relationship between antigen binding to toxin receptors on immune cells after immunization and subsequent antibody responses.

Receptor-enhanced immunogenicity of anthrax protective antigen is primarily mediated by capillary morphogenesis Protein-2.

Anthrax protective antigen (PA), the receptor-binding component of anthrax toxin, elicits toxin-neutralizing antibodies which provide protection against anthrax disease. PA binds to two mammalian receptors, capillary morphogenesis protein-2 (CMG2) and tumor endothelial marker-8 (TEM8). We previously observed that binding of PA to its receptors plays a role in eliciting a strong toxin-neutralizing antibody response.

Secretion of Pertussis Toxin from .

Production and secretion of pertussis toxin (PT) is essential for the virulence of . Due to the large oligomeric structure of PT, transport of the toxin across bacterial membrane barriers represents a significant hurdle that the bacteria must overcome in order to maintain pathogenicity. During the secretion process, PT undergoes a two-step transport process.

Improving the stability of recombinant anthrax protective antigen vaccine.

Development of recombinant protective antigen (rPA)-based anthrax vaccines has been hindered by a lack of stability of the vaccines associated with spontaneous deamidation of asparagine (Asn) residues of the rPA antigen during storage. In this study, we explored the role that two deamidation-prone Asn residues located directly adjacent to the receptor binding site of PA, Asn and Asn, play in the stability of rPA-based anthrax vaccines. We modified these residues to glutamine (Gln) and generated rPA(N713Q/N719Q), since Gln would not be expected to deamidate on a time scale relevant to vaccine storage.

Comparison of the immune response during acute and chronic Staphylococcus aureus infection.

Staphylococcus aureus bacteria are able to grow in a planktonic state that is associated with acute infections and in biofilms that are associated with chronic infections. Acute infections, such as skin infections, are often self-limiting. However, chronic infections, such as implant infections, can be difficult to clear and may require surgical intervention.

Role of the Antigen Capture Pathway in the Induction of a Neutralizing Antibody Response to Anthrax Protective Antigen.

Toxin neutralizing antibodies represent the major mode of protective immunity against a number of toxin-mediated bacterial diseases, including anthrax; however, the cellular mechanisms that lead to optimal neutralizing antibody responses remain ill defined. Here we show that the cellular binding pathway of anthrax protective antigen (PA), the binding component of anthrax toxin, determines the toxin neutralizing antibody response to this antigen. PA, which binds cellular receptors and efficiently enters antigen-presenting cells by receptor-mediated endocytosis, was found to elicit robust anti-PA IgG and toxin neutralizing antibody responses.

Towards replacement of the acellular pertussis vaccine safety test: Comparison of in vitro cytotoxic activity and in vivo activity in mice.

Because of the exquisite sensitivity of the murine histamine sensitization test (HIST) in detecting minute amounts of active pertussis toxin (PTx), this animal-based test has been used to assure the safety of acellular pertussis vaccines in the United States and other countries around the world. Prompted by humane considerations, efforts are underway to find a suitable in vitro replacement assay that has critical attributes comparable to that of the HIST. In this study, we compared the sensitivity of the in vivo HIST with an in vitro Chinese Hamster Ovary (CHO) cell-based assay.

Mechanistic Analysis of the Effect of Deamidation on the Immunogenicity of Anthrax Protective Antigen.

The spontaneous modification of proteins, such as deamidation of asparagine residues, can significantly affect the immunogenicity of protein-based vaccines. Using a "genetically deamidated" form of recombinant protective antigen (rPA), we have previously shown that deamidation can decrease the immunogenicity of rPA, the primary component of new-generation anthrax vaccines. In this study, we investigated the biochemical and immunological mechanisms by which deamidation of rPA might decrease the immunogenicity of the protein.

RNA-Seq Analysis of the Host Response to Staphylococcus aureus Skin and Soft Tissue Infection in a Mouse Model.

Staphylococcus aureus is a leading cause of skin and soft tissue infections (SSTI), which are primarily self-limiting. We conducted a comprehensive analysis of the host transcriptome during a S. aureus SSTI to provide insight on the protective mechanisms that thwart these infections.

Clinical evaluation of pertussis vaccines: US Food and Drug Administration regulatory considerations.

The resurgence of pertussis in the United States has stimulated considerable public health interest in developing new vaccination strategies to improve control of pertussis. The purpose of this article is to review the US Food and Drug Administration's regulatory framework for the prelicensure clinical evaluation of preventive vaccines and the clinical approaches that have been used to demonstrate effectiveness of US-licensed vaccines containing an acellular pertussis component.

Pertussis pathogenesis--what we know and what we don't know.

Pertussis is a worldwide public health threat. Bordetella pertussis produces multiple virulence factors that have been studied individually, and many have recently been found to have additional biological activities. Nevertheless, how they interact to cause the disease pertussis remains unknown.

Role of antibodies in protection elicited by active vaccination with genetically inactivated alpha hemolysin in a mouse model of staphylococcus aureus skin and soft tissue infections.

Due to the emergence of highly virulent community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections, S. aureus has become a major threat to public health. A majority of CA-MRSA skin and soft tissue infections in the United States are caused by S.

Immunogenicity analysis of Staphylococcus aureus clumping factor A genetic variants.

The staphylococcal adhesin clumping factor A (ClfA) has a variant amino acid sequence, generating the potential for alterations in epitope structure and immunogenicity of this vaccine candidate. We demonstrated for two recombinant ClfA(40-531) (a slightly truncated version of the fibrinogen-binding domain of ClfA containing amino acids 40 to 531) genetic variants that strain-specific epitopes are immunodominant. This work indicates that immune responses elicited by ClfA may, at least in part, be dependent on the strain of origin of the ClfA.

Evaluation of genetically inactivated alpha toxin for protection in multiple mouse models of Staphylococcus aureus infection.

Staphylococcus aureus is a major human pathogen and a leading cause of nosocomial and community-acquired infections. Development of a vaccine against this pathogen is an important goal. While S.

Use of site-directed mutagenesis to model the effects of spontaneous deamidation on the immunogenicity of Bacillus anthracis protective antigen.

Long-term stability is a desired characteristic of vaccines, especially anthrax vaccines, which must be stockpiled for large-scale use in an emergency situation; however, spontaneous deamidation of purified vaccine antigens has the potential to adversely affect vaccine immunogenicity over time. In order to explore whether spontaneous deamidation of recombinant protective antigen (rPA)--the major component of new-generation anthrax vaccines--affects vaccine immunogenicity, we created a "genetically deamidated" form of rPA using site-directed mutagenesis to replace six deamidation-prone asparagine residues, at positions 408, 466, 537, 601, 713, and 719, with either aspartate, glutamine, or alanine residues. We found that the structure of the six-Asp mutant rPA was not significantly altered relative to that of the wild-type protein as assessed by circular dichroism (CD) spectroscopy and biological activity.

Structural and immunological analysis of anthrax recombinant protective antigen adsorbed to aluminum hydroxide adjuvant.

New anthrax vaccines currently under development are based on recombinant protective antigen (rPA) and formulated with aluminum adjuvant. Because long-term stability is a desired characteristic of these vaccines, an understanding of the effects of adsorption to aluminum adjuvants on the structure of rPA is important. Using both biophysical and immunological techniques, we compared the structure and immunogenicity of freshly prepared rPA-Alhydrogel formulations to that of formulations stored for 3 weeks at either room temperature or 37°C in order to assess the changes in rPA structure that might occur upon long-term storage on aluminum adjuvant.

Licensure of vaccines using the Animal Rule.

A number of new vaccines are being developed against microbial pathogens that might be used as bioweapons. For some of these vaccines, because of the lack of endemic disease and the lethal nature of the disease, human efficacy studies would not be ethical or feasible to conduct. In such cases, a regulation, known as the 'Animal Rule', can be used which allows the United States Food and Drug Administration to consider appropriate animal studies as evidence of effectiveness of a vaccine.

Analysis of defined combinations of monoclonal antibodies in anthrax toxin neutralization assays and their synergistic action.

Antibodies against the protective antigen (PA) component of anthrax toxin play an important role in protection against disease caused by Bacillus anthracis. In this study, we examined defined combinations of PA-specific monoclonal antibodies for their ability to neutralize anthrax toxin in cell culture assays. We observed additive, synergistic, and antagonistic effects of the antibodies depending on the specific antibody combination examined and the specific assay used.

Analysis of antibody responses to protective antigen-based anthrax vaccines through use of competitive assays.

The licensed anthrax vaccine and many of the new anthrax vaccines being developed are based on protective antigen (PA), a nontoxic component of anthrax toxin. For this reason, an understanding of the immune response to PA vaccination is important. In this study, we examined the antibody response elicited by PA-based vaccines and identified the domains of PA that contribute to that response in humans as well as nonhuman primates (NHPs) and rabbits, animal species that will be used to generate efficacy data to support approval of new anthrax vaccines.

Comparison of three anthrax toxin neutralization assays.

Different types of anthrax toxin neutralization assays have been utilized to measure the antibody levels elicited by anthrax vaccines in both nonclinical and clinical studies. In the present study, we sought to determine whether three commonly used toxin neutralization assays-J774A.1 cell-, RAW 264.

Analysis of the Fc gamma receptor-dependent component of neutralization measured by anthrax toxin neutralization assays.

Anthrax toxin neutralization assays are used to measure functional antibody levels elicited by anthrax vaccines in both preclinical and clinical studies. In this study, we investigated the magnitude and molecular nature of Fc gamma (Fcgamma) receptor-dependent toxin neutralization observed in commonly used forms of the anthrax toxin neutralization assay. Significantly more Fcgamma receptor-dependent neutralization was observed in the J774A.

Role of the N-terminal amino acid of Bacillus anthracis lethal factor in lethal toxin cytotoxicity and its effect on the lethal toxin neutralization assay.

The cytotoxic activity of lethal factor (LF), a critical reagent used in the cell-based lethal toxin neutralization assay to assess anthrax vaccines, was shown to depend on the identity of its N-terminal amino acid, which plays a role in the targeting of LF to the proteasome for degradation. These results demonstrate that care must be taken to ensure that LF preparations used in standardized cell-based assays are not altered at their N-terminal ends.

Stabilization of the pertussis toxin secretion apparatus by the C terminus of PtlD.

Pertussis toxin (PT) is secreted from Bordetella pertussis by a type IV secretion system, known as the Ptl transporter, that comprises nine different proteins, PtlA to PtlI. In this study, we found that PtlD is required for the stability of three Ptl proteins, PtlE, PtlF, and PtlH. A region limited to the C-terminal 72 amino acids of PtlD (amino acids 392 to 463) was sufficient for maintaining the stability of PtlE, PtlF, and PtlH, although this region was not sufficient to support secretion of the toxin.