Protective antigen domain 4 of Bacillus anthracis as a candidate for use as vaccine for anthrax.

Existing research for using the protective antigen (PA) of Bacillus anthracis as a vaccine component shows that protection against anthrax may be obtained using fragments of this protein. The aim of the research is to check whether the selected protein fragment of the protective antigen (domain 4) encoded by an appropriate nucleotide sequence of gene pag of B. anthracis, was expressed in the bacterial system of E. coli. In order to examine the selected sequence of the pag gene, a PCR reaction and a highly effective TOPO cloning strategy were used, followed by purification of the recombinant proteins and their detection by a western-blot method. In the planning of the PA4 antigen expression a higher level of effectiveness in production of small protein - domain 4 - was anticipated. As a result, the 139 amino acids protein fragment of B. anthracis PA (domain 4) was isolated. The research may have found the basis for in vivo research aimed at finding potential anthrax vaccine components.

Development of a novel multiepitope chimeric vaccine against anthrax.

Bacillus anthracis (BA), the etiological agent of anthrax, secretes protective antigen (PA), lethal factor (LF), and edema factor (EF) as major virulence mediators. Amongst these, PA-based vaccines are most effective for providing immunity against BA, but their low shelf life limits their usage. Previous studies showed that B-cell epitopes, ID II and ID III present in PA domain IV possess higher toxin neutralization activity and elicit higher antibody titer than ID I. Moreover, N-terminal region of both LF and EF harbors PA-binding sites which share 100% identity with each other. Here, in this study, we have developed an epitope-based chimeric vaccine (ID-LFn) comprising ID II-ID III region of PA and N-terminal region of LF. We have also evaluated its protective efficacy as well as stability and found it to be more stable than PA-based vaccine. Binding reactivities of ID-LFn with anti-PA/LF/EF antibodies were determined by ELISA. The stability of chimeric vaccine was assessed using circular dichroism spectroscopy. ID-LFn response was characterized by toxin neutralization, lymphocyte proliferation isotyping and cytokine profiling. The protective efficacy was analyzed by challenging ID-LFn-immunized mice with B. anthracis (pXO1+ and pXO2+). ID-LFn was found to be significantly stable as compared to PA. Anti-ID-LFn antibodies recognized PA, LF as well as EF. The T-cell response and the protective efficacy of ID-LFn were found to be almost similar to PA. ID-LFn exhibits equal protective efficacy in mice and possesses more stability as compared to PA along with the capability of recognizing PA, LF and EF at the same time. Thus, it can be considered as an improved vaccine against anthrax with better shelf life. ID-LFn, a novel multiepitope chimeric anthrax vaccine: ID-LFn comprises of immunodominant epitopes of domain 4 of PA and N-terminal homologous stretch of LF and EF. The administration of this protein as a vaccine provides protection against anthrax.

Neutralizing antibody and functional mapping of Bacillus anthracis protective antigen-The first step toward a rationally designed anthrax vaccine.

Anthrax is defined by the Centers for Disease Control and Prevention as a Category A pathogen for its potential use as a bioweapon. Current prevention treatments include Anthrax Vaccine Adsorbed (AVA). AVA is an undefined formulation of Bacillus anthracis culture supernatant adsorbed to aluminum hydroxide. It has an onerous vaccination schedule, is slow and cumbersome to produce and is slightly reactogenic. Next-generation vaccines are focused on producing recombinant forms of anthrax toxin in a well-defined formulation but these vaccines have been shown to lose potency as they are stored. In addition, studies have shown that a proportion of the antibody response against these vaccines is focused on non-functional, non-neutralizing regions of the anthrax toxin while some essential functional regions are shielded from eliciting an antibody response. Rational vaccinology is a developing field that focuses on designing vaccine antigens based on structural information provided by neutralizing antibody epitope mapping, crystal structure analysis, and functional mapping through amino acid mutations. This information provides an opportunity to design antigens that target only functionally important and conserved regions of a pathogen in order to make a more optimal vaccine product. This review provides an overview of the literature related to functional and neutralizing antibody epitope mapping of the Protective Antigen (PA) component of anthrax toxin.

Host immunity to Bacillus anthracis lethal factor and other immunogens: implications for vaccine design.

Infections of humans with Bacillus anthracis are an issue with respect to the biothreat both to civilians and military personnel, infections of individuals by infected livestock in endemic regions and, recently, infections of intravenous drug users injecting anthrax-contaminated heroin. Existing vaccination regimens are reliant on protective antigen neutralization induced by repeated boosts with the AVA or AVP vaccines. However, there is ongoing interest in updated approaches in light of the intensive booster regime and extent of reactogenicity inherent in the current protocols. Several other immunogens from the B. anthracis proteome have been characterized in recent years, including lethal factor. Lethal factor induces strong CD4 T-cell immunity and encompasses immunodominant epitopes of relevance across diverse HLA polymorphisms. Taken together, recent studies emphasize the potential benefits of vaccines able to confer synergistic immunity to protective antigen and to other immunogens, targeting both B-cell and T-cell repertoires.

Vaccines: facing complex problems with the promise of immunology.

Highly renowned in the vaccines world, Stanley A. Plotkin has worked at many leading institutions throughout his career, and is Emeritus Professor of the University of Pennsylvania and Adjunct Professor of the Johns Hopkins University. In 1991, Plotkin joined Sanofi Pasteur and worked there from 1991 to 1997, and now works as principal of Vaxconsult, LLC as a consultant to vaccine manufacturers, biotechnology companies and non-profit research organizations. Plotkin has served as chairman of the Infectious Diseases Committee and the AIDS Task Force of the American Academy of Pediatrics, liaison member of the Advisory Committee on Immunization Practices, and Chairman of the Microbiology and Infectious Diseases Research Committee of the National Institutes of Health. He has been a recipient of numerous prestigious medals and awards throughout his career, and his bibliography includes over 700 articles and several books, including the standard textbook on vaccines. He has worked extensively on the development and application of many vaccines including anthrax, oral polio, rabies, varicella and cytomegalovirus. He is also codeveloper of the newly licensed pentavalent rotavirus and is well-known for developing the rubella vaccine, now in standard use throughout the world.

Plant-produced hepatitis B core protein chimera carrying anthrax protective antigen domain-4.

The hepatitis B core antigen (HBcAg) can generate a strong immune response and is recognized as an effective carrier for foreign epitopes. The domain-4 epitope of the anthrax protective antigen (PA-D4) plays an essential role in generating protective immunity against virulent Bacillus anthracis. Here we report the successful production of a recombinant protein comprised of the antigenic PA-D4 integrated into the c/e1 loop of HBcAg in transgenic low-alkaloid Nicotiana tabacum. Sera of mice injected with the plant-derived purified HB/PA-D4 protein exhibited significant anti-PA- and anti-HBcAg-specific IgG titers; however, formation of virus-like particles (VLP) was not observed. These data support the feasibility of producing complex protein chimeras in plants.

2-D reference map of Bacillus anthracis vaccine strain A16R proteins.

Bacillus anthracis has always been an important pathogen because it can cause lethal inhalational anthrax, and may be used as a bioweapon or by bioterrorists. In this study, a 2-DE reference map and database of B. anthracis A16R was constructed. In total, 534 spots were processed, and 406 spots representing 299 proteins were identified. Gel-estimated pIs and molecular masses mostly matched well with their theoretical predictions, but some discrepancies also existed. Spot and protein corresponding analysis revealed that post-translational modifications might be common in B. anthracis. Through the MASCOT search, the similarity of B. anthracis, B. cereus and B. thuringiensis was further verified by protein level and a possible annotation error in B. anthracis strain Ames 0581 genome was found. Proteins of energy metabolism, fatty acid and phospholipid metabolism, protein synthesis, and cellular processes represented a large part of the most abundant proteins. At the same time, 27 hypothetical proteins were experimentally proved. There were 28 proteins also identified as spore composition in recently spore-related research, which indicated that they might play some roles in different phases such as growth, sporulation and outgrowth. Maps and information about all identified proteins are available on the Internet at http://www.mpiib-berlin.mpg.de/2D-PAGE and http://www.proteomics.com.cn.

Efficacy of non-toxic deletion mutants of protective antigen from Bacillus anthracis.

Current human anthrax vaccines available in the United States and Europe consist of alum-precipitated supernatant material from cultures of a toxigenic, nonencapsulated strain of Bacillus anthracis. The major component of human anthrax vaccine that confers protection is protective antigen (PA). A second-generation human vaccine using the recombinant PA (rPA) is being developed. In this study, to prevent the toxicity and the degradation of the native rPA by proteases, we constructed two PA variants, delPA (163-168) and delPA (313-314), that lack trypsin (S(163)-R(164)-K(165)-K(166)-R(167)-S(168)) or chymotrypsin cleavage sequence (F(313)-F(314)), respectively. These proteins were expressed in Bacillus brevis 47-5Q. The delPAs were fractionated from the culture supernatant of B. brevis by ammonium sulfate at 70% saturation, followed by anion exchange chromatography on a Hitrap Q, Hiload 16/60 superdex 200 gel filtration column and phenyl sepharose hydrophobic interaction column. In accordance with previous reports, both delPA proteins combined with lethal factor protein did not show any cytotoxicity on J774A.1 cells. The delPA (163-168) and delPA (313-314) formulated either in Rehydragel HPA or MPL-TDM-CWS (Ribi-Trimix), elicited a comparable amount of anti-PA and neutralizing antibodies to those of native rPA in guinea pigs, and confers full protection of guinea pigs from 50xLD50 of fully virulent B. anthracis spore challenges. Ribi-Trimix was significantly more effective in inducing anti-PA and neutralizing antibodies than Rehydragel HPA. These results indicate the possibility of delPA (163-168) and delPA (313-314) proteins being developed into nontoxic, effective and stable recombinant vaccine candidates.

Anthrax vaccine: short-term safety experience in humans.

Bacillus anthracis is the major terrorist and biological warfare agent of concern to civilian and military medical planners. The licensed anthrax vaccine, adsorbed (AVA) is believed to be an effective prophylactic medical countermeasure against this threat. Our objective in this report was to expand the safety database for this vaccine by assessing data on self-reported, short-term safety of AVA during more than 25 years of use, measured by local and systemic adverse events temporally associated with the administration of AVA. A minority of AVA recipients reported systemic and injection site reactions. Females reported a higher incidence of injection site and systemic adverse events than males. Data show a difference in incidence of local reactions between lots. A prospective, randomized, placebo-controlled study to actively examine reactogenicity is needed to more completely define the extent and nature of reactions associated with receipt of AVA in humans as well as to confirm the gender lot differences in local reaction rates.