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Biomedical subjects

S F Little

Publications and source records attributed to S F Little.

At least 19 recordsLinked to original sources
PubMed2006

Comparative vaccine efficacy of different isoforms of recombinant protective antigen against Bacillus anthracis spore challenge in rabbits.

The next-generation human anthrax vaccine developed by the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) is based upon purified Bacillus anthracis recombinant protective antigen (rPA) adsorbed to aluminum hydroxide adjuvant (Alhydrogel). In addition to being safe, and effective, it is important that such a vaccine be fully characterized. Four major protein isoforms detected in purified rPA by native PAGE during research and development were reduced to two primary isoforms in bulk material produced by an improved process performed under Good Manufacturing Practices (GMP). Analysis of both rPA preparations by a protein-isoaspartyl-methyl-transferase assay (PIMT) revealed the presence of increasing amounts of iso-aspartic acid correlating with isoform content and suggesting deamidation as the source of rPA charge heterogeneity. Additional purification of GMP rPA by anion exchange chromatography separated and enriched the two principal isoforms. The in vitro and in vivo biological activities of each isoform were measured in comparison to the whole GMP preparation. There was no significant difference in the biological activity of each isoform compared to GMP rPA when analyzed in the presence of lethal factor using a macrophage lysis assay. Vaccination with the two individual isoforms revealed no differences in cytotoxicity neutralization antibody titers when compared to the GMP preparation although one isoform induced more anti-PA IgG antibody than the GMP material. Most importantly, each of the two isoforms as well as the whole GMP preparation protected 90-100% of rabbits challenged parenterally with 129 LD50 of B. anthracis Ames spores. The equivalent biological activity and vaccine efficacy of the two isoforms suggests that further processing to separate isoforms is unnecessary for continued testing of this next-generation anthrax vaccine.

Animals↗
PubMed2005

Duration of protection of rabbits after vaccination with Bacillus anthracis recombinant protective antigen vaccine.

Long-term protection of rabbits that had been vaccinated with two doses of a recombinant protective antigen (rPA) vaccine was examined against an aerosol spore challenge with the Ames isolate of Bacillus anthracis at 6 and 12 months. At 6 months after the primary injection, survival was 74.1% (20/27) with quantitative ELISA titer of 22.3 microg of anti-rPA IgG per millilitre and toxin neutralizing antibody (TNA) assay titer of 332. At 12 months after the primary injection, only 37.5% (9/24) of the rabbits were protected with quantitative ELISA titer of 19.8 microg of anti-rPA IgG per millilitre and TNA assay titer of 286. There was a significant loss of protection (p = 0.0117) and a significant difference in survival curves (p = 0.0157) between the 6- and 12-month groups. When ELISA or TNA assay titer, gender, and challenge dose were entered into a forward logistic regression model, week 26 ELISA titer (p = 0.0236) and week 13 TNA assay titer (p = 0.0147) for the 6-month group, and week 26 ELISA titer (p = 0.0326) and week 8 TNA assay titer (p = 0.0190) for the 12-month group, were significant predictors of survival. Neither gender nor challenge dose were identified as having a statistically significant effect on survival. Booster vaccinations with rPA may be required for the long-term protection of rabbits against anthrax.

Animals↗
PubMed2004

Development of an in vitro-based potency assay for anthrax vaccine.

The potency assay currently used to evaluate consistency of manufacture for the anthrax vaccine is contingent upon meeting specified parameters after statistical analysis of the percent survival and time to death of vaccinated guinea pigs after challenge with spores of a virulent strain of Bacillus anthracis. During the development of a new anthrax vaccine based upon recombinant protective antigen (rPA) adsorbed to aluminum hydroxide gel (Alhydrogel), we found that the serological response of female A/J mice, as measured by a quantitative anti-rPA IgG ELISA, may be an effective method to monitor a manufacturer's consistency for rPA-based vaccines. An advantage of the proposed in vitro-based potency assay is that it will not need stringent biosafety containment measures as required by the current guinea pig potency assay.

Animals↗
PubMed2004

Defining a serological correlate of protection in rabbits for a recombinant anthrax vaccine.

In these studies, a serological correlate of protection against anthrax was identified in New Zealand white (NZW) rabbits that had been given one or two injections of various amounts of recombinant protective antigen (rPA) combined with aluminum hydroxide adjuvant (Alhydrogel). Rabbits were subsequently challenged by the aerosol route with spores of the Ames isolate of Bacillus anthracis. Results suggested that the antibody response, as determined by the quantitative anti-rPA IgG ELISA and toxin neutralizing antibody (TNA) assay, were significant predictors (P<0.0015) of protection against a B. anthracis aerosol spore challenge in rabbits.

Adjuvants, Immunologic↗
PubMed2004

Evaluation of an anti-rPA IgG ELISA for measuring the antibody response in mice.

A recombinant protective antigen (rPA)-based enzyme-linked immunosorbent assay (ELISA) was developed to measure the serological response of female A/J mice after inoculation with the new rPA-based anthrax vaccine. Several fundamental parameters of the ELISA were evaluated: specificity, precision, accuracy, linearity, and stability. Experimental results suggested that the quantitative anti-rPA IgG ELISA could be used to measure antibody levels in female A/J mice and may be useful as a potency assay to monitor consistency of manufacture of a rPA-based vaccine for planned clinical trials.

Animals↗
PubMed2002

Anthrax vaccine efficacy in golden Syrian hamsters.

The efficacy of a licensed human anthrax vaccine (anthrax vaccine adsorbed, AVA) was tested in golden Syrian hamsters against a virulent Bacillus anthracis spore challenge. Groups of golden Syrian hamsters were vaccinated at either 0 and 4 weeks or 0, 4 and 8 weeks, then challenged subcutaneously (s.c.) at 10 weeks with spores of various B. anthracis isolates. Although ELISA and toxin neutralization assays demonstrated high titers, none of the AVA-vaccinated hamsters were protected from challenge or demonstrated a significantly extended time to death compared to that of control animals. The results of the study demonstrate that the golden Syrian hamster is not an appropriate model for investigating human anthrax vaccine efficacy.

Animals↗
PubMed2001

In vitro correlate of immunity in a rabbit model of inhalational anthrax.

A serological correlate of vaccine-induced immunity was identified in the rabbit model of inhalational anthrax. Animals were inoculated intramuscularly at 0 and 4 weeks with varying doses of Anthrax Vaccine Adsorbed (AVA) ranging from a human dose to a 1:256 dilution in phosphate-buffered saline (PBS). At 6 and 10 weeks, both the quantitative anti-protective antigen (PA) IgG ELISA and the toxin-neutralizing antibody (TNA) assays were used to measure antibody levels to PA. Rabbits were aerosol-challenged at 10 weeks with a lethal dose (84-133 LD(50)) of Bacillus anthracis spores. All the rabbits that received the undiluted and 1:4 dilution of vaccine survived, whereas those receiving the higher dilutions of vaccine (1:16, 1:64 and 1:256) had deaths in their groups. Results showed that antibody levels to PA at both 6 and 10 weeks were significant (P<0.0001) predictors of survival.

Administration, Inhalation↗
PubMed1999

Molecular pathogenesis of Bacillus anthracis infection.

This review summarizes the current knowledge pertaining to the pathogenesis of infection with Bacillus anthracis relative to the two exotoxins and the capsule. Emphasis is given to the structure and activities of the individual components of the exotoxins, their interaction with cells, and the response of macrophages to lethal toxin. Finally, results from vaccination studies are reviewed.

Animals↗
PubMed1998

Comparative efficacy of experimental anthrax vaccine candidates against inhalation anthrax in rhesus macaques.

The authors examined the efficacy of Bacillus anthracis protective antigen (PA) combined with adjuvants as vaccines against an aerosol challenge of virulent anthrax spores in rhesus macaques. Adjuvants tested included i) aluminum hydroxide (Alhydrogel), ii) saponin QS-21 and iii) monophosphoryl lipid A (MPL) in squalene/lecithin/Tween 80 emulsion (SLT). Animals were immunized once with either 50 micrograms of recombinant PA plus adjuvant, or with Anthrax Vaccine Adsorbed (AVA), the licensed human anthrax vaccine. The serological response to PA was measured by enzyme linked immunosorbent assay. Lymphocyte proliferation and serum neutralization of in vitro lethal toxin cytotoxicity were also assayed. In all vaccine groups, anti-PA IgM and IgG titers peaked at 2 weeks and 4-5 weeks postimmunization, respectively. Five weeks postimmunization, animals in all vaccine groups demonstrated PA-specific lymphocyte proliferation and sera that neutralized in vitro cytotoxicity. Six weeks after immunization, the animals were challenged by aerosol with approximately 93 LD50 of virulent anthrax spores. Animals were bled daily for 1 week to monitor bacteremia, and deaths were recorded. Anti-PA ELISA titers in all groups of immunized animals were substantially increased 2 weeks after challenge. One dose of each vaccine provided significant protection (> 90%) against inhalation anthrax in the rhesus macaques.

Administration, Inhalation↗
PubMed1998

Fermentation, purification, and characterization of protective antigen from a recombinant, avirulent strain of Bacillus anthracis.

Bacillus anthracis, the etiologic agent for anthrax, produces two bipartite, AB-type exotoxins, edema toxin and lethal toxin. The B subunit of both exotoxins is an M(r) 83,000 protein termed protective antigen (PA). The human anthrax vaccine currently licensed for use in the United States consists primarily of this protein adsorbed onto aluminum oxyhydroxide. This report describes the production of PA from a recombinant, asporogenic, nontoxigenic, and nonencapsulated host strain of B. anthracis and the subsequent purification and characterization of the protein product. Fermentation in a high-tryptone, high-yeast-extract medium under nonlimiting aeration produced 20 to 30 mg of secreted PA per liter. Secreted protease activity under these fermentation conditions was low and was inhibited more than 95% by the addition of EDTA. A purity of 88 to 93% was achieved for PA by diafiltration and anion-exchange chromatography, while greater than 95% final purity was achieved with an additional hydrophobic interaction chromatography step. The purity of the PA product was characterized by reversed-phase high-pressure liquid chromatography, sodium dodecyl sulfate (SDS)-capillary electrophoresis, capillary isoelectric focusing, native gel electrophoresis, and SDS-polyacrylamide gel electrophoresis. The biological activity of the PA, when combined with excess lethal factor in the macrophage cell lysis assay, was comparable to previously reported values.

Antigens, Bacterial↗
PubMed1997

Passive protection by polyclonal antibodies against Bacillus anthracis infection in guinea pigs.

The protective effects of polyclonal antisera produced by injecting guinea pigs with protective antigen (PA), the chemical anthrax vaccine AVA, or Sterne spore vaccine, as well as those of toxin-neutralizing monoclonal antibodies (MAbs) produced against PA, lethal factor, and edema factor, were examined in animals infected with Bacillus anthracis spores. Only the anti-PA polyclonal serum significantly protected the guinea pigs from death, with 67% of infected animals surviving. Although none of the MAbs was protective, one PA MAb caused a significant delay in time to death. Our findings demonstrate that antibodies produced against only PA can provide passive protection against anthrax infection in guinea pigs.

Animals↗
PubMed1994

Structure-function analysis of Bacillus anthracis edema factor by using monoclonal antibodies.

Edema toxin of Bacillus anthracis is composed of protective antigen (PA) and edema factor (EF), a calcium- and calmodulin-dependent adenylate cyclase. At least five different antigenic regions of EF were identified using a competitive-binding, enzyme-linked immunosorbent assay of paired monoclonal antibodies (mAbs). Two mAbs, 9F5 and 7G10, inhibited the binding of 125I-EF to cell-bound PA. However, only 9F5 inhibited the elongation response of Chinese hamster ovary cells in the presence of edema toxin. Cleavage of EF at the two aspartic acid-proline residues by acid hydrolysis resulted in three fragments: a C-terminal 17 kDa fragment, a central 53 kDa fragment, and an N-terminal 18 kDa fragment. Immunoblots of EF cleaved by formic acid mapped mAbs 9F5 and 7G10 to the N-terminal 18 kDa fragment, mAb 1E6 to the C-terminal 17 kDa fragment, and the remaining 7 mAbs to the central 53 kDa fragment. mAbs 7G10 and 9F5 defined an antigenic region within amino acids 1-156 of EF which is involved in interaction with PA in forming edema toxin.

Adenylyl Cyclase Inhibitors↗
PubMed1992

Functional characterization of protease-treated Bacillus anthracis protective antigen.

Characterization of the functional domains of Bacillus anthracis protective antigen (PA, 83-kDa), the common cellular binding molecule for both anthrax edema toxin and anthrax lethal toxin, is important for understanding the mechanism of entry and action of the anthrax toxins. In this study, we generated both biologically active (facilitates killing of J774A.1 cells in combination with lethal factor, LF) and inactive preparations of PA by protease treatment. Limited proteolytic digestion of PA in vitro with trypsin generated a 20-kDa fragment and a biologically active 63-kDa fragment. In contrast, limited digestion of PA with chymotrypsin yielded a preparation containing 37- and 47-kDa fragments defective for biological activity. Treatment with both chymotrypsin and trypsin generated three major fragments, 20, "17," and 47 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This PA preparation was also biologically inactive. To investigate the nature of the defect resulting from chymotrypsin treatment, we assayed PA preparations for the ability to bind to the cellular receptor and to bind and internalize 125I-LF. All radiolabeled PA preparations bound with specificity to J774A.1 cells and exhibited affinities similar to native 83-kDa PA. Once bound to the cell surface receptor, both trypsin-treated PA and chymotrypsin/trypsin-treated PA specifically bound 125I-LF with high affinity. Finally, these PA preparations delivered 125I-LF to a Pronase-resistant cellular compartment in a time- and temperature-dependent fashion. Thus, the biological defect exhibited by chymotrypsin-treated PA is not at the level of cell binding or internalization but at a step later, such as toxin routing or processing by J774A.1 cells. These protease-treated preparations of PA should prove useful in both elucidating the intracellular processing of anthrax lethal toxin and determining the structure-function relationship of PA and LF.

Animals↗
PubMed1992

Immunization against anthrax with Bacillus anthracis protective antigen combined with adjuvants.

The protective efficacy of immunization against anthrax with Bacillus anthracis protective antigen (PA) combined with different adjuvants was tested in Hartley guinea pigs and CBA/J and A/J mice. Adjuvant components derived from microbial products that were tested included threonyl-muramyl dipeptide (threonyl-MDP); monophosphoryl lipid A (MPL); trehalose dimycolate (TDM); and the delipidated, deproteinized, cell wall skeleton (CWS) from either Mycobacterium phlei or the BCG strain of Mycobacterium bovis. Non-microbially derived adjuvants tested included aluminum hydroxide and the lipid amine CP-20,961. In guinea pigs, all adjuvants and adjuvant mixtures enhanced antibody titers to PA as well as survival after a parenteral challenge of virulent B. anthracis Ames spores. In contrast, PA alone or combined with either aluminum hydroxide or CP-20,961 failed to protect mice. Vaccines containing PA combined with threonyl-MDP or MPL-TDM-CWS protected a majority of female CBA/J mice. Statistical analysis of survival data in the guinea pigs indicated that PA-MPL-CWS and PA-MPL-TDM-CWS were more efficacious than the currently licensed human anthrax vaccine.

Adjuvants, Immunologic↗
PubMed1991

Location of receptor-binding region of protective antigen from Bacillus anthracis.

The pag gene, which codes for protective antigen (PA), a common component of the lethal and edema toxins of Bacillus anthracis, was cloned and expressed in Escherichia coli. Nested deletions of pag were generated into the C-terminus coding region. Recombinant proteins were analyzed by Western blot with either an anti-PA polyclonal antisera or two monoclonal antibodies that neutralized lethal toxin and edema toxin activities by inhibiting the binding of PA to cell receptors. Localization of the receptor binding domain within the C-terminal region of PA was suggested by the inability of the monoclonal antibodies 3B6 and 14B7 to recognize the recombinant proteins expressed by C-terminal deletions of the pag gene.

Antigens, Bacterial↗
PubMed1990

Immunization against anthrax with aromatic compound-dependent (Aro-) mutants of Bacillus anthracis and with recombinant strains of Bacillus subtilis that produce anthrax protective antigen.

The safety and efficacy of five prototype, live anthrax vaccines were studied in Hartley guinea pigs and CBA/J and A/J mice. Two of the strains, Bacillus anthracis FD111 and FD112, are Aro- mutants derived by Tn916 mutagenesis of B. anthracis UM23-1. Bacillus subtilis PA1 and PA2 contain a recombinant plasmid, pPA101 or pPA102, respectively, that carries the gene from B. anthracis encoding synthesis of protective antigen (PA). The final strain, B. subtilis PA7, was isolated in this study from B. subtilis DB104 transformed with pPA101. All five strains were less virulent in guinea pigs and A/J and CBA/J mice than the toxinogenic, nonencapsulated B. anthracis veterinary vaccine Sterne strain. A/J and CBA/J inbred mice represent strains that are innately susceptible and resistant, respectively, to the Sterne strain. These differences in susceptibility are due to differences in ability to produce complement component 5. In guinea pigs, immunization with PA1 or PA2 vegetative cells or PA7 spores protected greater than or equal to 95% from an intramuscular spore challenge with the virulent, "vaccine-resistant" B. anthracis Ames strain. Strain PA2 vegetative cells and strain PA7 spores were as effective as the Sterne strain in Sterne-resistant CBA/J mice, protecting 70% of the mice from Ames strain spore challenge. Immunization with FD111 or FD112 vegetative cells fully protected guinea pigs from challenge. Immunization with FD111 cells protected up to 100% of CBA/J mice and up to 70% of A/J mice.

Animals↗
PubMed1990

Production and characterization of monoclonal antibodies against the lethal factor component of Bacillus anthracis lethal toxin.

The lethal toxin of Bacillus anthracis consists of two components, protective antigen and lethal factor. Protective antigen is cleaved after binding to cell receptors, yielding a receptor-bound fragment that binds lethal factor. Sixty-one monoclonal antibodies to the lethal factor protein have been characterized for specificity, antibody subtype, and ability to neutralize lethal toxin. Three monoclonal antibodies (10G3, 2E7, and 3F6) neutralized lethal toxin in Fisher 344 rats. However, in a macrophage cytolysis assay, monoclonal antibodies 10G3, 2E7, 10G4, 10D4, 13D10, and 1D8, but not 3F6, were found to neutralize lethal toxin. Binding studies showed that five of the monoclonal antibodies that neutralized lethal toxin in the macrophage assay (10G3, 2E7, 10G4, 10D4, and 13D10) did so by inhibiting the binding of lethal factor to the protective antigen fragment bound to cells. Monoclonal antibody 1D8, which was also able to neutralize lethal toxin activity after lethal factor was prebound to cell-bound protective antigen, only partially inhibited binding of lethal factor to protective antigen. Monoclonal antibody 3F6 did not inhibit the binding of lethal factor to protective antigen. A competitive-binding enzyme-linked immunosorbent assay showed that at least four different antigenic regions on lethal factor were recognized by these seven neutralizing hybridomas. The anomalous behavior of 3F6 suggests that it may induce a conformational change in lethal factor. Differences in neutralizing activity of monoclonal antibodies were related to their relative affinity and epitope specificity and the type of assay.

Animals↗