Morbidity and Mortality Weekly Report [Morb. Mortal. Weekly Rep.], vol. 49, no. 16, pp. 341-345, Apr 2000

Concerns about the potential use of anthrax as a biologic weapon prompted the U.S. Department of Defense (DoD) to announce on December 15, 1997, anthrax vaccination of all U.S. military personnel. This effort is coordinated by the Anthrax Vaccine Immunization Program (AVIP). AVIP plans a phased vaccination process to achieve total force protection against anthrax by 2004. The current phase of implementation includes vaccination of all service members and mission-essential DoD civilian employees assigned or deployed to high-threat areas. On the basis of program monitoring, as of April 12, 2000, 425,976 service members had received 1,620,793 doses of anthrax vaccine adsorbed (AVA) (Bioport, Inc., Lansing, Michigan). Some service members have cited concerns about vaccine safety and efficacy in their decision to refuse vaccination, despite the possibility of administrative or disciplinary actions. To assess anthrax vaccination safety, DoD has conducted surveys of vaccinated personnel. This report describes three completed or ongoing surveys. The findings indicate that rates of local reactions were higher in women than men and that no patterns of unexpected local or systemic adverse events have been identified.

Turnbull, PCB

Current Opinion in Infectious Diseases [Curr. Opin. Infect. Dis.], vol. 13, no. 2, pp. 113-120, Apr 2000

Anthrax vaccination has become a `hot' topic. On the one hand, fears that Iraq holds secret caches of anthrax-based weaponry, that other countries may be developing or may have developed similar devices, or that hard-line groups may make their own anthrax-based devices for bioterrorist attacks have focused official attention on the need for means of protection, principally, though, for the military. On the other hand, the unsolved issues of the Gulf War illnesses have left elements of doubt in the minds of some as to the possible role of anthrax (among other) vaccines in this syndrome, and have drawn attention to the shortage of pre-clinical, clinical, pharmacological and safety data on the existing UK and US anthrax vaccines. In the middle are those hotly debating the US and Canadian policies of mandatory anthrax immunization for military personnel or, in the case of the UK policy of voluntary immunization, simply voting with their feet. Compounding matters have been the publicized failures of the US vaccine production facility and the less publicized UK problems of supply. Meanwhile, those in genuine at-risk occupations are left unsure whether, if they can get the vaccine at all, they really want it. Despite two decades of elegant science aimed at formulating alternative vaccines to overcome all the problems of efficacy, safety and supply, such an alternative is at least five years away, and the current status is that we must live with the old vaccines or not vaccinate.

Price, BM; Liner, AL; Park, S; Leppla, SH; Mateczun, A; Galloway, DR*

Infection and Immunity [Infect. Immun.], vol. 69, no. 7, pp. 4509-4515, Jul 2001

The ability of genetic vaccination to protect against a lethal challenge of anthrax toxin was evaluated. BALB/c mice were immunized via gene gun inoculation with eucaryotic expression vector plasmids encoding either a fragment of the protective antigen (PA) or a fragment of lethal factor (LF). Plasmid pCLF4 contains the N- terminal region (amino acids [aa] 10 to 254) of Bacillus anthracis LF cloned into the pCI expression plasmid. Plasmid pCPA contains a biologically active portion (aa 175 to 764) of B. anthracis PA cloned into the pCI expression vector. One-micrometer-diameter gold particles were coated with plasmid pCLF4 or pCPA or a 1:1 mixture of both and injected into mice via gene gun (1 mu g of plasmid DNA/injection) three times at 2-week intervals. Sera were collected and analyzed for antibody titer as well as antibody isotype. Significantly, titers of antibody to both PA and LF from mice immunized with the combination of pCPA and pCLF4 were four to five times greater than titers from mice immunized with either gene alone. Two weeks following the third and final plasmid DNA boost, all mice were challenged with 5 50% lethal doses of lethal toxin (PA plus LF) injected intravenously into the tail vein. All mice immunized with pCLF4, pCPA, or the combination of both survived the challenge, whereas all unimmunized mice did not survive. These results demonstrate that DNA-based immunization alone can provide protection against a lethal toxin challenge and that DNA immunization against the LF antigen alone provides complete protection.

Cohen, S; Mendelson, I; Altboum, Z; Kobiler, D; Elhanany, E; Bino, T; Leitner, M; Inbar, I; Rosenberg, H; Gozes, Y; Barak, R; Fisher, M; Kronman, C; Velan, B; Shafferman, A

Infection and Immunity [Infect. Immun.], vol. 68, no. 8, pp. 4549-4558, Aug 2000

Several highly attenuated spore-forming nontoxinogenic and nonencapsulated Bacillus anthracis vaccines differing in levels of expression of recombinant protective antigen (rPA) were constructed. Biochemical analyses (including electrospray mass spectroscopy and N terminus amino acid sequencing) as well as biological and immunological tests demonstrated that the rPA retains the characteristics of native PA. A single immunization of guinea pigs with 5 x 10 super(7) spores of one of these recombinant strains, MASC-10, expressing high levels of rPA ( greater than or equal to 100 mu g/ml) from a constitutive heterologous promoter induced high titers of neutralizing anti-PA antibodies. This immune response was long lasting (at least 12 months) and provided protection against a lethal challenge of virulent (Vollum) anthrax spores. The recombinant B. anthracis spore vaccine appears to be more efficacious than the vegetative cell vaccine. Furthermore, while results clearly suggest a direct correlation between the level of expression of PA and the potency of the vaccine, they also suggest that some B. anthracis spore-associated antigen(s) may contribute in a significant manner to protective immunity.

Pittman, PR; Mangiafico, JA; Rossi, CA; Cannon, TL; Gibbs, PH; Parker, GW; Friedlander, AM

Vaccine, vol. 19, no. 2-3, pp. 213-216, 15 Sep 2000

The influence of dosing interval on the human antibody response to anthrax vaccine adsorbed (AVA) was evaluated in two retrospective serological studies. In both studies, the interval between the first two doses was 2, 3 or 4 weeks. In the first study, banked sera were selected from 89 at-risk individuals at a mean time of 13 days after the second dose of vaccine. In the second study, banked sera were selected from 51 at-risk individuals at a mean time of 48 days following the first dose of AVA. In both studies, the geometric mean anti-protective antigen IgG antibody titer increased significantly as the interval between the two doses increased from 2 to 4 weeks (p = 0.0005-0.029). In the first study, the seroconversion rate also increased as the interval between the first two doses increased (p = 0.0034). A prospective, randomized study has been completed and is being analyzed to confirm these findings.

Reuveny, S; White, MD; Adar, YY; Kafri, Y; Altboum, Z; Gozes, Y; Kobiler, D; Shafferman, A; Velan*, B

Infection and Immunity [Infect. Immun.], vol. 69, no. 5, pp. 2888-2893, May 2001

Vaccination by anthrax protective antigen (PA)-based vaccines requires multiple immunization, underlying the need to develop more efficacious vaccines or alternative vaccination regimens. In spite of the vast use of PA-based vaccines, the definition of a marker for protective immunity is still lacking. Here we describe studies designed to help define such markers. To this end we have immunized guinea pigs by different methods and monitored the immune response and the corresponding extent of protection against a lethal challenge with anthrax spores. Active immunization was performed by a single injection using one of two methods: (i) vaccination with decreasing amounts of PA and (ii) vaccination with constant amounts of PA that had been thermally inactivated for increasing periods. In both studies a direct correlation between survival and neutralizing-antibody titer was found (r = 0.92 and 0.95, respectively). Most significantly, in the two protocols a similar neutralizing- antibody titer range provided 50% protection. Furthermore, in a complementary study involving passive transfer of PA hyperimmune sera to naive animals, a similar correlation between neutralizing-antibody titers and protection was found. In all three immunization studies, neutralization titers of at least 300 were sufficient to confer protection against a dose of 40 50% lethal doses (LD sub(50)) of virulent anthrax spores of the Vollum strain. Such consistency in the correlation of protective immunity with anti-PA antibody titers was not observed for antibody titers determined by an enzyme-linked immunosorbent assay. Taken together, these results clearly demonstrate that neutralizing antibodies to PA constitute a major component of the protective immunity against anthrax and suggest that this parameter could be used as a surrogate marker for protection.

Fellows, PF; Linscott, MK; Ivins, BE; Pitt, MLM; Rossi, CA; Gibbs, PH; Friedlander, AM

Vaccine, vol. 19, no. 23-24, pp. 3241-3247, 30 Apr 2001

The efficacy of a licensed human anthrax vaccine (Anthrax Vaccine Adsorbed (AVA)) was tested in guinea pigs, rabbits, and rhesus macaques against spore challenge by Bacillus anthracis isolates of diverse geographical origin. Initially, groups of Hartley guinea pigs were vaccinated at 0 and 4 weeks with AVA, then challenged intramuscularly at 10 weeks with spores from 33 isolates of B. anthracis. Survival among the vaccinated groups varied from 6 to 100%, although there were no differences in mean time to death among the groups. There was no correlation between isolate virulence and variable number tandem repeat category or protective antigen genotype identified. New Zealand white rabbits were then vaccinated with AVA at 0 and 4 weeks, and challenged at 10 weeks by aerosol with spores from six of the isolates that were highly virulent in vaccinated guinea pigs. AVA completely protected the rabbits from four of the isolates, and protected 90% of the animals from the other two isolates. Subsequently, two of these six isolates were then used to challenge rhesus macaques, previously vaccinated with AVA at 0 and 4 weeks, and challenged at 10 weeks by aerosol. AVA protected 80 and 100% of the animals from these two isolates. These studies demonstrated that, although AVA confers variable protection against different B. anthracis isolates in guinea pigs, it is highly protective against these same isolates in both rabbits and rhesus macaques.

Cataldi, A; Mock, M; Bentancor, L

Journal of Applied Microbiology [J. Appl. Microbiol.], vol. 88, no. 4, pp. 648-654, Apr 2000

Three Bacillus anthracis strains, formerly used as anti-anthrax vaccine strains in Argentina, were characterized from genetic and pathogenic perspectives. Southern blotting and PCR with pXO1 and pXO2 probes and primers, as well as pathogenicity and protection tests in guinea pigs and mice, were performed. Two of the B. anthracis strains contained both pXO1 and pXO2 plasmids, as did the fully virulent strains, while the third was a Sterne-type strain (pXO1 super(+) , pXO2 super(-)). The three strains were, however, markedly less pathogenic than a wild-type virulent strain. The methodology applied here may be used to characterize other B. anthracis strains.

Lu, Y; Friedman, R; Kushner, N; Doling, A; Thomas, L; Touzjian, N; Starnbach, M; Lieberman, J

Proceedings of the National Academy of Sciences, USA [Proc. Natl. Acad. Sci. USA], vol. 97, no. 14, pp. 8027-8032, 5 Jul 2000

Bacillus anthrax lethal toxin can be engineered to deliver foreign proteins to the cytosol for antigen presentation to CD8 T cells. Vaccination with modified toxins carrying 8-9 amino acid peptide epitopes induces protective immunity in mice. To evaluate whether large protein antigens can be used with this system, recombinant constructs encoding several HIV antigens up to 500 amino acids were produced. These candidate HIV vaccines are safe in animals and induce CD8 T cells in mice. Constructs encoding gag p24 and nef stimulate gag-specific CD4 proliferation and a secondary cytotoxic T lymphocyte response in HIV-infected donor peripheral blood mononuclear cells in vitro. These results lay the foundation for future clinical vaccine studies.

Zhang, Ye; Kida, Yutaka; Kuwano, Koichi; Misumi, Yoshio; Ikehara, Yukio; Arai, Sumio

Microbiology and Immunology [Microbiol. Immunol.], vol. 45, no. 2, pp. 119-125, 2001

Anthrax toxin lethal factor (LF) in combination with anthrax toxin protective antigen (PA) was endocytosed and translocated to the cytosol of mammalian cells. Residues 1-255 of anthrax toxin lethal factor (LFn) was fused to a cytotoxic T lymphocyte (CTL) epitope of an influenza virus. For processing the toxins, PA must be cleaved into a 63-kDa fragment (PA63) by furin, which is a subtilisin-like processing endo-protease expressed by many eukaryotic cells. To test the ability of cells treated with the LFn fusion protein plus PA to deliver the epitope, CTL assay was performed. Two types of cell lines were identified, one was able to deliver CTL epitope while the other failed to efficiently deliver the epitope. To further elucidate the differences between these cells, the role of furin in these cells was examined. Disruption of the furin gene reduced its ability to deliver the CTL epitope. Furin expression in cells capable of efficiently delivering CTL epitope was quantitatively higher than in cells unable to deliver the epitope. The results suggest that furin plays a critical role in delivery of the CTL epitope of LFn fusion protein.

Snyder, JW

Clinical Microbiology Newsletter [Clin. Microbiol. Newsl.], vol. 23, no. 7, pp. 51-54, 1 Apr 2001

Anthrax continues to be a significant threat as a biological weapon for use in both military and domestic acts of bioterrorism. The current controversy and skepticism that surround the safety of the anthrax vaccine continue to be major topics of discussion since the Persian Gulf War. In 1970, the Food and Drug Administration (FDA) licensed a vaccine to protect humans who might be exposed to anthrax. This is the same vaccine that was used in the Gulf War and is currently being administered to military personnel. Although the vaccine has a proven safety record with over 30 years of use by thousands of military personnel, woolworkers, and veterinarians, the debate over its safety has come to the forefront in recent years. As a result of this concern, and with the recent FDA citation for quality control violations of the only licensed commercial producer of human vaccine in the United States, BioPort Corporation in Lansing, Michigan, Congress ordered an in-depth investigation into the safety of and the alleged link between the vaccine and illnesses reported by military personnel following the Persian Gulf War.

Chauhan, V; Singh, A; Waheed, SM; Singh, S; Bhatnagar, R*

Biochemical and Biophysical Research Communications [Biochem. Biophys. Res. Commun.], vol. 283, no. 2, pp. 308-315, 4 May 2001

The fatal bacterial infection caused by inhalation of the Bacillus anthracis spores results from the synthesis of protein toxins--protective antigen (PA), lethal factor (LF), and edema factor (EF)--by the bacterium. PA is the target-cell binding protein and is common to the two effector molecules, LF and EF, which exert their toxic effects once they are translocated to the cytosol by PA. PA is the major component of vaccines against anthrax since it confers protective immunity. The large-scale production of recombinant protein-based anthrax vaccines requires overexpression of the PA protein. We have constitutively expressed the protective antigen protein in E. coli DH5 alpha strain. We have found no increase in degradation of PA when the protein is constitutively expressed and no plasmid instability was observed inside the expressing cells. We have also scaled up the expression by bioprocess optimization using batch culture technique in a fermentor. The protein was purified using metal-chelate affinity chromatography. Approximately 125 mg of recombinant protective antigen (rPA) protein was obtained per liter of batch culture. It was found to be biologically and functionally fully active in comparison to PA protein from Bacillus anthracis. This is the first report of constitutive overexpression of protective antigen gene in E. coli. Copyright 2001 Academic Press.

Swanson-Biearman, B; Krenzelok, EP*

Journal of Toxicology: Clinical Toxicology [J. Toxicol.: Clin. Toxicol.], vol. 39, no. 1, pp. 81-84, 2001

Background: Anthrax is an acute infectious disease caused by the spore-forming bacterium Bacillus anthracis. Due to the current world threat of unpredictable biological terrorism, the Department of Defense has mandated the systematic vaccination of all US military personnel against this warfare agent. Many may experience a mild flu-like illness and soreness at the injection site, but systemic reactions are rare. Case Report: We report a delayed and potentially serious life-threatening adverse reaction to anthrax vaccine. A previously healthy 34-year-old male was transported to the emergency department with dyspnea, diaphoresis, pallor, and urticarial wheals on his face, arms, and torso after the administration of the third dose of anthrax vaccine. All symptoms resolved after pharmacological intervention and the patient was discharged. Pharmaco-epidemiological data indicate that 30% of anthrax vaccine recipients experience mild local reactions. With large numbers of military personnel being vaccinated, emergency physicians may encounter more vaccine-related adverse reactions.

Journal of Toxicology: Clinical Toxicology [J. Toxicol.: Clin. Toxicol.], vol. 39, no. 1, pp. 85-100, 2001

These recommendations concern the use of aluminum hydroxide adsorbed cell-free anthrax vaccine (Anthrax Vaccine Adsorbed [AVA], BioPort Corporation, Lansing, MI) in the United States for protection against disease caused by Bacillus anthracis. In addition, information is included regarding the use of chemoprophylaxis against B. anthracis.

Fasanella, A; Losito, S; Trotta, T; Adone, R; Massa, S; Ciuchini, F; Chiocco, D

Vaccine, vol. 19, no. 30, pp. 4214-4218, 20 Jul 2001

In Italy, an attenuated Bacillus anthracis strain, named `Carbosap', is used for immunization against ovine and bovine anthrax. Analysis on `Carbosap', Sterne vaccine strain F34 and Pasteur vaccine strain SS104, were performed using primers specific for the sequences, encoding the toxic factors, located on plasmids pXO1 and pXO2 and primers specific for the chromosome. The results obtained from polymerase chain reaction (PCR) assay revealed the presence of both plasmids pXO1 and pXO2 in `Carbosap' strain. This study showed that the `Carbosap' vaccine strain has a different plasmid pattern in comparison to Pasteur vaccine strain SS104 and Sterne vaccine strain F34.