Appropriation and commercialization of the Pasteur anthrax vaccine

Whereas Pasteur patented the biotechnological processes that he invented between 1857 and 1873 in the agro-food domain, he did not file any patents on the artificial vaccine preparation processes that he subsequently developed. This absence of patents can probably be explained by the 1844 patent law in France that established the non-patentable status of pharmaceutical preparations and remedies, including those for use in veterinary medicine. Despite the absence of patents, the commercial exploitation of the anthrax vaccine in the 1880s and 1890s led to a technical and commercial monopoly by Pasteur's laboratory as well as the founding of a commercial company to diffuse the vaccine abroad. Pasteur repeatedly refused to transfer his know-how and anthrax vaccine production methods to foreign laboratories, on the grounds that he wished to control the quality of the vaccines produced. Indeed, it was relatively difficult to transfer a method that was not yet perfectly stabilized in the early 1880s. Pasteur also wanted to maintain the monopoly of his commercial company and to increase the profits from vaccine sales so that the Institut Pasteur could be financially independent. The 'Pasteur anthrax vaccine' operating licences are described and analysed in detail in this article.     

Engineered chloroplasts as vaccine factories to combat bioterrorism

Bacillus anthracis is ideal for making biological weapons, but the licensed anthrax vaccine is unsuitable for widespread public administration. Recombinant subunit-vaccine candidates offer potential alternatives, and plant-based production systems facilitate the inexpensive bulking of target antigens. A recent report demonstrates expression of anthrax protective antigen in tobacco chloroplasts--this material is immunogenic and protective when injected into mice. Provided an economic purification scheme can be developed, this technology holds promise for an improved vaccine.     

Treatment and Prophylaxis of Anthrax by New Neurosecretory Cytokines

In 1881, Louis Pasteur described the Bacillus anthracis vaccine, which plays an important role for the treatment and prophylaxis of anthrax. Currently, treatment for anthrax infection involves the use of several different antibiotics, used in combination with vaccines, which possess potential virulence in white mice and guinea pigs. We discovered several new immunomodulators cytokines (polypeptides) produced by the neurosecretory cells of hypothalamus, some of which can be used as drugs for the treatment and prophylaxis of the anthrax. The proline-rich polypeptides, which consist from 10 to 15 amino acids and four proline residues, are of the special interest; one of them (PRP-1), which consist of 15 amino acids and has the following primary structure ALa-GLy-ALa-Pro-GLu-Pro-Ala-GLu-Pro-Ala-GLn-Pro-GLy-Val-Tyr (AGAPEPAEPAQPGVY) possesses antibacterial activity, and a new proline-rich peptide described by Galoyan and called G _x -NH₂. Both were tested for treatment against the anthrax bacillus or anthrax strain N55 vaccine in guinea pigs and mice in vivo, and in vitro preparations. The results of experiments show that these hypothalamic neurosecretory cytokines have a strong prophylaxis and therapeutic properties towards animals infected by episodic strain of anthrax and anthrax vaccine N55. The conventional concepts concerning the function of hypothalamic neurosecretion and hypothalamic mechanisms of adaptation have to be reconsidered.     

Assessment of immunity and allergy after vaccination with dry combined anthrax vaccine

Study of humoral immune response and allergy in recipients of dry combined anthrax vaccine was performed. Immune response was assessed by antibody titers to protective antigen and by index of preventive properties of blood serum (PPS) of recipients. Relation of index of PPS and antibody titers in blood serum of the donors was established. Distribution of erythrocyte antigens in recipients of live dry and combined anthraxvaccines depending on blood group, Rh-factor, and age was studied. It has been shown that 80% of recipients of dry combined anthrax vaccine formed potent immunity with its high level lasted for 8 months. Study of allergenic properties of the combined anthrax vaccine using registration of neutrophils chemiluminescence in vivo showed low level of sensitization of vacinees.     

Analysis of anthrax and plague biowarfare vaccine interactions with human monocyte-derived dendritic cells

The anti-biowarfare anthrax and plague vaccines require repeated dosing to achieve adequate protection. To test the hypothesis that this limited immunogenicity results from the nature of vaccine interactions with the host innate immune system, we investigated molecular and cellular interactions between vaccines, dendritic cells (DCs), and T cells and explored the potential for adjuvants (pertussis) to boost induction of host immunity. Human monocyte-derived DCs were matured in the presence of vaccines and analyzed for their ability to induce Th1/Th2 development from naive T cells, expression of cell surface maturation/costimulation molecules, and cytokine production. The vaccines showed different behavior patterns. Although the plague vaccine is equivalent to control maturation factors in maturation and stimulation of DCs and induces strong MLR and Th outgrowth, the anthrax vaccine is a poor inducer of DC maturation, as indicated by low levels of HLA-DR, CD86, and CD83 induction and minimal proinflammatory cytokine production. Interestingly, however, anthrax vaccine-treated DCs stimulate Th1 and Th2 outgrowth and a limited MLR response. There was no sustained negative modulatory effects of the anthrax vaccine on DCs, and its limited stimulatory effects could be overridden by coculture with pertussis. These results were supported by analysis of anthrax vaccine recall responses in subjects vaccinated using pertussis as an adjuvant, who demonstrate anthrax-specific effector T cell responses. These data show that the anthrax vaccine is a suboptimal DC stimulus that may in part explain the observation that it requires repeated administration in vivo and offer a rational basis for the use of complementary DC-maturing adjuvants in combined immunotherapy.     

The improvement of immunobiological preparations against leptospirosis. An experimental study of a new concentrated purified vaccine against icterohemorrhagic leptospirosis for human immunization

The findings of the study of immunological structure of the population in regions endemic for leptospirosis indicate that the immune status of humans makes it impossible to obtain titrated blood sera for the preparation of antileptospirosis immunoglobulin. The data obtained in the study of the immunobiological properties of a new concentrated vaccine against icterohemorrhagic leptospirosis show the possibility of using this vaccine for the immunization of donors with the aim of obtaining blood sera to be used as raw material for the production of immunobiological preparations.     

A novel immunogenic spore coat-associated protein in Bacillus anthracis: characterization via proteomics approaches and a vector-based vaccine system

New generation anthrax vaccines have been actively explored with the aim of enhancing efficacies and decreasing undesirable side effects that could be caused by licensed vaccines. Targeting novel antigens and/or eliminating the requirements for multiple needle injections and adjuvants are major objectives in the development of new anthrax vaccines. Using proteomics approaches, we identified a spore coat-associated protein (SCAP) in Bacillus anthracis. An Escherichia coli vector-based vaccine system was used to determine the immunogenicity of SCAP. Mice generated detectable SCAP antibodies three weeks after intranasal immunization with an intact particle of ultraviolet (UV)-irradiated E. coli vector overproducing SCAP. The production of SCAP antibodies was detected via western blotting and SCAP-spotted antigen-arrays. The adjuvant effect of a UV-irradiated E. coli vector eliminates the necessity of boosting and the use of other immunomodulators which will foster the screening and manufacturing of new generation anthrax vaccines. More importantly, the immunogenic SCAP may potentially be a new candidate for the development of anthrax vaccines.     

A study of the physiology of Bacillus anthracis Sterne during manufacture of the UK acellular anthrax vaccine

AIM: To analyse the growth of Bacillus anthracis during simulations of the UK anthrax vaccine manufacturing process. METHODS AND RESULTS: Simulated vaccine production runs were performed using the toxigenic, acapsulate Sterne 34F(2) strain of B. anthracis in semi-defined medium. After rising during the logarithmic growth phase, the pH of the culture starts to fall at about 18 h from pH 8.7 to reach <7.6 at 26 h, coincident with consumption of glucose and optimal production of protective antigen (PA; 7.89 g ml(-1), SD 1.0) and lethal factor (LF; 1.85 g ml(-1), SD 0.29). No increased breakdown of toxin antigens was seen over the 26-32 h period. When glucose was exhausted, amino acids (principally serine) were utilized as an alternative carbon source. Sporulation was not observed during the 32 h. CONCLUSIONS: PA and LF, the principal constituents in the UK anthrax vaccine, undergo little degradation during vaccine fermentation. The vaccine manufacturing process is robust and reproducible. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first detailed analysis of the manufacturing process used for the UK acellular anthrax vaccine; insight gained into the process will support continued and safe vaccine manufacture.     

Antigen delivery by attenuated Bacillus anthracis: new prospects in veterinary vaccines

This report summarizes the recent investigations on the use of Bacillus anthracis as a live vector for delivery of antigens. Recombinant strains were constructed by engineering the current live Sterne vaccine. This vaccine, used to prevent anthrax in cattle, causes side-effects due to anthrax toxin activities. Bacteria producing a genetically detoxified toxin factor were devoid of lethal effects and were as protective as the Sterne strain against experimental anthrax. Moreover, B. anthracis expressing a foreign antigen controlled by an in vivo inducible promoter were able to generate either antibody or cellular protective responses against heterologous diseases.     

Effect of X-irradiation on the course of experimental vaccine anthrax

The author studied the course of vaccine anthrax infection in irradiated rabbits. The experiments show that infection of irradiated rabbits with a vaccine strain can give rise to a disease bacteriologically, clinically, histologically and biochemically identical with typical anthrax and that anthrax toxin can be demonstrated in the plasma of dead rabbits. The main cause of anthrax sepsis is not raised sensitivity to the toxin, but the high degree of proliferation of the microorganism in the irradiated organism. The significance of phagocytosis as a defence against vaccine anthrax infection and the significance of the capsule or of another somatic substance for the development of the anthrax syndrome are discussed.     

Monoclonal antibodies to B.anthracis protective antigen are capable to neutralize and to enhance the anthrax lethal toxin action in vitro

Anthrax belongs to highly dangerous infections of man and animals. No effective treatment methods for pulmonary types of the disease have been yet developed. The existing anthrax vaccines were designed decades ago and need improvement to fit the large-scale vaccination of population. At the same time, the immunological properties of the anthrax vaccine main component, i.e. of the protective agent, have been poorly studied. We obtained, within the present case study, a panel of mouse monoclonal antibodies to the protective agent and investigated the properties of the highest-affine panel representatives. An unusual phenomenon was detected, which is related with enhancement of the anthrax toxin action on the mouse macrophage-like cell-line in presence of the 1F2 monoclonal antibody. The remaining analyzed antibodies, i.e. 6G8 and 6G7, were found to neutralize effectively the toxin action. The enhancing and neutralizing antibodies were proven to be specific to different domains of the protective antigen and to recognize epitopes in its composition. The antibody-mediated enhancement of the anthrax lethal action is a convincing argument for further development of a new-generation anthrax vaccine. Definition of the linear antigen determinants for neutralizing antibodies in the protective antigens is an important step in the development of the next-generation anthrax vaccine.     

Anthrax vaccination strategies

The biological attack conducted through the US postal system in 2001 broadened the threat posed by anthrax from one pertinent mainly to soldiers on the battlefield to one understood to exist throughout our society. The expansion of the threatened population placed greater emphasis on the reexamination of how we vaccinate against Bacillus anthracis. The currently-licensed Anthrax Vaccine, Adsorbed (AVA) and Anthrax Vaccine, Precipitated (AVP) are capable of generating a protective immune response but are hampered by shortcomings that make their widespread use undesirable or infeasible. Efforts to gain US Food and Drug Administration (FDA) approval for licensure of a second generation recombinant protective antigen (rPA)-based anthrax vaccine are ongoing. However, this vaccine’s reliance on the generation of a humoral immune response against a single virulence factor has led a number of scientists to conclude that the vaccine is likely not the final solution to optimal anthrax vaccine design. Other vaccine approaches, which seek a more comprehensive immune response targeted at multiple components of the B. anthracis organism, are under active investigation. This review seeks to summarize work that has been done to build on the current PA-based vaccine methodology and to evaluate the search for future anthrax prophylaxis strategies.     

Immunological properties of the preparations of the anti-anthrax antigen]

The results of examination of immunological properties of the preparations of anthrax protective antigen on laboratory animals (guinea pigs) confirmed the efficacy of using the lactic-peptone medium for obtaining the anthrax protective antigen. Incubation for 18 hours at 37 degrees C of the strain-producer (STI-1) and a double immunization scheme with the antigen obtained proved to be the most rational conditions for inducing the immunological response in the vaccinated laboratory animals. Three fractions of the anthrax protective antigen obtained possessed weaker immunobiological properties than the whole preparation of this antigen.     

Anthrax vaccine design: strategies to achieve comprehensive protection against spore,bacillus, and toxin

The successful use of Bacillus anthracis as a lethal biological weapon has prompted renewed research interest in the development of more effective vaccines against anthrax. The disease consists of three critical components: spore, bacillus, and toxin, elimination of any of which confers at least partial protection against anthrax. Current remedies rely on postexposure antibiotics to eliminate bacilli and pre- and postexposure vaccination to target primarily toxins. Vaccines effective against toxin have been licensed for human use, but need improvement. Vaccines against bacilli have recently been developed by us and others. Whether effective vaccines will be developed against spores is still an open question. An ideal vaccine would confer simultaneous protection against spores, bacilli, and toxins. One step towards this goal is our dually active vaccine, designed to destroy both bacilli and toxin. Existing and potential strategies towards potent and effective anthrax vaccines are discussed in this review.     

水氮互作下直播稻群体质量与氮素利用特征的关系

探讨水氮耦合下直播稻群体质量和氮素利用特征可为直播水稻丰产高效生产提供理论基础和实践依据。以杂交稻‘F优498’为试验材料,设置淹水灌溉(W₁)、干湿交替灌溉(W₂)、旱种(W₃)3种灌水方式和基肥∶蘖肥∶穗肥分别为5∶3∶2(N₁)、3∶3∶4(N₂)、3∶1∶6(N₃)3种氮肥运筹模式,以不施氮处理(N₀)为对照,研究水氮互作对直播稻群体质量和氮素利用特征的影响,并探讨水氮互作下直播稻群体质量构建与氮素利用特征和产量的关系。结果表明:灌溉方式和氮肥运筹对直播稻各生育时期干物质积累、稻谷收获指数、抽穗及抽穗20 d高效叶(上3叶)干重、结实期群体透光率、氮素积累总量、氮肥表观利用率、氮肥偏生产力、氮肥生理利用率和产量均存在显著的互作效应。综合直播稻群体质量、产量和氮肥利用特征,各灌溉方式下,氮肥后移比例均以占总量的20%～40%(N₁～N₂)为宜,氮肥后移比例达到总量的60%(N₃)和W₃处理均会显著降低直播稻群体质量、产量、氮肥农学利用率和氮肥偏生产力。相关分析表明,水氮互作下直播稻产量和氮素利用特征与最终有效分蘖数、结实期干物质积累、其余叶(除上三叶以外叶片)干重减少量、总叶片干重减少量,以及群体中部和基部受光率均呈显著或极显著正相关。干湿交替灌溉(W₂)可以提高直播稻茎蘖成穗率、各时期干物质积累、稻谷收获指数、氮肥吸收总量、氮肥农学利用率和最终产量,配合N₂的氮肥运筹模式可优化调控直播稻群体质量,实现高产与氮肥高效利用的协调统一,为本试验最优组合。     

Nasal immunization with the mixture of PA63, LF, and a PGA conjugate induced strong antibody responses against all three antigens

A new generation anthrax vaccine is expected to target not only the anthrax protective antigen (PA) protein, but also other virulent factors of Bacillus anthracis. It is also expected to be amenable for rapid mass immunization of a large number of people. This study aimed to address these needs by designing a prototypic triantigen nasal anthrax vaccine candidate that contained a truncated PA (rPA63), the anthrax lethal factor (LF), and the capsular poly-gamma-D-glutamic acid (gammaDPGA) as the antigens and a synthetic double-stranded RNA (dsRNA), polyriboinosinic-polyribocytodylic acid (poly(I:C)) as the adjuvant. This study identified the optimal dose of nasal poly(I:C) in mice, demonstrated that nasal immunization of mice with the LF was capable of inducing functional anti-LF antibodies (Abs), and showed that nasal immunization of mice with the prototypic triantigen vaccine candidate induced strong immune responses against all three antigens. The immune responses protected macrophages against an anthrax lethal toxin challenge in vitro and enabled the immunized mice to survive a lethal dose of anthrax lethal toxin challenge in vivo. The anti-PGA Abs were shown to have complement-mediated bacteriolytic activity. After further optimization, this triantigen nasal vaccine candidate is expected to become one of the newer generation anthrax vaccines.     

A Single-Dose PLGA Encapsulated Protective Antigen Domain 4 Nanoformulation Protects Mice against Bacillus anthracis Spore Challenge

Bacillus anthracis, the etiological agent of anthrax, is a major bioterror agent. Vaccination is the most effective prophylactic measure available against anthrax. Currently available anthrax vaccines have issues of the multiple booster dose requirement, adjuvant-associated side effects and stability. Use of biocompatible and biodegradable nanoparticles to deliver the antigens to immune cells could solve the issues associated with anthrax vaccines. We hypothesized that the delivery of a stable immunogenic domain 4 of protective antigen (PAD4) of Bacillus anthracis encapsulated in a poly (lactide-co-glycolide) (PLGA) - an FDA approved biocompatible and biodegradable material, may alleviate the problems of booster dose, adjuvant toxicity and stability associated with anthrax vaccines. We made a PLGA based protective antigen domain 4 nanoparticle (PAD4-NP) formulation using water/oil/water solvent evaporation method. Nanoparticles were characterized for antigen content, morphology, size, polydispersity and zeta potential. The immune correlates and protective efficacy of the nanoparticle formulation was evaluated in Swiss Webster outbred mice. Mice were immunized with single dose of PAD4-NP or recombinant PAD4. The PAD4-NP elicited a robust IgG response with mixed IgG1 and IgG2a subtypes, whereas the control PAD4 immunized mice elicited low IgG response with predominant IgG1 subtype. The PAD4-NP generated mixed Th1/Th2 response, whereas PAD4 elicited predominantly Th2 response. When we compared the efficacy of this single-dose vaccine nanoformulation PAD4-NP with that of the recombinant PAD4 in providing protective immunity against a lethal challenge with Bacillus anthracis spores, the median survival of PAD4-NP immunized mice was 6 days as compared to 1 day for PAD4 immunized mice (p<0.001). Thus, we demonstrate, for the first time, the possibility of the development of a single-dose and adjuvant-free protective antigen based anthrax vaccine in the form of PAD4-NP. Further work in this direction may produce a better and safer candidate anthrax vaccine.     

Investigation of Anthrax Cases in North-East China, 2010-2014

We determined the genotypes of seven Bacillus anthracis strains that were recovered from nine anthrax outbreaks in North-East China from 2010 to 2014, and two approved vaccine strains that are currently in use in China. The causes of these cases were partly due to local farmers being unaware of the presence of anthrax, and butchers with open wounds having direct contact with anthrax-contaminated meat products. The genotype of five of the seven recovered strains was A.Br.001/002 sub-lineage, which was concordant with previously published research. The remaining two cases belongs to the A.Br.Ames sub-lineage. Both of these strains displayed an identical SNR pattern, which was the first time that this genotype was identified in North-East China. Strengthening education in remote villages of rural China is an important activity aimed at fostering attempts to prevent and control anthrax. The genotype of the vaccine strain Anthrax Spore Vaccine No.II was A.Br.008/009 and A.Br.001/002 for the vaccine strain Anthrax Spore Vaccine Non-capsulated. Further studies of their characteristics are clearly warranted.     

Prospects of the use of bacteriophage-based virus-like particles in the creation of anthrax vaccines

The profitability of vaccine production is less than that of other pharmaceutical goods worldwide. Thus, the cost of the vaccine substance determines the range of vaccines available for use. This is of particular importance for veterinary vaccines. In this review, we have surveyed the published data on exploited vaccines and concluded that the immunogenicity of antigen substances based on whole virions is higher than that of soluble antigens. The physiological basis of this phenomenon remains unknown; however, it may explain why most of the described recombinant vaccines have not yet been put into practice. All practically implemented antiviral vaccines (except that for hepatitis B) are based on viral substances produced by conventional cultural technologies. In light of this observation, an approach to the development of a universal platform for recombinant vaccines produced in the form of virus-like particles is suggested. To this end, a technique of designing fused bifunctional derivatives of bacteriophage proteins containing antigens of interest should be involved. The approach is depicted with the use of the protective anthrax antigen, a conventional vaccine antigen.     

Recent progress in the development of anthrax vaccines

Bacillus anthracis is the etiological agent of anthrax. Although anthrax is primarily an epizootic disease; humans are at risk for contracting anthrax. The potential use of B. anthracis spores as biowarfare agent has led to immense attention. Prolonged vaccination schedule of current anthrax vaccine and variable protection conferred; often leading to failure of therapy. This highlights the need for alternative anthrax countermeasures. A number of approaches are being investigated to substitute or supplement the existing anthrax vaccines. These relied on expression of Protective antigen (PA), the key protective immunogen; in bacterial or plant systems; or utilization of attenuated strains of B. anthracis for immunization. Few studies have established potential of domain IV of PA for immunization. Other targets including the spore, capsule, S-layer and anthrax toxin components have been investigated for imparting protective immunity. It has been shown that co-immunization of PA with domain I of lethal factor that binds PA resulted in higher antibody responses. Of the epitope based vaccines, the loop neutralizing determinant, in particular; elicited robust neutralizing antibody response and conferred 97% protection upon challenge. DNA vaccination resulted in varying degree of protection and seems a promising approach. Additionally, the applicability of monoclonal and therapeutic antibodies in the treatment of anthrax has also been demonstrated. The recent progress in the direction of anthrax prophylaxis has been evaluated in this review.