Antibiotic Therapy of Plague: A Review.

Plague-a deadly disease caused by the bacterium Yersinia pestis-is still an international public health concern. There are three main clinical forms: bubonic plague, septicemic plague, and pulmonary plague. In all three forms, the symptoms appear suddenly and progress very rapidly. Early antibiotic therapy is essential for countering the disease. Several classes of antibiotics (e.g., tetracyclines, fluoroquinolones, aminoglycosides, sulfonamides, chloramphenicol, rifamycin, and ß-lactams) are active in vitro against the majority of Y. pestis strains and have demonstrated efficacy in various animal models. However, some discrepancies have been reported. Hence, health authorities have approved and recommended several drugs for prophylactic or curative use. Only monotherapy is currently recommended; combination therapy has not shown any benefits in preclinical studies or case reports. Concerns about the emergence of multidrug-resistant strains of Y. pestis have led to the development of new classes of antibiotics and other therapeutics (e.g., LpxC inhibitors, cationic peptides, antivirulence drugs, predatory bacteria, phages, immunotherapy, host-directed therapy, and nutritional immunity). It is difficult to know which of the currently available treatments or therapeutics in development will be most effective for a given form of plague. This is due to the lack of standardization in preclinical studies, conflicting data from case reports, and the small number of clinical trials performed to date.

Immunotropic Properties of an Experimental Synthetic Selenium-Organic Compound.

We studied immunotropic properties of synthetic selenium-organic preparation 2,6-dipyridinium-9-selenabicyclo[3.3.1]nonyl dibromide (974zh). The experimental preparation reduced the cAMP/cGMP ratio, which indicated an increase in proliferative activity of cells of immunocompetent organs (thymus and spleen) in experimental animals. It was shown that 974zh intensified the immune response to Yersinia pestis EV thereby increasing the resistance to the plague agent.

Mutated and bacteriophage T4 nanoparticle arrayed F1-V immunogens from Yersinia pestis as next generation plague vaccines.

Pneumonic plague is a highly virulent infectious disease with 100% mortality rate, and its causative organism Yersinia pestis poses a serious threat for deliberate use as a bioterror agent. Currently, there is no FDA approved vaccine against plague. The polymeric bacterial capsular protein F1, a key component of the currently tested bivalent subunit vaccine consisting, in addition, of low calcium response V antigen, has high propensity to aggregate, thus affecting its purification and vaccine efficacy. We used two basic approaches, structure-based immunogen design and phage T4 nanoparticle delivery, to construct new plague vaccines that provided complete protection against pneumonic plague. The NH2-terminal ß-strand of F1 was transplanted to the COOH-terminus and the sequence flanking the ß-strand was duplicated to eliminate polymerization but to retain the T cell epitopes. The mutated F1 was fused to the V antigen, a key virulence factor that forms the tip of the type three secretion system (T3SS). The F1mut-V protein showed a dramatic switch in solubility, producing a completely soluble monomer. The F1mut-V was then arrayed on phage T4 nanoparticle via the small outer capsid protein, Soc. The F1mut-V monomer was robustly immunogenic and the T4-decorated F1mut-V without any adjuvant induced balanced TH1 and TH2 responses in mice. Inclusion of an oligomerization-deficient YscF, another component of the T3SS, showed a slight enhancement in the potency of F1-V vaccine, while deletion of the putative immunomodulatory sequence of the V antigen did not improve the vaccine efficacy. Both the soluble (purified F1mut-V mixed with alhydrogel) and T4 decorated F1mut-V (no adjuvant) provided 100% protection to mice and rats against pneumonic plague evoked by high doses of Y. pestis CO92. These novel platforms might lead to efficacious and easily manufacturable next generation plague vaccines.

CpG oligodeoxynucleotides augment the murine immune response to the Yersinia pestis F1-V vaccine in bubonic and pneumonic models of plague.

The current U.S. Department of Defense candidate plague vaccine is a fusion between two Yersinia pestis proteins: the F1 capsular protein, and the low calcium response (Lcr) V-protein. We hypothesized that an immunomodulator, such as CpG oligodeoxynucleotide (ODN)s, could augment the immune response to the plague F1-V vaccine in a mouse model for plague. CpG ODNs significantly augmented the antibody response and efficacy of a single dose of the plague vaccine in murine bubonic and pneumonic models of plague. In the latter study, we also found an overall significant augmentation the immune response to the individual subunits of the plague vaccine by CpG ODN 2006. In a long-term, prime-boost study, CpG ODN induced a significant early augmentation of the IgG response to the vaccine. The presence of CpG ODN induced a significant increase in the IgG2a subclass response to the vaccine up to 5 months after the boost. Our studies showed that CpG ODNs significantly augmented the IgG antibody response to the plague vaccine, which increased the probability of survival in murine models of plague (P<0.0001).

Lipid A mimetics are potent adjuvants for an intranasal pneumonic plague vaccine.

An effective intranasal (i.n.) vaccine against pneumonic plague was developed. The formulation employed two synthetic lipid A mimetics as adjuvant combined with Yersinia pestis-derived V- and F1-protective antigens. The two nontoxic lipid A mimetics, classed as amino-alkyl glucosaminide 4-phosphates (AGPs) are potent ligands for the Toll-like receptor (TLR) 4. Using a murine (BALB/c) pneumonic plague model, we showed a single i.n. application of the vaccine provided 63% protection within 21 days against a Y. pestis CO92 100 LD50 challenge. Protection reached 100% by 150 days. Using a homologous i.n. 1 degrees /2 degrees dose regimen, with the boost administered at varying times, 63% protection was achieved within 7 days and 100% protection was achieved by 21 days after the first immunization. Little or no protection was observed in animals that received antigens alone, and no protection was observed when the vaccine was administered to BALB/c TLR4 mutant mice. Vaccine-induced serum IgG titers to F1 and V-antigen were reflected in high titers for IgG1 and IgG2a, the latter reflecting a bias for a cell-mediated (TH1) immune response. This intranasal vaccine showed 90% protection in Sprague-Dawley rats challenged with 1000 LD50. We conclude that lipid A mimetics are highly effective adjuvants for an i.n. plague vaccine.

Purification and protective efficacy of monomeric and modified Yersinia pestis capsular F1-V antigen fusion proteins for vaccination against plague.

The F1-V vaccine antigen, protective against Yersinia pestis, exhibits a strong tendency to multimerize that affects larger-scale manufacture and characterization. In this work, the sole F1-V cysteine was replaced with serine by site-directed mutagenesis for characterization of F1-V non-covalent multimer interactions and protective potency without participation by disulfide-linkages. F1-V and F1-V(C424S) proteins were overexpressed in Escherichia coli, recovered using mechanical lysis/pH-modulation and purified from urea-solubilized soft inclusion bodies, using successive ion-exchange, ceramic hydroxyapatite, and size-exclusion chromatography. This purification method resulted in up to 2mg/g of cell paste of 95% pure, mono-disperse protein having < or =0.5 endotoxin units per mg by a kinetic chromogenic limulus amoebocyte lysate reactivity assay. Both F1-V and F1-V(C424S) were monomeric at pH 10.0 and progressively self-associated as pH conditions decreased to pH 6.0. Solution additives were screened for their ability to inhibit F1-V self-association at pH 6.5. An L-arginine buffer provided the greatest stabilizing effect. Conversion to >500-kDa multimers occurred between pH 6.0 and 5.0. Conditions for efficient F1-V adsorption to the cGMP-compatible alhydrogel adjuvant were optimized. Side-by-side evaluation for protective potency against subcutaneous plague infection in mice was conducted for F1-V(C424S) monomer; cysteine-capped F1-V monomer; cysteine-capped F1-V multimer; and a F1-V standard reported previously. After a two-dose vaccination with 2 x 20 microg of F1-V, respectively, 100%, 80%, 80%, and 70% of injected mice survived a subcutaneous lethal plague challenge with 10(8) LD(50)Y. pestis CO92. Thus, vaccination with F1-V monomer and multimeric forms resulted in significant, and essentially equivalent, protection.

Treatment of plague: promising alternatives to antibiotics.

Plague still poses a significant threat to human health, and interest has been renewed recently in the possible use of Yersinia pestis as a biological weapon by terrorists. The septicaemic and pneumonic forms are always lethal if untreated. Attempts to treat this deadly disease date back to the era of global pandemics, when various methods were explored. The successful isolation of the plague pathogen led to the beginning of more scientific approaches to the treatment and cure of plague. This subsequently led to specific antibiotic prophylaxis and therapy for Y. pestis. The use of antibiotics such as tetracycline and streptomycin for the treatment of plague has been embraced by the World Health Organization Expert Committee on Plague as the 'gold standard' treatment. However, concerns regarding the development of antibiotic-resistant Y. pestis strains have led to the exploration of alternatives to antibiotics. Several investigators have looked into the use of alternatives, such as immunotherapy, non-pathogen-specific immunomodulatory therapy, phage therapy, bacteriocin therapy, and treatment with inhibitors of virulence factors. The alternative therapies reported in this review should be further investigated by comprehensive studies of their clinical application for the treatment of plague.

[The experimental validation of the advantages of combined emergency (fluoroquinolones) and specific (EV Nalr) prevention of plague versus their sequential use]. / Eksperimental'noe obosnovanie preimushchestv kombinirovannoi ékstrennoi (ftorkhinolony) i spetsificheskoi (EV Nalr) profilaktiki chumy pered ikh posledovatel'nym primeneniem.

Mice immunization with reference vaccine at the early stage of plague infection provided animals survival and prolonged mean survival period up to 2-5 days. Ciprofloxacin, ofloxacin and pefloxacin prevents development of post vaccine immunity at white mice, immunized by reference vaccine strain EV. Nalidixic acid and norfloxacin effect on post vaccine immunity was lower. Use of immunogenic strain EV Nafr (resistant to nalidixic acid and fluoroquinolones) provided antiplague immunity formation at the background of fluoroquinolones prophylaxis. Ciprofloxacin, ofloxacin and pefloxacin used for plague prophylaxis at white mice infected with Yersinia pestis (about 1000 LD50) inhibited postinfective immunity development. Nalidixic acid and norfloxacin didn't demonstrate such effect. Urgent (fluoroquinolones) and specific (EV Nalr) combined prophylaxis was evaluated as more effective for a 5-day period and provided the development of antiplague immunity.

[The evaluation of the efficacy of immunization against plague by the modulation in the index of the macrophagal transformation of of the peripheral blood monocytes]. / Otsenka éffektivnosti immunizatsii protiv chumy po moduliatsii pokazatelia makrofagal'noi transformatsii monotsitov perifericheskoi krovi.

The subcutaneous and aerogenous immunization of experimental animals with live dried antiplague vaccine has been shown to modulate the macrophagal transformation index of peripheral blood monocytes (PBM) which correlates with the resistance of the animals to the causative agent of plague. This finding allows to recommend the use of PBM for the evaluation of the effectiveness of immunization with antiplague vaccine.

[Combined (associated) vaccination against quarantinable infections]. / Kompleksnaia (assotsiirovannaia) vaktsinatsiia protiv karantinnykh infektsii.

The presence of several active foci of infection of different etiology is an indication for complex (combined) immunization against these diseases. The scheme of complex (combined) immunization against plague, cholera and yellow fever has been experimentally substantiated and successfully tested on volunteers.

[Thymosin alpha-1 and hybrid proteins consisting of tumor necrosis factor-alpha and thymosin alpha-1 enhance the efficacy of vaccination against the causative agent of plague]. / Timozin al'fa-1 i gibridnye belki, sostoiashchie iz faktora nekroza opukholei al'fa i timozina al'fa-1, uvelichivaiut éffektivnost' vaktsinatsii protiv vozbuditelia chumy.

The influence of the preparation of chemical thymosin alpha 1 (T), recombinant thymosin alpha 1 (rT), tumor necrosis factor (TNF) and hybrid proteins on their basis (T-TNF, TNF-T and T-TNF-T) on the effectiveness of immunization against Y.pestis have been studied. The preparations of T and hybrid proteins exhibit immunostimulating action, enhancing specific immunity when injected at different periods of the vaccinal process against Y.pestis virulent strain 231 in experiments on mice and guinea pigs. The highest effectiveness and reproducibility of results is observed after the use of hybrid protein T-TNF-T. An increase in immunity after the use of the preparations of hybrid proteins is accompanied by the activation of its T-cell element. The influence of rT on the restoration of the immune system of white mice after their exposure to sublethal doses of gamma radiation has been shown.