A Bacteriophage T4 Nanoparticle-Based Dual Vaccine against Anthrax and Plague.

Bacillus anthracis and Yersinia pestis, the causative agents of anthrax and plague, respectively, are two of the deadliest pathogenic bacteria that have been used as biological warfare agents. Although Biothrax is a licensed vaccine against anthrax, no Food and Drug Administration-approved vaccine exists for plague. Here, we report the development of a dual anthrax-plague nanoparticle vaccine employing bacteriophage (phage) T4 as a platform. Using an in vitro assembly system, the 120- by 86-nm heads (capsids) of phage T4 were arrayed with anthrax and plague antigens fused to the small outer capsid protein Soc (9 kDa). The antigens included the anthrax protective antigen (PA) (83 kDa) and the mutated (mut) capsular antigen F1 and the low-calcium-response V antigen of the type 3 secretion system from Y. pestis (F1mutV) (56 kDa). These viral nanoparticles elicited robust anthrax- and plague-specific immune responses and provided complete protection against inhalational anthrax and/or pneumonic plague in three animal challenge models, namely, mice, rats, and rabbits. Protection was demonstrated even when the animals were simultaneously challenged with lethal doses of both anthrax lethal toxin and Y. pestis CO92 bacteria. Unlike the traditional subunit vaccines, the phage T4 vaccine uses a highly stable nanoparticle scaffold, provides multivalency, requires no adjuvant, and elicits broad T-helper 1 and 2 immune responses that are essential for complete clearance of bacteria during infection. Therefore, phage T4 is a unique nanoparticle platform to formulate multivalent vaccines against high-risk pathogens for national preparedness against potential bioterror attacks and emerging infections.IMPORTANCE Following the deadly anthrax attacks of 2001, the Centers for Disease Control and Prevention (CDC) determined that Bacillus anthracis and Yersinia pestis that cause anthrax and plague, respectively, are two Tier 1 select agents that pose the greatest threat to the national security of the United States. Both cause rapid death, in 3 to 6 days, of exposed individuals. We engineered a virus nanoparticle vaccine using bacteriophage T4 by incorporating key antigens of both B. anthracis and Y. pestis into one formulation. Two doses of this vaccine provided complete protection against both inhalational anthrax and pneumonic plague in animal models. This dual anthrax-plague vaccine is a strong candidate for stockpiling against a potential bioterror attack involving either one or both of these biothreat agents. Further, our results establish the T4 nanoparticle as a novel platform to develop multivalent vaccines against pathogens of high public health significance.

A lipid A-based TLR4 mimetic effectively adjuvants a Yersinia pestis rF-V1 subunit vaccine in a murine challenge model.

Vaccination can significantly reduce worldwide morbidity and mortality to infectious diseases, thereby reducing the health burden as a result of microbial infections. Effective vaccines contain three components: a delivery system, an antigenic component of the pathogen, and an adjuvant. With the growing use of purely recombinant or synthetic antigens, there is a need to develop novel adjuvants that enhance the protective efficacy of a vaccine against infection. Using a structure-activity relationship (SAR) model, we describe here the synthesis of a novel TLR4 ligand adjuvant compound, BECC438, by bacterial enzymatic combinatorial chemistry (BECC). This compound was identified using an in vitro screening pipeline consisting of (i) NFκB activation and cytokine production by immortalized cell lines, (ii) cytokine production by primary human PBMCs, and (iii) upregulation of surface costimulatory markers by primary human monocyte-derived dendritic cells. Using this SAR screening regimen, BECC438 was shown to produce an innate immune activation profile comparable to the well-characterized TLR4 agonist adjuvant compound, phosphorylated hexa-acyl disaccharide (PHAD). To evaluate the in vivo adjuvant activity of BECC438, we used the known protective Yersinia pestis (Yp) antigen, rF1-V, in a murine prime-boost vaccination schedule followed by lethal challenge. In addition to providing protection from lethal challenge, BECC438 stimulated production of higher levels of rF1-V-specific total IgG as compared to PHAD after both prime and boost vaccinations. Similar to PHAD, BECC438 elicited a balanced IgG1/IgG2c response, indicative of active TH2/TH1-driven immunity. These data demonstrate that the novel BECC-derived TLR4L adjuvant, BECC438, elicits cytokine profiles in vitro similar to PHAD, induces high antigen-specific immune titers and a TH1-associated IgG2c immune titer skew, and protects mice against a lethal Yp challenge.

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.

Characterization of systemic and pneumonic murine models of plague infection using a conditionally virulent strain.

Yersinia pestis causes bubonic and pneumonic plague in humans. The pneumonic infection is the most severe and invariably fatal if untreated. Because of its high virulence, ease of delivery and precedent of use in warfare, Y. pestis is considered as a potential bioterror agent. No licensed plague vaccine is currently available in the US. Laboratory research with virulent strains requires appropriate biocontainment (i.e., Biosafety Level 3 (BSL-3) for procedures that generate aerosol/droplets) and secure facilities that comply with federal select agent regulations. To assist in the identification of promising vaccine candidates during the early phases of development, we characterized mouse models of systemic and pneumonic plague infection using the Y. pestis strain EV76, an attenuated human vaccine strain that can be rendered virulent in mice under in vivo iron supplementation. Mice inoculated intranasally or intravenously with Y. pestis EV76 in the presence of iron developed a systemic and pneumonic plague infection that resulted in disease and lethality. Bacteria replicated and severely compromised the spleen, liver and lungs. Susceptibility was age dependent, with younger mice being more vulnerable to pneumonic infection. We used these models of infection to assess the protective capacity of newly developed Salmonella-based plague vaccines. The protective outcome varied depending on the route and dose of infection. Protection was associated with the induction of specific immunological effectors in systemic/mucosal compartments. The models of infection described could serve as safe and practical tools for identifying promising vaccine candidates that warrant further potency evaluation using fully virulent strains in BSL-3 settings.

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.

[Immunogenicity of B-antigen in experimental plague and pseudotuberculosis].

The results of the evaluation of the immunogenic properties of B-antigen, earlier identified in the culture fluid of Yersinia pseudotuberculosis submerged culture, with respect to experimental plague and pseudotuberculosis are presented. B-antigen has been shown to produce protective effect in guinea pigs and, probably, hamadryas baboons, but not in white mice infected with the causative agent of plague. Immunizaton with B-antigen protects guinea pigs from primary pneumonic plague caused by both capsule-forming and noncapsular Y. pestis virulent strains. Passive immunization with antibodies to B-antigen induces limitedly pronounced protective effect in guinea pigs and is not effective for white mice with respect to experimental plague. No active or passive protection of white mice or guinea pigs, infected with Y. pseudotuberculosis cultures, has been achieved by the injection of B-antigen or antibodies to it.

[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.

[Combined (associated) immunization against typhoid, typhus and plague]. / Kompleksnaia (assotsiirovannaia) immunizatsiia protiv briushnogo i sypnogo tifov i chumy.

The article substantiates epidemiological expediency of complex (associated) immunization of servicemen and population against typhoid, typhus and plague in polyetiological zones of these infections, and also in cases of simultaneous proliferation of these diseases. For simultaneous preventive vaccination against these infections a complex immunization scheme was experimentally substantiated and clinically approved. It is based on national commercial vaccines and ensures a simultaneous administration of 2-3 vaccine preparations by hypodermic syringe or jet injection. Typhoid and typhus vaccines are injected under one shoulder-blade, and plague vaccine is injected under another shoulder-blade. This complex vaccine is harmless, moderately reactogenic, develops expressing immunity which have the same protective features as monovaccines alone. This scheme is recommended for use in anti-epidemic practice.