Hereditary hemochromatosis restores the virulence of plague vaccine strains.

Nonpigmented Yersinia pestis (pgm) strains are defective in scavenging host iron and have been used in live-attenuated vaccines to combat plague epidemics. Recently, a Y. pestis pgm strain was isolated from a researcher with hereditary hemochromatosis who died from laboratory-acquired plague. We used hemojuvelin-knockout (Hjv(-/-)) mice to examine whether iron-storage disease restores the virulence defects of nonpigmented Y. pestis. Unlike wild-type mice, Hjv(-/-) mice developed lethal plague when challenged with Y. pestis pgm strains. Immunization of Hjv(-/-) mice with a subunit vaccine that blocks Y. pestis type III secretion generated protection against plague. Thus, individuals with hereditary hemochromatosis may be protected with subunit vaccines but should not be exposed to live-attenuated plague vaccines.

Polymorphisms in the lcrV gene of Yersinia enterocolitica and their effect on plague protective immunity.

Current efforts to develop plague vaccines focus on LcrV, a polypeptide that resides at the tip of type III secretion needles. LcrV-specific antibodies block Yersinia pestis type III injection of Yop effectors into host immune cells, thereby enabling phagocytes to kill the invading pathogen. Earlier work reported that antibodies against Y. pestis LcrV cannot block type III injection by Yersinia enterocolitica strains and suggested that lcrV polymorphisms may provide for escape from LcrV-mediated plague immunity. We show here that polyclonal or monoclonal antibodies raised against Y. pestis KIM D27 LcrV (LcrV(D27)) bind LcrV from Y. enterocolitica O:9 strain W22703 (LcrV(W22703)) or O:8 strain WA-314 (LcrV(WA-314)) but are otherwise unable to block type III injection by Y. enterocolitica strains. Replacing the lcrV gene on the pCD1 virulence plasmid of Y. pestis KIM D27 with either lcrV(W22703) or lcrV(WA-314) does not affect the ability of plague bacteria to secrete proteins via the type III pathway, to inject Yops into macrophages, or to cause lethal plague infections in mice. LcrV(D27)-specific antibodies blocked type III injection by Y. pestis expressing lcrV(W22703) or lcrV(WA-314) and protected mice against intravenous lethal plague challenge with these strains. Thus, although antibodies raised against LcrV(D27) are unable to block the type III injection of Y. enterocolitica strains, expression of lcrV(W22703) or lcrV(WA-314) in Y. pestis did not allow these strains to escape LcrV-mediated plague protective immunity in the intravenous challenge model.

Unique mode of lipogenic activation in rat preputial sebocytes.

Lipoprotein delivery of fatty acids and cholesterol is linked with peroxisome proliferator-activated receptor (PPAR) activation in adipocytes and macrophages. We postulated that similar interactions exist in sebaceous epithelial cells (sebocytes) in which PPAR activation induces differentiation. High-density lipoprotein (HDL) and very low-density lipoprotein (VLDL) markedly enhanced sebocyte differentiation above that found with PPAR agonists and were more potent than explicable by their lipid content. The PPARγ antagonist GW5393 reduced sebocyte differentiation to all PPAR isoform agonists, HDL and VLDL, suggesting that the lipoprotein effect on differentiation occurs partially through activation of PPARγ. Furthermore, we found that sebocytes expressed a unique pattern of lipogenic genes. Our results demonstrate that HDL and VLDL are the most potent inducers of sebocyte differentiation tested to date, and these actions are partially inhibited by PPAR antagonists. This suggests that substrates provided by lipoproteins are targeted to sebocytes and affect their own disposition via PPAR activation.

Prevention of pneumonic plague in mice, rats, guinea pigs and non-human primates with clinical grade rV10, rV10-2 or F1-V vaccines.

Yersinia pestis causes plague, a disease with high mortality in humans that can be transmitted by fleabite or aerosol. A US Food and Drug Administration (FDA)-licensed plague vaccine is currently not available. Vaccine developers have focused on two subunits of Y. pestis: LcrV, a protein at the tip of type III secretion needles, and F1, the fraction 1 pilus antigen. F1-V, a hybrid generated via translational fusion of both antigens, is being developed for licensure as a plague vaccine. The rV10 vaccine is a non-toxigenic variant of LcrV lacking residues 271-300. Here we developed Current Good Manufacturing Practice (cGMP) protocols for rV10. Comparison of clinical grade rV10 with F1-V did not reveal significant differences in plague protection in mice, guinea pigs or cynomolgus macaques. We also developed cGMP protocols for rV10-2, a variant of rV10 with an altered affinity tag. Immunization with rV10-2 adsorbed to aluminum hydroxide elicited antibodies against LcrV and conferred pneumonic plague protection in mice, rats, guinea pigs, cynomolgus macaques and African Green monkeys. The data support further development of rV10-2 for FDA Investigational New Drug (IND) authorization review and clinical testing.

Plague in Guinea pigs and its prevention by subunit vaccines.

Human pneumonic plague is a devastating and transmissible disease for which a Food and Drug Administration-approved vaccine is not available. Suitable animal models may be adopted as a surrogate for human plague to fulfill regulatory requirements for vaccine efficacy testing. To develop an alternative to pneumonic plague in nonhuman primates, we explored guinea pigs as a model system. On intranasal instillation of a fully virulent strain, Yersinia pestis CO92, guinea pigs developed lethal lung infections with hemorrhagic necrosis, massive bacterial replication in the respiratory system, and blood-borne dissemination to other organ systems. Expression of the Y. pestis F1 capsule was not required for the development of pulmonary infection; however, the capsule seemed to be important for the establishment of bubonic plague. The mean lethal dose (MLD) for pneumonic plague in guinea pigs was estimated to be 1000 colony-forming units. Immunization of guinea pigs with the recombinant forms of LcrV, a protein that resides at the tip of Yersinia type III secretion needles, or F1 capsule generated robust humoral immune responses. Whereas LcrV immunization resulted in partial protection against pneumonic plague challenge with 250 MLD Y. pestis CO92, immunization with recombinant F1 did not. rV10, a vaccine variant lacking LcrV residues 271-300, elicited protection against pneumonic plague, which seemed to be based on conformational antibodies directed against LcrV.

Amino acid residues 196-225 of LcrV represent a plague protective epitope.

LcrV, a protein that resides at the tip of the type III secretion needles of Yersinia pestis, is the single most important plague protective antigen. Earlier work reported monoclonal antibody MAb 7.3, which binds a conformational epitope of LcrV and protects experimental animals against lethal plague challenge. By screening monoclonal antibodies directed against LcrV for their ability to protect immunized mice against bubonic plague challenge, we examined here the possibility of additional protective epitopes. MAb BA5 protected animals against plague, neutralized the Y. pestis type III secretion pathway and promoted opsonophagocytic clearance of bacteria in blood. LcrV residues 196-225 were necessary and sufficient for MAb BA5 binding. Compared to full-length LcrV, a variant lacking its residues 196-225 retained the ability of eliciting plague protection. These results identify LcrV residues 196-225 as a linear epitope that is recognized by the murine immune system to confer plague protection.

Yersinia pestis IS1541 transposition provides for escape from plague immunity.

Yersinia pestis is perhaps the most feared infectious agent due to its ability to cause epidemic outbreaks of plague disease in animals and humans with high mortality. Plague infections elicit strong humoral immune responses against the capsular antigen (fraction 1 [F1]) of Y. pestis, and F1-specific antibodies provide protective immunity. Here we asked whether Y. pestis generates mutations that enable bacterial escape from protective immunity and isolated a variant with an IS1541 insertion in caf1A encoding the F1 outer membrane usher. The caf1A::IS1541 insertion prevented assembly of F1 pili and provided escape from plague immunity via F1-specific antibodies without a reduction in virulence in mouse models of bubonic or pneumonic plague. F1-specific antibodies interfere with Y. pestis type III transport of effector proteins into host cells, an inhibitory effect that was overcome by the caf1A::IS1541 insertion. These findings suggest a model in which IS1541 insertion into caf1A provides for reversible changes in envelope structure, enabling Y. pestis to escape from adaptive immune responses and plague immunity.

Pneumonic plague pathogenesis and immunity in Brown Norway rats.

The Brown Norway rat was recently described as a bubonic plague model that closely mimics human disease. We therefore evaluated the Brown Norway rat as an alternative small animal model for pneumonic plague and characterized both the efficacy and potency of vaccine candidates. When infected by intranasal instillation, these rats rapidly developed fatal pneumonic plague within 2 to 4 days of infection. Plague disease was characterized by severe alveolar edema and vascular hemorrhage in the lung in addition to fulminant necrotizing pneumonia caused by massive bacterial replication and inflammation. Twenty-four hours before death, animals developed systemic disease with an apparent delayed inflammatory response. We evaluated the ability of the protective antigen, LcrV, and a mutant derivative, V10, to protect these rats from pneumonic plague. Both were highly effective vaccines because complete protection was observed at challenge doses of 7500 LD(50). Antibody analyses suggested stronger potency of V10 immune sera compared with LcrV in the passive transfer of immunity to bubonic plague, with multiple neutralizing epitopes in LcrV. Taken together, these data demonstrate the effectiveness of inhibiting type III secretion in the prevention of pneumonic plague in rats and reveal critical contributions from both the cellular and humoral immune systems. Thus, the Brown Norway rat is an appealing alternative small animal model for the study of pneumonic plague pathogenesis and immunity.

Toll-like receptor 6 drives differentiation of tolerogenic dendritic cells and contributes to LcrV-mediated plague pathogenesis.

Educating dendritic cells (DC) to become tolerogenic DC, which promote regulatory IL-10 immune responses, represents an effective immune evasion strategy for pathogens. Yersinia pestis virulence factor LcrV is reported to induce IL-10 production via interaction with Toll-like receptor (TLR) 2. However, TLR2-/- mice are not protected against subcutaneous plague infection. Using complementary in vitro and in vivo approaches and LcrV as a model, we show that TLR6 associates with TLR2 to induce tolerogenic DC and regulatory type-1 T cells selectively secreting IL-10. In contrast, TLR1 heterodimerizes with TLR2 to promote proinflammatory IL-12p40 cytokine, producing DC and inflammatory T cell differentiation. LcrV specifically hijacks the TLR2/6 pathway to stimulate IL-10 production, which blocks host protective inflammatory responses. These results explain why TLR2 can mediate both pro- and anti-inflammatory responses and identify TLR6 as a distinct receptor driving regulatory IL-10 responses.

Immunization with recombinant V10 protects cynomolgus macaques from lethal pneumonic plague.

Vaccine and therapeutic strategies that prevent infections with Yersinia pestis have been sought for over a century. Immunization with live attenuated (nonpigmented) strains and immunization with subunit vaccines containing recombinant low-calcium-response V antigen (rLcrV) and recombinant F1 (rF1) antigens are considered effective in animal models. Current antiplague subunit vaccines in development for utilization in humans contain both antigens, either as equal concentrations of the two components (rF1 plus rLcrV) or as a fusion protein (rF1-rLcrV). Here, we show that immunization with either purified rLcrV (a protein at the tip of type III needles) or a variant of this protein, recombinant V10 (rV10) (lacking amino acid residues 271 to 300), alone or in combination with rF1, prevented pneumonic lesions and disease pathogenesis. In addition, passive immunization studies showed that specific antibodies of macaques immunized with rLcrV, rV10, or rF1, either alone or in combination, conferred protection against bubonic plague challenge in mice. Finally, we found that when we compared the reactivities of anti-rLcrV and anti-rV10 immune sera from cynomolgus macaques, BALB/c mice, and brown Norway rats with LcrV-derived peptides, rV10, but not rLcrV immune sera, lacked antibodies recognizing linear LcrV oligopeptides.

Yersinia pestis caf1 variants and the limits of plague vaccine protection.

Yersinia pestis, the highly virulent agent of plague, is a biological weapon. Strategies that prevent plague have been sought for centuries, and immunization with live, attenuated (nonpigmented) strains or subunit vaccines with F1 (Caf1) antigen is considered effective. We show here that immunization with live, attenuated strains generates plague-protective immunity and humoral immune responses against F1 pilus antigen and LcrV. Y. pestis variants lacking caf1 (F1 pili) are not only fully virulent in animal models of bubonic and pneumonic plague but also break through immune responses generated with live, attenuated strains or F1 subunit vaccines. In contrast, immunization with purified LcrV, a protein at the tip of type III needles, generates protective immunity against the wild-type and the fully virulent caf1 mutant strain, in agreement with the notion that LcrV can elicit vaccine protection against both types of virulent plague strains.

Immunogenicity and protective immunity against bubonic plague and pneumonic plague by immunization of mice with the recombinant V10 antigen, a variant of LcrV.

In contrast to Yersinia pestis LcrV, the recombinant V10 (rV10) variant (lacking residues 271 to 300) does not suppress the release of proinflammatory cytokines by immune cells. Immunization with rV10 generates robust antibody responses that protect mice against bubonic plague and pneumonic plague, suggesting that rV10 may serve as an improved plague vaccine.

Sebaceous epithelial cell differentiation requires cyclic adenosine monophosphate generation.

The cyclic adenosine monophosphate (cAMP) generator choleratoxin is known to promote the growth of sebaceous epithelial cells (sebocytes) in monolayer culture in classical serum-containing media. Now that sebocytes can be grown in serum-free medium, we have examined whether choleratoxin or other cAMP generators are required for differentiation of rat preputial sebocytes in response to specific ligand activators of peroxisome proliferator-activated receptors (PPARs). Unexpectedly, choleratoxin reduced sebocyte proliferation. However, sebocyte differentiation in response to specific PPARalpha and PPARgamma agonists required a cAMP generator such as choleratoxin, and this response was suppressed by a protein kinase A inhibitor. In contrast, the stable prostacyclin analog, carbaprostacyclin (cPGI2), a PPARalpha,delta agonist that also generates cAMP, stimulated differentiation independently of choleratoxin. Furthermore, unlike the selective PPARalpha and PPARgamma agonists, cPGI2 stimulated both sebocyte DNA synthesis and proliferation. These data are compatible with the evidence that prostacyclin has the additional effect of generating cAMP. In addition, we addressed the possibility that choleratoxin may act as a surrogate for beta-adrenergic catecholamines in generating cAMP. In contrast with choleratoxin, both alpha- and beta-adrenergic catecholamines stimulated sebocyte growth and interfered with the choleratoxin effect on differentiation. These data suggest ligand-dependent, complex interactions between cAMP and the other signal transduction pathways involved in sebocyte growth and development.