pestis

pestis. In conclusion, we have further optimized a gentamicin protection assay to specifically examine novel anti-F1 and anti-LcrV monoclonal antibodies. system that facilitates translocation of virulence factors into the host cell, using two functional assays. Anti-F1 and anti-LcrV monoclonal antibodies both increased bacterial uptake by macrophages, with greater uptake observed in the presence of antibodies that were protective in the mouse pneumonic plague model. In addition, the protective anti-F1 and anti-LcrV antibodies produced unique cytokine signatures that were also associated with in vivo protection. These antibody-dependent characteristics from in vitro functional assays will be useful in down-selecting efficacious novel antibodies that can be used for treatment of plague. KEYWORDS:Yersinia pestis, mice, immunity, antibody treatment, HS-173 antibody screening, plague == Introduction == Yersinia pestisis a gram-negative bacteria that is classified as a Tier 1 select agent by the United States Department of Health and Human Services due to its ability to cause rapidly fatal infections in both humans and animals. While bubonic plague is the most common form of the disease, pneumonic plague is usually a greater concern in the context of biodefense strategies.1,2Y. pestisis a major biothreat due to its capacity for aerosol dissemination and its contagious nature in the pneumonic form. The illness can be treated with several different classes of antibiotics, including aminoglycosides (e.g., streptomycin) and fluoroquinolones (e.g., ciprofloxacin), if accurate diagnosis is made early after contamination and antibiotic therapy is initiated without delay.3However, antibiotic treatment options could become limited if the bacteria are engineered to become resistant or naturally acquire antibiotic resistance. The recent plague outbreak in Madagascar, which included cases of naturally acquired antibiotic resistance, emphasizes the need for novel therapeutics that can be used either alone or in combination with other medical countermeasures.47 Two protective antigens have been used to make subunit vaccines, including the Fraction 1 (F1) and HS-173 the low-calcium response V (LcrV) antigens.8,9The F1 protein is encoded by thecaf1gene located on a large pMT plasmid and is expressed at 37C. It inhibits the uptake of the bacteria by macrophages, creating an anti-phagocytic capsule.10It is also thought to play a role in bacterial transmission because it was shown to inhibit the adhesion of the bacteria to human epithelial cells.11However, strains ofY. pestis(e.g., C12 strain) have been recognized that are F1-unfavorable and still retain virulence in mice,1215emphasizing the need to additionally target another conserved and protective antigen that would be found in F1-unfavorable strains, such as the LcrV protein.16LcrV is encoded around the pCD1 plasmid and is a major virulence factor that is necessary for proper assembly of the translocation pore of the type 3 secretion system (T3SS) injectisome complex. This antigen facilitates translocation of the virulence factors called Yops into target cells, which results in the inhibition of phagocytosis and induction of apoptosis.17The LcrV antigen has also been demonstrated to be a multifunctional protein as it has been shown to have immunomodulatory effects in both in vivo and in vitro settings as well as the ability to translocate into host cells.1820The protective epitope of the LcrV antigen has been mapped by several groups and includes amino acids 135 to 275.17,21 Active immunization with recombinant LcrV protein has been shown to confer protection against both bubonic and pneumonic models of plague caused by either an encapsulated (F1+) strain HS-173 CO92 or non-encapsulated (F1-) strain C12,22although the level of protection against non-encapsulated strains remains equivocal. Combining both F1 and LcrV antigens resulted in improved protection in mice infected withY. pestis.16Targeting these two antigens increases the likelihood of protecting against emerging or engineeredY. pestisisolates that may be F1-unfavorable despite the known heterogeneity among the LcrV proteins from different isolates.23,24Researchers in the United States have predominantly pursued a recombinant fusion-protein strategy (i.e., F1-V),2527whereas experts in the United Kingdom focused their efforts on a vaccine with both unique protein entities (i.e., F1 + V).2831However, to date, there is no FDA-approved vaccine against Erg plague. Vaccine studies using the F1 and LcrV antigens suggest that antibodies play a role in the acquired protection. In addition, HS-173 passive protection has been accomplished using antibodies directed against either antigen.21,30,3238Passive immunization with two monoclonal antibodies (mAb) generated againstY. pestisLcrV (mAb 7.3) or F1 (mAb F104-A-G1) antigens are highly protective in mouse models of bubonic and pneumonic plague when administered prophylactically or 48 hours post-infection, either alone or in.