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1.  Persistence of cell-mediated immunity three decades after vaccination with the live vaccine strain of Francisella tularensis 
European journal of immunology  2011;41(4):974-980.
The efficacy of many vaccines against intracellular bacteria depends on the generation of cell-mediated immunity, but studies to determine the duration of immunity are usually confounded by re-exposure. The causative agent of tularemia, Francisella tularensis, is rare in most areas and, therefore, tularemia vaccination is an interesting model for studies of the longevity of vaccine-induced cell-mediated immunity. Here lymphocyte proliferation and cytokine production in response to F. tularensis were assayed in two groups of 16 individuals, vaccinated 1-3 or 27-34 years previously. As compared to naïve individuals, vaccinees of both groups showed higher proliferative responses and, out of 17 cytokines assayed, higher levels of MIP-1β, IFN-γ, IL-10, and IL-5 in response to recall stimulation. The responses were very similar in the two groups of vaccinees. A statistical model was developed to predict the immune status of the individuals and by use of two parameters, proliferative responses and levels of IFN-γ, 91.1% of the individuals were correctly classified. Using flow cytometry analysis, we demonstrated that during recall stimulation, expression of IFN-γ by CD4+CCR7+, CD4+CD62L+, CD8+CCR7+, and CD8+CD62L+ cells significantly increased in samples from vaccinated donors. In conclusion, cell-mediated immunity was found to persist three decades after tularemia vaccination without evidence of decline.
PMCID: PMC3516913  PMID: 21442618
Francisella tularensis; vaccination; persistence; cell-mediated immunity
2.  Immunoproteomic analysis of the human antibody response to natural tularemia infection with Type A or Type B strains or LVS vaccination 
Francisella tularensis is pathogenic for many mammalian species including humans, causing a spectrum of diseases called tularemia. The highly virulent Type A strains have associated mortality rates of up to 60% if inhaled. An attenuated live vaccine strain (LVS) is the only vaccine to show efficacy in humans, but suffers several barriers to licensure, including the absence of a correlate of protection. An immunoproteomics approach was used to survey the repertoire of antibodies in sera from individuals who had contracted tularemia during two outbreaks and individuals from two geographical areas who had been vaccinated with NDBR Lot 11 or Lot 17 LVS. These data showed a large overlap in the antibodies generated in response to tularemia infection or LVS vaccination. A total of seven proteins were observed to be reactive with 60 % or more sera from vaccinees and convalescents. A further four proteins were recognised by 30–60 % of the sera screened. These proteins have the potential to serve as markers of vaccination or candidates for subunit vaccines.
PMCID: PMC3184521  PMID: 21873113
Francisella tularensis; live vaccine strain; vaccine; immunoproteomics; tularemia
3.  Establishment of a Swiss Webster Mouse Model of Pneumonic Plague To Meet Essential Data Elements under the Animal Rule 
A recombinant vaccine (rF1V) is being developed for protection against pneumonic plague. This study was performed to address essential data elements to establish a well-characterized Swiss Webster mouse model for licensing the rF1V vaccine using the FDA's Animal Rule. These elements include the documentation of challenge material characteristics, aerosol exposure parameters, details of the onset and severity of clinical signs, pathophysiological response to disease, and relevance to human disease. Prior to animal exposures, an evaluation of the aerosol system was performed to determine and understand the variability of the aerosol exposure system. Standardized procedures for the preparation of Yersinia pestis challenge material also were developed. The 50% lethal dose (LD50) was estimated to be 1,966 CFU using Probit analysis. Following the LD50 determination, pathology was evaluated by exposing mice to a target LD99 (42,890 CFU). Mice were euthanized at 12, 24, 36, 48, 60, and 72 h postexposure. At each time point, samples were collected for clinical pathology, detection of bacteria in blood and tissues, and pathology evaluations. A general increase in incidence and severity of microscopic findings was observed in the lung, lymph nodes, spleen, and liver from 36 to 72 h postchallenge. Similarly, the incidence and severity of pneumonia increased throughout the study; however, some mice died in the absence of pneumonia, suggesting that disease progression does not require the development of pneumonia. Disease pathology in the Swiss Webster mouse is similar to that observed in humans, demonstrating the utility of this pneumonic plague model that can be used by researchers investigating plague countermeasures.
PMCID: PMC3318273  PMID: 22336286
4.  Advanced Development of the rF1V and rBV A/B Vaccines: Progress and Challenges 
The development of vaccines for microorganisms and bacterial toxins with the potential to be used as biowarfare and bioterrorism agents is an important component of the US biodefense program. DVC is developing two vaccines, one against inhalational exposure to botulinum neurotoxins A1 and B1 and a second for Yersinia pestis, with the ultimate goal of licensure by the FDA under the Animal Rule. Progress has been made in all technical areas, including manufacturing, nonclinical, and clinical development and testing of the vaccines, and in assay development. The current status of development of these vaccines, and remaining challenges are described in this chapter.
PMCID: PMC3199075  PMID: 22028978
5.  Inhalational Botulism in Rhesus Macaques Exposed to Botulinum Neurotoxin Complex Serotypes A1 and B1▿ †  
A recombinant botulinum vaccine (rBV A/B) is being developed for protection against inhalational intoxication with botulinum neurotoxin (BoNT) complex serotype A, subtype A1 (BoNT/A1), and BoNT serotype B, subtype B1 (BoNT/B1). A critical component for evaluating rBV A/B efficacy will be the use of animal models in which the pathophysiology and dose-response relationships following aerosol exposure to well-characterized BoNT are thoroughly understood and documented. This study was designed to estimate inhaled 50% lethal doses (LD50) and to estimate 50% lethal exposure concentrations relative to time (LCt50) in rhesus macaques exposed to well-characterized BoNT/A1 and BoNT/B1. During the course of this study, clinical observations, body weights, clinical hematology results, clinical chemistry results, circulating neurotoxin levels, and telemetric parameters were documented to aid in the understanding of disease progression. The inhaled LD50 and LCt50 for BoNT/A1 and BoNT/B1 in rhesus macaques were determined using well-characterized challenge material. Clinical observations were consistent with the recognized pattern of botulism disease progression. A dose response was demonstrated with regard to the onset of these clinical signs for both BoNT/A1 and BoNT/B1. Dose-related changes in physiologic parameters measured by telemetry were also observed. In contrast, notable changes in body weight, hematology, and clinical chemistry parameters were not observed. Circulating levels of BoNT/B1 were detected in animals exposed to the highest levels of BoNT/B1; however, BoNT/A1 was not detected in the circulation at any aerosol exposure level. The rhesus macaque aerosol challenge model will be used for future evaluations of rBV A/B efficacy against inhalational BoNT/A1 and BoNT/B1 intoxication.
PMCID: PMC2944462  PMID: 20660138
6.  Differential ability of novel attenuated targeted deletion mutants of Francisella tularensis subspecies tularensis strain SCHU S4 to protect mice against aerosol challenge with virulent bacteria: effects of host background and route of immunization 
Vaccine  2009;28(7):1824.
Francisella tularensis subspecies tularensis is a highly virulent facultative intracellular pathogen of humans and a potential biological weapon. A live vaccine strain, F. tularensis LVS, was developed more than 50 years ago by pragmatic attenuation of a strain of the less virulent holarctica subspecies. LVS was demonstrated to be highly effective in human volunteers who were exposed to intradermal challenge with fully virulent subsp. tularensis, but was less effective against aerosol exposure. LVS faces regulatory hurdles that to date have prevented its licensure for general use. Therefore, a better defined and more effective vaccine is being sought. To this end we have created gene deletion mutants in the virulent subsp. tularensis strain and tested them for their ability to elicit a protective immune response against systemic or aerosol challenge with the highly virulent wild-type subsp. tularensis strain, SCHU S4. Both oral and Intradermal (ID) primary vaccination routes were assessed in BALB/c and C3H/HeN mice as was oral boosting. One SCHU S4 mutant missing the heat shock gene, clpB, was significantly more attenuated than LVS whereas a double deletion mutant missing genes FTT0918 and capB was as attenuated as LVS. In general mice immunized with SCHU S4ΔclpB were significantly better protected against aerosol challenge than mice immunized with LVS. A single ID immunization of BALB/c mice with SCHU S4ΔclpB was at least as effective as any other regimen examined. Mice immunized with SCHU S4Δ0918ΔcapB were generally protected to a similar degree as mice immunized with LVS. A preliminary examination of immune responses to vaccination with LVS, SCHU S4ΔclpB, or SCHU S4Δ0918ΔcapB provided no obvious correlate to their relative efficacies.
PMCID: PMC2822029  PMID: 20018266
7.  Immunoproteomics Analysis of the Murine Antibody Response to Vaccination with an Improved Francisella tularensis Live Vaccine Strain (LVS) 
PLoS ONE  2010;5(4):e10000.
Francisella tularensis subspecies tularensis is the causative agent of a spectrum of diseases collectively known as tularemia. An attenuated live vaccine strain (LVS) has been shown to be efficacious in humans, but safety concerns have prevented its licensure by the FDA. Recently, F. tularensis LVS has been produced under Current Good Manufacturing Practice (CGMP guidelines). Little is known about the immunogenicity of this new vaccine preparation in comparison with extensive studies conducted with laboratory passaged strains of LVS. Thus, the aim of the current work was to evaluate the repertoire of antibodies produced in mouse strains vaccinated with the new LVS vaccine preparation.
Methodology/Principal Findings
In the current study, we used an immunoproteomics approach to examine the repertoire of antibodies induced following successful immunization of BALB/c versus unsuccessful vaccination of C57BL/6 mice with the new preparation of F. tularensis LVS. Successful vaccination of BALB/c mice elicited antibodies to nine identified proteins that were not recognized by antisera from vaccinated but unprotected C57BL/6 mice. In addition, the CGMP formulation of LVS stimulated a greater repertoire of antibodies following vaccination compared to vaccination with laboratory passaged ATCC LVS strain. A total of 15 immunoreactive proteins were identified in both studies, however, 16 immunoreactive proteins were uniquely reactive with sera from the new formulation of LVS.
This is the first report characterising the antibody based immune response of the new formulation of LVS in the widely used murine model of tularemia. Using two mouse strains, we show that successfully vaccinated mice can be distinguished from unsuccessfully vaccinated mice based upon the repertoire of antibodies generated. This opens the door towards downselection of antigens for incorporation into tularemia subunit vaccines. In addition, this work also highlights differences in the humoral immune response to vaccination with the commonly used laboratory LVS strain and the new vaccine formulation of LVS.
PMCID: PMC2848853  PMID: 20368994
8.  An improved Francisella tularensis Live Vaccine Strain (LVS) is well tolerated and highly immunogenic when administered to rabbits in escalating doses using various immunization routes 
Vaccine  2008;26(14):1773-1785.
Tularemia is a severe disease for which there is no licensed vaccine. An attenuated F. tularensis live vaccine strain (LVS) was protective when administered to humans but safety concerns precluded its licensure and use in large scale immunization. An improved F. tularensis LVS preparation was produced under current Good Manufacturing Practice (cGMP) guidelines for evaluation in clinical trials. Preclinical safety, tolerability and immunogenicity were investigated in rabbits that received LVS in escalating doses (1x105 to 1x109 CFU) by the intradermal, subcutaneous or percutaneous (scarification) route. This improved LVS formulation was well tolerated at all doses; no death or adverse clinical signs were observed and necropsies showed no signs of pathology. No live organisms were detected in liver or spleen. Transient local reactogenicity was observed after scarification injection. Erythema and edema developed after intradermal injection in the highest dose cohorts. High levels of F. tularensis-specific IgM, IgG and IgA developed early after immunization, in a dose-dependent fashion. Scarification elicited higher levels of IgA. Antibodies elicited by LVS also recognized F. tularensis Schu-S4 antigens and there was a significant correlation between antibody titers measured against both LVS and Schu-S4. The ELISA titers also correlated closely with those measured by microagglutination. This is the first report describing comprehensive toxicological and immunological studies of F. tularensis LVS in rabbits. This animal model, which closely resembles human disease, proved adequate to assess safety and immunogenicity of F. tularensis vaccine candidates. This new LVS vaccine preparation is being evaluated in human clinical studies.
PMCID: PMC2678717  PMID: 18308432
Tularemia and vaccines; F. tularensis vaccines; F. tularensis and antibody responses; tularemia and rabbits
9.  Biological Activity of an Intravenous Preparation of Human Vaccinia Immune Globulin in Mouse Models of Vaccinia Virus Infection 
The biological activity of a new intravenous (i.v.) preparation of human vaccinia immune globulin (VIGIV) was evaluated in two mouse models of vaccinia virus (VV) infection. In a mouse tail lesion model, female CD-1 mice were inoculated i.v. with 7 × 104 PFU of VV to produce >10 lesions per tail 8 days later. In a mouse lethality model, female severe combined immunodeficient (SCID) mice were inoculated i.v. with 3 × 104 PFU of VV to produce 100% mortality within 45 days. The ability of VIGIV to reduce tail lesion formation in CD-1 mice and mortality in SCID mice was determined by (i) pretreatment of a lethal VV dose with VIGIV prior to i.v. inoculation into SCID mice and (ii) i.v. administration of VIGIV to CD-1 and SCID mice the day before and up to 8 days after VV infection. VIGIV reduced the proportion of CD-1 mice with >10 tail lesions in a dose-related manner when VIGIV was given 1 day before and up to 1 day after VV inoculation. The pretreatment of VV with VIGIV prolonged survival and decreased mortality. VIGIV (100 and 400 mg/kg) prolonged survival when given up to 4 days after VV inoculation, and the 400-mg/kg dose reduced the mortality rate by 80% when given the day before or immediately after VV inoculation. The biological activity of VIGIV was demonstrated in both the immunocompetent and immunocompromised murine models. The timing of treatment relative to VV inoculation appeared to be important for the demonstration of VIGIV's biological activity.
PMCID: PMC1168682  PMID: 15980330

Results 1-9 (9)