Promiscuous \u3cem\u3eCoxiella burnetii\u3c/em\u3e CD4 Epitope Clusters Associated With Human Recall Responses Are Candidates for a Novel T-Cell Targeted Multi-Epitope Q Fever Vaccine

Coxiella burnetii, the causative agent of Q fever, is a Gram-negative intracellular bacterium transmitted via aerosol. Regulatory approval of the Australian whole-cell vaccine Q-VAX® in the US and Europe is hindered by reactogenicity in previously exposed individuals. The aim of this study was to identify and rationally select C. burnetii epitopes for design of a safe, effective, and less reactogenic T-cell targeted human Q fever vaccine. Immunoinformatic methods were used to predict 65 HLA class I epitopes and 50 promiscuous HLA class II C. burnetii epitope clusters, which are conserved across strains of C. burnetii. HLA binding assays confirmed 89% of class I and 75% of class II predictions, and 11 HLA class II epitopes elicited IFNγ responses following heterologous DNA/DNA/peptide/peptide prime-boost immunizations of HLA-DR3 transgenic mice. Human immune responses to the predicted epitopes were characterized in individuals naturally exposed to C. burnetii during the 2007–2010 Dutch Q fever outbreak. Subjects were divided into three groups: controls with no immunological evidence of previous infection and individuals with responses to heat-killed C. burnetii in a whole blood IFNγ release assay (IGRA) who remained asymptomatic or who experienced clinical Q fever during the outbreak. Recall responses to C. burnetii epitopes were assessed by cultured IFNγ ELISpot. While HLA class I epitope responses were sparse in this cohort, we identified 21 HLA class II epitopes that recalled T-cell IFNγ responses in 10–28% of IGRA+ subjects. IGRA+ individuals with past asymptomatic and symptomatic C. burnetii infection showed a comparable response pattern and cumulative peptide response which correlated with IGRA responses. None of the peptides elicited reactogenicity in a C. burnetii exposure-primed guinea pig model. These data demonstrate that a substantial proportion of immunoinformatically identified HLA class II epitopes show long-lived immunoreactivity in naturally infected individuals, making them desirable candidates for a novel human multi-epitope Q fever vaccine

VaxCelerate II: Rapid development of a self-assembling vaccine for Lassa fever

Development of effective vaccines against emerging infectious diseases (EID) can take as much or more than a decade to progress from pathogen isolation/identification to clinical approval. As a result, conventional approaches fail to produce field-ready vaccines before the EID has spread extensively. Lassa is a prototypical emerging infectious disease endemic to West Africa for which no successful vaccine is available. We established the VaxCelerate Consortium to address the need for more rapid vaccine development by creating a platform capable of generating and pre-clinically testing a new vaccine against specific pathogen targets in less than 120 d. A self-assembling vaccine is at the core of the approach. It consists of a fusion protein composed of the immunostimulatory Mycobacterium tuberculosis heat shock protein 70 (MtbHSP70) and the biotin binding protein, avidin. Mixing the resulting protein (MAV) with biotinylated pathogen-specific immunogenic peptides yields a self-assembled vaccine (SAV). To meet the time constraint imposed on this project, we used a distributed R&D model involving experts in the fields of protein engineering and production, bioinformatics, peptide synthesis/design and GMP/GLP manufacturing and testing standards. SAV immunogenicity was first tested using H1N1 influenza specific peptides and the entire VaxCelerate process was then tested in a mock live-fire exercise targeting Lassa fever virus. We demonstrated that the Lassa fever vaccine induced significantly increased class II peptide specific interferon-γ CD4+ T cell responses in HLA-DR3 transgenic mice compared to peptide or MAV alone controls. We thereby demonstrated that our SAV in combination with a distributed development model may facilitate accelerated regulatory review by using an identical design for each vaccine and by applying safety and efficacy assessment tools that are more relevant to human vaccine responses than current animal models

Promiscuous Coxiella burnetii CD4 Epitope Clusters Associated With Human Recall Responses Are Candidates for a Novel T-Cell Targeted Multi-Epitope Q Fever Vaccine

Coxiella burnetii, the causative agent of Q fever, is a Gram-negative intracellular bacterium transmitted via aerosol. Regulatory approval of the Australian whole-cell vaccine Q-VAX® in the US and Europe is hindered by reactogenicity in previously exposed individuals. The aim of this study was to identify and rationally select C. burnetii epitopes for design of a safe, effective, and less reactogenic T-cell targeted human Q fever vaccine. Immunoinformatic methods were used to predict 65 HLA class I epitopes and 50 promiscuous HLA class II C. burnetii epitope clusters, which are conserved across strains of C. burnetii. HLA binding assays confirmed 89% of class I and 75% of class II predictions, and 11 HLA class II epitopes elicited IFNγ responses following heterologous DNA/DNA/peptide/peptide prime-boost immunizations of HLA-DR3 transgenic mice. Human immune responses to the predicted epitopes were characterized in individuals naturally exposed to C. burnetii during the 2007–2010 Dutch Q fever outbreak. Subjects were divided into three groups: controls with no immunological evidence of previous infection and individuals with responses to heat-killed C. burnetii in a whole blood IFNγ release assay (IGRA) who remained asymptomatic or who experienced clinical Q fever during the outbreak. Recall responses to C. burnetii epitopes were assessed by cultured IFNγ ELISpot. While HLA class I epitope responses were sparse in this cohort, we identified 21 HLA class II epitopes that recalled T-cell IFNγ responses in 10–28% of IGRA+ subjects. IGRA+ individuals with past asymptomatic and symptomatic C. burnetii infection showed a comparable response pattern and cumulative peptide response which correlated with IGRA responses. None of the peptides elicited reactogenicity in a C. burnetii exposure-primed guinea pig model. These data demonstrate that a substantial proportion of immunoinformatically identified HLA class II epitopes show long-lived immunoreactivity in naturally infected individuals, making them desirable candidates for a novel human multi-epitope Q fever vaccine

Q-vaxcelerate: A distributed development approach for a new Coxiella burnetii vaccine

Development of vaccines that are both safe and effective remains a costly and time-consuming challenge. To accelerate the pace of development and improve the efficacy and safety of candidate vaccines for both existing and emerging infectious agents, we have used a distributed development approach. This features the managed integration of individual expert groups having the requisite vaccine platforms, pre-clinical models, assays, skills and knowledge pertinent to a specific pathogen into a single, end-to-end development team capable of producing a new vaccine tailored to that particular agent. Distributed development focuses on integrating existing effort across multiple institutions rather than developing new capabilities or consolidating resources within an individual organization. Previously we have used the distributed development strategy to generate vaccine candidates for emerging viral diseases. Coxiella burnetii is a highly infectious and resilient bacterium and the causative agent of Q fever. Treatment for Q fever can require months of antibiotics. The current vaccine for Q-fever is only approved in Australia and requires prescreening due to the potential for severe reactogenicity in previously exposed individuals. Here we discuss Q-VaxCelerate, a distributed development consortium for the development of a new vaccine to prevent Q fever

Antimicrobial photodynamic therapy mediated by methylene blue and potassium iodide to treat urinary tract infection in a female rat model

Drug-resistant urinary tract infections (UTIs) are difficult and sometimes impossible to treat. Many UTIs are caused by uropathogenic Escherichia coli (UPEC). We developed an intact rat model of UTI, by catheterizing female rats and introducing a bioluminescent UPEC strain into the female rat bladder which lasted for up to six days. We recently showed that antimicrobial photodynamic inactivation (aPDI) of a bacterial infection mediated by the well-known phenothiazinium salt, methylene blue (MB) could be strongly potentiated by addition of the non-toxic salt potassium iodide (KI). In the intact rat model we introduced MB into the bladder by catheter, followed by KI solution and delivered intravesicular illumination with a diffusing fiber connected to a 1 W 660 nm laser. Bioluminescent imaging of the bacterial burden was carried out during the procedure and for 6 days afterwards. Light-dose dependent loss of bioluminescence was observed with the combination of MB followed by KI, but recurrence of infection was seen the next day in some cases. aPDT with MB + KI gave a significantly shorter duration of infection compared to untreated controls. aPDT with MB alone was the least effective. No signs of aPDT damage to the bladder lining were detected. This procedure to treat urinary tract infections without antibiotics by using already approved pharmaceutical substances (MB and KI) may have clinical applicability, either initially as a stand-alone therapy, or as an adjunct to antibiotic therapy by a rapid and substantial reduction of the bacterial burden

A pilot clinical trial of a near-infrared laser vaccine adjuvant: safety, tolerability, and cutaneous immune cell trafficking.

Many vaccines require adjuvants to enhance immunogenicity, but there are few safe and effective intradermal (i.d.) adjuvants. Murine studies have validated the potency of laser illumination of skin as an adjuvant for i.d. vaccination with advantages over traditional adjuvants. We report a pilot clinical trial of low-power, continuous-wave, near-infrared laser adjuvant treatment, representing the first human trial of the safety, tolerability, and cutaneous immune cell trafficking changes produced by the laser adjuvant. In this trial we demonstrated a maximum tolerable energy dose of 300 J/cm2 to a spot on the lower back. The irradiated spot was biopsied 4 h later, as was a control spot. Paired biopsies were submitted for histomorphologic and immunohistochemical evaluation in a blinded fashion as well as quantitative PCR analysis for chemokines and cytokines. Similar to prior murine studies, highly significant reductions in CD1a+ Langerhans cells in the dermis and CD11c+ dermal dendritic cells were observed, corresponding to the increased migratory activity of these cells; changes in the epidermis were not significant. There was no evidence of skin damage. The laser adjuvant is a safe, well-tolerated adjuvant for i.d. vaccination in humans and results in significant cutaneous immune cell trafficking.-Gelfand, J. A., Nazarian, R. M., Kashiwagi, S., Brauns, T., Martin, B., Kimizuka, Y., Korek, S., Botvinick, E., Elkins, K., Thomas, L., Locascio, J., Parry, B., Kelly, K. M., Poznansky, M. C. A pilot clinical trial of a near-infrared laser vaccine adjuvant: safety, tolerability, and cutaneous immune cell trafficking

A pilot clinical trial of a near-infrared laser vaccine adjuvant: safety, tolerability, and cutaneous immune cell trafficking.

Many vaccines require adjuvants to enhance immunogenicity, but there are few safe and effective intradermal (i.d.) adjuvants. Murine studies have validated the potency of laser illumination of skin as an adjuvant for i.d. vaccination with advantages over traditional adjuvants. We report a pilot clinical trial of low-power, continuous-wave, near-infrared laser adjuvant treatment, representing the first human trial of the safety, tolerability, and cutaneous immune cell trafficking changes produced by the laser adjuvant. In this trial we demonstrated a maximum tolerable energy dose of 300 J/cm2 to a spot on the lower back. The irradiated spot was biopsied 4 h later, as was a control spot. Paired biopsies were submitted for histomorphologic and immunohistochemical evaluation in a blinded fashion as well as quantitative PCR analysis for chemokines and cytokines. Similar to prior murine studies, highly significant reductions in CD1a+ Langerhans cells in the dermis and CD11c+ dermal dendritic cells were observed, corresponding to the increased migratory activity of these cells; changes in the epidermis were not significant. There was no evidence of skin damage. The laser adjuvant is a safe, well-tolerated adjuvant for i.d. vaccination in humans and results in significant cutaneous immune cell trafficking.-Gelfand, J. A., Nazarian, R. M., Kashiwagi, S., Brauns, T., Martin, B., Kimizuka, Y., Korek, S., Botvinick, E., Elkins, K., Thomas, L., Locascio, J., Parry, B., Kelly, K. M., Poznansky, M. C. A pilot clinical trial of a near-infrared laser vaccine adjuvant: safety, tolerability, and cutaneous immune cell trafficking