Assessment of CD200R Activation in Combination with Doxycycline in a Model of Melioidosis.

Antimicrobial resistance continues to be a global issue. Pathogens, such as Burkholderia pseudomallei, have evolved mechanisms to efflux certain antibiotics and manipulate the host response. New treatment strategies are therefore required, such as a layered defense approach. Here, we demonstrate, using biosafety level 2 (BSL-2) and BSL-3 in vivo murine models, that combining the antibiotic doxycycline with an immunomodulatory drug that targets the CD200 axis is superior to antibiotic treatment in combination with an isotype control. CD200-Fc treatment alone significantly reduces bacterial burden in lung tissue in both the BSL-2 and BSL-3 models. When CD200-Fc treatment is combined with doxycycline to treat the acute BSL-3 model of melioidosis, there is a 50% increase in survival compared with relevant controls. This benefit is not due to increasing the area under the concentration-time curve (AUC) of the antibiotic, suggesting the immunomodulatory nature of CD200-Fc treatment is playing an important role by potentially controlling the overactive immune response seen with many lethal bacterial infections. IMPORTANCE Traditional treatments for infectious disease have focused on the use of antimicrobial compounds (e.g. antibiotics) that target the infecting organism. However, timely diagnosis and administration of antibiotics remain crucial to ensure efficacy of these treatments especially for the highly virulent biothreat organisms. The need for early antibiotic treatment, combined with the increasing emergence of antibiotic resistant bacteria, means that new therapeutic strategies are required for organisms that cause rapid, acute infections. Here, we show that a layered defense approach, where an immunomodulatory compound is combined with an antibiotic, is better than an antibiotic combined with a relevant isotype control following infection with the biothreat agent Burkholderia pseudomallei. This approach has the potential to be truly broad spectrum and since the strategy includes manipulation of the host response it's application could be used in the treatment of a wide range of diseases.

Vaccines for emerging pathogens: from research to the clinic. Part two.

For this two-part Clinical & Experimental Immunology Review Series, Guest Editor E. Diane Williamson invited experts in their fields to contribute articles on the status of vaccine research and development for emerging pathogens. Part One addressed our progress in developing vaccines for emerging and re-emerging viruses and considered current work on effective vaccines for bacterial pathogens. In this edition of the journal, we are pleased to present Part Two, in which our authors emphasise the importance of vaccine formulation and of the use of effective delivery vehicles, as well as the prospects for licensure for current candidate vaccines.

Antibody-mediated protection against MERS-CoV in the murine model.

Murine antisera with neutralising activity for the coronavirus causative of Middle East respiratory syndrome (MERS) were induced by immunisation of Balb/c mice with the receptor binding domain (RBD) of the viral Spike protein. The murine antisera induced were fully-neutralising in vitro for two separate clinical strains of the MERS coronavirus (MERS-CoV). To test the neutralising capacity of these antisera in vivo, susceptibility to MERS-CoV was induced in naive recipient Balb/c mice by the administration of an adenovirus vector expressing the human DPP4 receptor (Ad5-hDPP4) for MERS-CoV, prior to the passive transfer of the RBD-specific murine antisera to the transduced mice. Subsequent challenge of the recipient transduced mice by the intra-nasal route with a clinical isolate of the MERS-CoV resulted in a significantly reduced viral load in their lungs, compared with transduced mice receiving a negative control antibody. The murine antisera used were derived from mice which had been primed sub-cutaneously with a recombinant fusion of RBD with a human IgG Fc tag (RBD-Fc), adsorbed to calcium phosphate microcrystals and then boosted by the oral route with the same fusion protein in reverse micelles. The data gained indicate that this dual-route vaccination with novel formulations of the RBD-Fc, induced systemic and mucosal anti-viral immunity with demonstrated in vitro and in vivo neutralisation capacity for clinical strains of MERS-CoV.

CD200R deletion promotes a neutrophil niche for Francisella tularensis and increases infectious burden and mortality.

Pulmonary immune control is crucial for protection against pathogens. Here we identify a pathway that promotes host responses during pulmonary bacterial infection; the expression of CD200 receptor (CD200R), which is known to dampen pulmonary immune responses, promotes effective clearance of the lethal intracellular bacterium Francisella tularensis. We show that depletion of CD200R in mice increases in vitro and in vivo infectious burden. In vivo, CD200R deficiency leads to enhanced bacterial burden in neutrophils, suggesting CD200R normally limits the neutrophil niche for infection. Indeed, depletion of this neutrophil niche in CD200R-/- mice restores F. tularensis infection to levels seen in wild-type mice. Mechanistically, CD200R-deficient neutrophils display significantly reduced reactive oxygen species production (ROS), suggesting that CD200R-mediated ROS production in neutrophils is necessary for limiting F. tularensis colonisation and proliferation. Overall, our data show that CD200R promotes the antimicrobial properties of neutrophils and may represent a novel antibacterial therapeutic target.

Vaccines for emerging pathogens: prospects for licensure.

Globally, there are a number of emerging pathogens. For most, there are no licensed vaccines available for human use, although there is ongoing research and development. However, given the extensive and increasing list of emerging pathogens and the investment required to bring vaccines into clinical use, the task is huge. Overlaid on this task is the risk of anti-microbial resistance (AMR) acquisition by micro-organisms which can endow a relatively harmless organism with pathogenic potential. Furthermore, climate change also introduces a challenge by causing some of the insect vectors and environmental conditions prevalent in tropical regions to begin to spread out from these traditional areas, thus increasing the risk of migration of zoonotic disease. Vaccination provides a defence against these emerging pathogens. However, vaccines for pathogens which cause severe, but occasional, disease outbreaks in endemic pockets have suffered from a lack of commercial incentive for development to a clinical standard, encompassing Phase III clinical trials for efficacy. An alternative is to develop such vaccines to request US Emergency Use Authorization (EUA), or equivalent status in the United States, Canada and the European Union, making use of a considerable number of regulatory mechanisms that are available prior to licensing. This review covers the status of vaccine development for some of the emerging pathogens, the hurdles that need to be overcome to achieve EUA or an equivalent regional or national status and how these considerations may impact vaccine development for the future, such that a more comprehensive stockpile of promising vaccines can be achieved.

Vaccines for emerging pathogens: from research to the clinic.

In this two-part series of reviews, we have invited experts in their fields to contribute articles on the status of vaccine research and development for emerging pathogens. This topic has been brought into sharp focus in recent years following significant outbreaks of viral diseases such as those causing severe acute respiratory syndrome and Middle East respiratory syndrome, as well as devastating outbreaks of diseases caused by the Ebola, Marburg, Zika and Lassa fever viruses, to name only a few examples. Additionally, bacterial infections leading to bubonic and pneumonic plague, most notably in Madagascar in 2018, as well as malaria in many tropical countries, melioidosis in south east Asia and tularaemia in northern Europe and North America, have incurred significant morbidity and mortality. In this review series, the life cycle of these pathogens and the epidemiology of disease have been reviewed in the context of potential points of intervention for the prevention of human infection. Many of the emerging pathogens are zoonoses and, as such, there is scope for intervention at the animal/insect/environmental reservoir. Other pathogens covered in this review series are considered to be re-emerging, such as multi-drug resistant tuberculosis.

Use of bioengineered human commensal gut bacteria-derived microvesicles for mucosal plague vaccine delivery and immunization.

Plague caused by the Gram-negative bacterium, Yersinia pestis, is still endemic in parts of the world today. Protection against pneumonic plague is essential to prevent the development and spread of epidemics. Despite this, there are currently no licensed plague vaccines in the western world. Here we describe the means of delivering biologically active plague vaccine antigens directly to mucosal sites of plague infection using highly stable microvesicles (outer membrane vesicles; OMVs) that are naturally produced by the abundant and harmless human commensal gut bacterium Bacteroides thetaiotaomicron (Bt). Bt was engineered to express major plague protective antigens in its OMVs, specifically Fraction 1 (F1) in the outer membrane and LcrV (V antigen) in the lumen, for targeted delivery to the gastrointestinal (GI) and respiratory tracts in a non-human primate (NHP) host. Our key findings were that Bt OMVs stably expresses F1 and V plague antigens, particularly the V antigen, in the correct, immunogenic form. When delivered intranasally V-OMVs elicited substantive and specific immune and antibody responses, both in the serum [immunoglobulin (Ig)G] and in the upper and lower respiratory tract (IgA); this included the generation of serum antibodies able to kill plague bacteria. Our results also showed that Bt OMV-based vaccines had many desirable characteristics, including: biosafety and an absence of any adverse effects, pathology or gross alteration of resident microbial communities (microbiotas); high stability and thermo-tolerance; needle-free delivery; intrinsic adjuvanticity; the ability to stimulate both humoral and cell-mediated immune responses; and targeting of primary sites of plague infection.

Dual route vaccination for plague with emergency use applications.

Here, we report a dual-route vaccination approach for plague, able to induce a rapid response involving systemic and mucosal immunity, whilst also providing ease of use in those resource-poor settings most vulnerable to disease outbreaks. This novel vaccine (VypVaxDuo) comprises the recombinant F1 and V proteins in free association. VypVaxDuo has been designed for administration via a sub-cutaneous priming dose followed by a single oral booster dose and has been demonstrated to induce early onset immunity 14 days after the primary immunisation; full protective efficacy against live organism challenge was achieved in Balb/c mice exposed to 2 × 104 median lethal doses of Yersinia pestis Co92, by the sub-cutaneous route at 25 days after the oral booster immunisation. This dual-route vaccination effectively induced serum IgG and serum and faecal IgA, specific for F1 and V, which constitute two key virulence factors in Y. pestis, and is therefore suitable for further development to prevent bubonic plague and for evaluation in models of pneumonic plague. This is an essential requirement for control of disease outbreaks in areas of the world endemic for plague and is supported further by the observed exceptional stability of the primary vaccine formulation in vialled form under thermostressed conditions (40 °C for 29 weeks, and 40 °C with 75% relative humidity for 6 weeks), meaning no cold chain for storage or distribution is needed. In clinical use, the injected priming dose would be administered on simple rehydration of the dry powder by means of a dual barrel syringe, with the subsequent single booster dose being provided in an enteric-coated capsule suitable for oral self-administration.

Exposure to anthrax toxin alters human leucocyte expression of anthrax toxin receptor 1.

Anthrax is a toxin-mediated disease, the lethal effects of which are initiated by the binding of protective antigen (PA) with one of three reported cell surface toxin receptors (ANTXR). Receptor binding has been shown to influence host susceptibility to the toxins. Despite this crucial role for ANTXR in the outcome of disease, and the reported immunomodulatory consequence of the anthrax toxins during infection, little is known about ANTXR expression on human leucocytes. We characterized the expression levels of ANTXR1 (TEM8) on human leucocytes using flow cytometry. In order to assess the effect of prior toxin exposure on ANTXR1 expression levels, leucocytes from individuals with no known exposure, those exposed to toxin through vaccination and convalescent individuals were analysed. Donors could be defined as either 'low' or 'high' expressers based on the percentage of ANTXR1-positive monocytes detected. Previous exposure to toxins appears to modulate ANTXR1 expression, exposure through active infection being associated with lower receptor expression. A significant correlation between low receptor expression and high anthrax toxin-specific interferon (IFN)-γ responses was observed in previously infected individuals. We propose that there is an attenuation of ANTXR1 expression post-infection which may be a protective mechanism that has evolved to prevent reinfection.

Protecting against plague: towards a next-generation vaccine.

The causative organism of plague is the bacterium Yersinia pestis. Advances in understanding the complex pathogenesis of plague infection have led to the identification of the F1- and V-antigens as key components of a next-generation vaccine for plague, which have the potential to be effective against all forms of the disease. Here we review the roles of F1- and V-antigens in the context of the range of virulence mechanisms deployed by Y. pestis, in order to develop a greater understanding of the protective immune responses required to protect against plague.

Conjugation of Y. pestis F1-antigen to gold nanoparticles improves immunogenicity.

The efficacy of 15 nm gold nanoparticles (AuNP) coated with Yersinia pestis F1-antigen, as an immunogen in mice, has been assessed. The nanoparticles were decorated with F1-antigen using N-hydroxysuccinimide and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride coupling chemistry. Mice given AuNP-F1 in alhydrogel generated the greatest IgG antibody response to F1-antigen when compared with mice given AuNP-F1 in PBS or given unconjugated F1-antigen in PBS or alhydrogel. Compared with unconjugated F1-antigen, the IgG2a response was enhanced in mice dosed with AuNP-F1 in PBS (p<0.05) but not in mice immunised with AuNP-F1 in alhydrogel. All treatment groups developed a memory response to F1-antigen, the polarity of which was inflenced by formulation in alhydrogel. The sera raised against F1-antigen coupled to AuNPs was able to competitively bind to rF1-antigen, displacing protective macaque sera.

The natural history and incidence of Yersinia pestis and prospects for vaccination.

Plague is an ancient, serious, infectious disease which is still endemic in regions of the modern world and is a potential biothreat agent. This paper discusses the natural history of the bacterium and its evolution into a flea-vectored bacterium able to transmit bubonic plague. It reviews the incidence of plague in the modern world and charts the history of vaccines which have been used to protect against the flea-vectored disease, which erupts as bubonic plague. Current approaches to vaccine development to protect against pneumonic, as well as bubonic, plague are also reviewed. The considerable challenges in achieving a vaccine which is licensed for human use and which will comprehensively protect against this serious human pathogen are assessed.

The role of immune correlates and surrogate markers in the development of vaccines and immunotherapies for plague.

One of the difficulties in developing countermeasures to biothreat agents is the challenge inherent in demonstrating their efficacy in man. Since the first publication of the Animal Rule by the FDA, there has been increased discussion of potential correlates of protection in animal models and their use to establish surrogate markers of efficacy in man. The latter need to be relatively easy to measure in assays that are at least qualified, if not validated, in order to derive a quantitative assessment of the clinical benefit conferred. The demonstration of safety and clinical benefit is essential to achieve regulatory approval for countermeasures for which clinical efficacy cannot be tested directly, as is the case for example, for biodefence vaccines. Plague is an ancient, serious infectious disease which is still endemic in regions of the modern world and is a potential biothreat agent. This paper discusses potential immune correlates of protection for plague, from which it may be possible to derive surrogate markers of efficacy, in order to predict the clinical efficacy of candidate prophylaxes and therapies.

Recombinant (F1+V) vaccine protects cynomolgus macaques against pneumonic plague.

Cynomolgus macaques, immunised at the 80 µg dose level with an rF1+rV vaccine (two doses, three weeks apart), were fully protected against pneumonic plague following inhalational exposure to a clinical isolate of Yersinia pestis (strain CO92) at week 8 of the schedule. At this time, all the immunised animals had developed specific IgG titres to rF1 and rV with geometric mean titres of 96.83±20.93 µg/ml and 78.59±12.07 µg/ml, respectively, for the 40 µg dose group; by comparison, the 80 µg dose group had developed titres of 114.4±22.1 and 90.8±15.8 µg/ml to rF1 and rV, respectively, by week 8. For all the immunised animals, sera drawn at week 8 competed with the neutralising and protective Mab7.3 for binding to rV antigen in a competitive ELISA, indicating that a functional antibody response to rV had been induced. All but one of the group immunised at the lower 40 µg dose-level were protected against infection; the single animal which succumbed had significantly reduced antibody responses to both the rF1 and rV antigens. Although a functional titre to rV antigen was detected for this animal, this was insufficient for protection, indicating that there may have been a deficiency in the functional titre to rF1 and underlining the need for immunity to both vaccine antigens to achieve protective efficacy against plague. This candidate vaccine, which has been evaluated as safe and immunogenic in clinical studies, has now been demonstrated to protect cynomolgus macaques, immunised in the clinical regimen, against pneumonic plague.

Plague.

Killed whole cell vaccines for plague were first produced as long ago as the late 1890s and modified versions of these are still used, with evidence that they are efficacious against bubonic plague. Renewed efforts with modern technology have yielded new candidate vaccines which are less reactogenic, can be produced in a conventional pharmaceutical manufacturing plant and are protective against the life-threatening pneumonic form of the disease. This paper reviews the progress towards an improved vaccine for plague and assesses the likely impact of a prophylactic vaccine for bubonic and pneumonic plague.

The fraction 1 and V protein antigens of Yersinia pestis activate dendritic cells to induce primary T cell responses.

The F1 and V antigens of Yersinia pestis, despite acting as virulence factors secreted by the organism during infection, also combine to produce an effective recombinant vaccine against plague, currently in clinical trial. The protective mechanisms induced by rF1 + rV probably involve interactions with dendritic cells (DC) as antigen uptake, processing and presenting cells. To study such interactions, naive ex vivo DC from bone marrow, spleen and lymph node were cultured with rF1, rV or combined antigens and demonstrated to secrete interleukin (IL)-4 and IL-12 into the culture supernatant. Cytokine production in response to pulsing was dependent on the maturity of the bone marrow-derived DC culture, so that pulsed 8-day-old cultures had accumulated significantly more intracellular IL-4 and IL-12 than unpulsed cells. DC, pulsed with rF1 + rV for 2-24 h, were able to prime naive autologous lymph node T cells to proliferate in an antigen dose-dependent manner, with an order of potency of 3d bone marrow-derived DC (BMDC) > 7d BMDC > splenic DC. Significantly, cell-free supernatants from rF1 + rV-pulsed BMDC and splenic DC were also able to induce specific primary responses effectively in naive T cells, suggesting that these supernatants contained stimulatory factor(s). This study suggests an important role for DC, or factors secreted by them, in the induction of protective immunity to plague by the rF1 and rV antigens.

Kinetics of the immune response to the (F1+V) vaccine in models of bubonic and pneumonic plague.

Protection against aerosol challenge with > 300 MLD of Yersinia pestis was observed 7 days after a single immunisation of mice with the F1+V vaccine. At day 60, mice were protected against injected challenge (10(7)MLD) in a vaccine dose-related manner. Recall responses to rV in splenocytes ex vivo at day 98 correlated significantly (p<0.001) with the immunising dose-level of V antigen; no memory response or anti-V serum IgG was detected in killed whole cell vaccine (KWCV) recipients. This may explain the susceptibility of KWCV recipients to aerosol challenge and the enhanced protection conferred by the F1+V sub-unit vaccine, particularly since the anti-F1 responses induced by either vaccine were similarly IgG1-polarised.

A biocompatible microdevice for core body temperature monitoring in the early diagnosis of infectious disease.

The early diagnosis of microbial infection is critical to the clinical instigation of effective post-exposure prophylaxis or therapy. However, diagnosis of infection is often attempted only when there are overt clinical signs, and for some of the serious human pathogens, this may jeopardize the efficacy of therapy. We have used a miniaturised sealed, implantable transponder incorporating a calibrated temperature sensor with an external receiver system, to monitor core body temperature (Tc) remotely. We have observed early changes in the diurnal rhythm of Tc, after infection of mice with bacterial pathogens. Changes in Tc preceded overt clinical signs by 3-10 h following challenge with Yersinia pestis, which causes acute infection, In contrast, changes in Tc were detected 11 days before clinical signs in mice exposed to Burkholderia pseudomallei, which causes a chronic syndrome. Significantly, mice pre-vaccinated against Y.pestis infection showed only slight and transient disruption to the diurnal rhythm for Tc, in the absence of clinical signs, when challenged with 10(6) median lethal doses of Y.pestis. This remote monitoring technology could be used to monitor changes in more than one physiological parameter and extrapolation of these data to the clinic would define the available therapeutic window in which diagnosis and post-exposure prophylaxis could be instigated, after a suspected exposure.

Immunogenicity of the rF1+rV vaccine for plague with identification of potential immune correlates.

The rF1+rV candidate sub-unit vaccine for plague, formulated by adsorption to alhydrogel, has been demonstrated to be immunogenic in the cynomolgus macaque in a clinically relevant dose-range (5-40 microg of each sub-unit) and regimen. Following two doses of vaccine, a specific IgG titre developed in a dose-related manner with predominance of the IgG1/IgG2 isotypes. Groups of macaques receiving only a single dose of vaccine at the 40 microg dose-level had a significantly reduced peak IgG response and faster decline to baseline. Serum collected at week 5 from 19 immunised animals competed with and displaced murine Mab7.3 from binding to the V antigen in vitro. By week 53 of the schedule, although absolute IgG titres had declined, 17/19 macaque sera tested contained competing antibody, indicating the durability of a functional immune response to rF1+rV in this species. Thirteen of these week 53 sera were passively transferred into groups of naive mice, and all conferred full or partial protection against subsequent challenge of the mice with plague. Generally, those sera which were most competitive with Mab 7.3 for binding to V antigen were fully protective by passive transfer, although one week-53 serum sample was fully protective by passive transfer but not active by competitive ELISA. The early development of protective immunity in macaques was also indicated from the protection conferred on naive mice by the passive transfer of immune macaque serum collected at 2-10 weeks of the immunisation schedule. Serum samples from representative macaques within this time period also inhibited the Yersinia-mediated cytotoxicity of J774 macrophages in a qualitative in vitro assay of type three secretion.

Distribution of productive antigen-processing activity for MHC class II presentation in macrophages.

We demonstrated that an epitope from the recombinant protective antigen (rPA) of Bacillus anthracis was presented by mature major histocompatibility complex class II (MHC-II) molecules, whereas an epitope from the recombinant virulent (rV) antigen of Yersinia pestis was presented by newly synthesized MHC-II. We addressed which endosomal compartments were involved in the antigen processing of each epitope. Bone-marrow-derived macrophages were subjected to subcellular fractionation; fractions were analysed for the expression of endosomal markers and used as a source of enzyme activity for the processing of rPA and rV antigens. The rPA epitope was productively processed by dense lysosomal fractions and light membrane fractions expressing early endosomal markers Rab5 and early endosomal antigen-1 as well as markers of antigen-presenting compartments (MHC-II, DM, DO and Ii chain). In contrast, the rV epitope was productively processed only by dense fractions with lysosomal activity. No productive antigen-processing activity was associated with fractions of intermediate density expressing Rab7 and Rab9, characteristic of late endosomes. The data suggest that endosomal compartments expressing Rab5 guanosine triphosphatase can productively process protein antigens for presentation by mature MHC class II molecules.