Prevention of bubonic and pneumonic plague using plant-derived vaccines

Yersinia pestis, the causative agent of bubonic and pneumonic plague, is an extremely virulent bacterium but there are currently no approved vaccines for protection against this organism. Plants represent an economical and safer alternative to fermentation-based expression systems for the production of therapeutic proteins. The recombinant plague vaccine candidates produced in plants are based on the two most immunogenic antigens of Y. pestis: the fraction-1 capsular antigen (F1) and the low calcium response virulent antigen (V) either in combination or as a fusion protein (F1–V). These antigens have been expressed in plants using all three known possible strategies: nuclear transformation, chloroplast transformation and plant-virus-based expression vectors. These plant-derived plague vaccine candidates were successfully tested in animal models using parenteral, oral, or prime/boost immunization regimens. This review focuses on the recent research accomplishments towards the development of safe and effective pneumonic and bubonic plague vaccines using plants as bioreactors.     

Vocal turn-taking in a non-human primate is learned during ontogeny

Conversational turn-taking is an integral part of language development, as it reflects a confluence of social factors that mitigate communication. Humans coordinate the timing of speech based on the behaviour of another speaker, a behaviour that is learned during infancy. While adults in several primate species engage in vocal turn-taking, the degree to which similar learning processes underlie its development in these non-human species or are unique to language is not clear. We recorded the natural vocal interactions of common marmosets (Callithrix jacchus) occurring with both their sibling twins and parents over the first year of life and observed at least two parallels with language development. First, marmoset turn-taking is a learned vocal behaviour. Second, marmoset parents potentially played a direct role in guiding the development of turn-taking by providing feedback to their offspring when errors occurred during vocal interactions similarly to what has been observed in humans. Though species-differences are also evident, these findings suggest that similar learning mechanisms may be implemented in the ontogeny of vocal turn-taking across our Order, a finding that has important implications for our understanding of language evolution.     

Evidence of an evolutionary precursor to human language affixation in a non-human primate

Human language, and grammatical competence in particular, relies on a set of computational operations that, in its entirety, is not observed in other animals. Such uniqueness leaves open the possibility that components of our linguistic competence are shared with other animals, having evolved for non-linguistic functions. Here, we explore this problem from a comparative perspective, asking whether cotton-top tamarin monkeys (Saguinus oedipus) can spontaneously (no training) acquire an affixation rule that shares important properties with our inflectional morphology (e.g. the rule that adds –ed to create the past tense, as in the transformation of walk into walk-ed). Using playback experiments, we show that tamarins discriminate between bisyllabic items that start with a specific ‘prefix’ syllable and those that end with the same syllable as a ‘suffix’. These results suggest that some of the computational mechanisms subserving affixation in a diversity of languages are shared with other animals, relying on basic perceptual or memory primitives that evolved for non-linguistic functions.     

The non-human primate model of tuberculosis

Non-human primates (NHPs) are used to model human disease owing to their remarkably similar genomes, physiology, and immune systems. Recently, there has been an increased interest in modeling tuberculosis (TB) in NHPs. Macaques are susceptible to infection with different strains of Mycobacterium tuberculosis (Mtb), producing the full spectrum of disease conditions, including latent infection, chronic progressive infection, and acute TB, depending on the route and dose of infection. Clearly, NHPs are an excellent model of human TB. While the initial aim of the NHP model was to allow preclinical testing of candidate vaccines and drugs, it is now also being used to study pathogenesis and immune correlates of protection. Recent advances in this field are discussed in this review. Key questions such as the effect of hypoxia on the biology of Mtb and the basis of reactivation of latent TB can now be investigated through the use of this model.     

An encapsulated Yersinia pseudotuberculosis is a highly efficient vaccine against pneumonic plague

Background

Plague is still a public health problem in the world and is re-emerging, but no efficient vaccine is available. We previously reported that oral inoculation of a live attenuated Yersinia pseudotuberculosis, the recent ancestor of Yersinia pestis, provided protection against bubonic plague. However, the strain poorly protected against pneumonic plague, the most deadly and contagious form of the disease, and was not genetically defined.

Methodology and Principal Findings

The sequenced Y. pseudotuberculosis IP32953 has been irreversibly attenuated by deletion of genes encoding three essential virulence factors. An encapsulated Y. pseudotuberculosis was generated by cloning the Y. pestis F1-encoding caf operon and expressing it in the attenuated strain. The new V674pF1 strain produced the F1 capsule in vitro and in vivo. Oral inoculation of V674pF1 allowed the colonization of the gut without lesions to Peyer''s patches and the spleen. Vaccination induced both humoral and cellular components of immunity, at the systemic (IgG and Th1 cells) and the mucosal levels (IgA and Th17 cells). A single oral dose conferred 100% protection against a lethal pneumonic plague challenge (33×LD₅₀ of the fully virulent Y. pestis CO92 strain) and 94% against a high challenge dose (3,300×LD₅₀). Both F1 and other Yersinia antigens were recognized and V674pF1 efficiently protected against a F1-negative Y. pestis.

Conclusions and Significance

The encapsulated Y. pseudotuberculosis V674pF1 is an efficient live oral vaccine against pneumonic plague, and could be developed for mass vaccination in tropical endemic areas to control pneumonic plague transmission and mortality.     

Bioluminescent tracking of colonization and clearance dynamics of plasmid-deficient Yersinia pestis strains in a mouse model of septicemic plague

Yersinia pestis 201 contains 4 plasmids pPCP1, pMT1, pCD1 and pCRY, but little is known about the effects of these plasmids on the dissemination of Y. pestis. We developed a plasmid-based luxCDABE bioreporter in Y. pestis 201, Y. pestis 201-pCD1⁺, Y. pestis 201-pMT1⁺, Y. pestis 201-pPCP1⁺, Y. pestis 201-pCRY⁺, Y. pestis 201-p⁻ and Yersinia pseudotuberculosis Pa36060 strains, and investigated their dissemination by bioluminescence imaging during primary septicemic plague in a mouse model. These strains mainly colonized the livers and spleens shortly after intravenous inoculation. Y. pestis 201-pMT1⁺ appeared to have a stronger ability to survive in the livers, spleens and blood, and to be more virulent than other plasmid-deficient strains. Y. pestis 201-pPCP1⁺ appeared to have a stronger ability to colonize lungs than other plasmid-deficient strains. Pa36060 has the strongest ability to colonize intestines and lungs. Y. pestis 201 has the strongest ability to survive in blood, and the strongest virulence. These results indicated that the plasmid pMT1 was an important determinant in the colonization of livers, spleens and blood, whereas the plasmid pPCP1 appeared to correlate with the colonization in lungs. The resistance to killing in mouse blood seemed to be the critical factor causing animal death.     

Vocal production mechanisms in a non-human primate: morphological data and a model

Human beings are thought to be unique amongst the primates in their capacity to produce rapid changes in the shape of their vocal tracts during speech production. Acoustically, vocal tracts act as resonance chambers, whose geometry determines the position and bandwidth of the formants. Formants provide the acoustic basis for vowels, which enable speakers to refer to external events and to produce other kinds of meaningful communication. Formant-based referential communication is also present in non-human primates, most prominently in Diana monkey alarm calls. Previous work has suggested that the acoustic structure of these calls is the product of a non-uniform vocal tract capable of some degree of articulation. In this study we test this hypothesis by providing morphological measurements of the vocal tract of three adult Diana monkeys, using both radiography and dissection. We use these data to generate a vocal tract computational model capable of simulating the formant structures produced by wild individuals. The model performed best when it combined a non-uniform vocal tract consisting of three different tubes with a number of articulatory manoeuvres. We discuss the implications of these findings for evolutionary theories of human and non-human vocal production.     

Primary pneumonic plague in the African Green monkey as a model for treatment efficacy evaluation

Background Primary pneumonic plague is rare among humans, but treatment efficacy may be tested in appropriate animal models under the FDA ‘Animal Rule’. Methods Ten African Green monkeys (AGMs) inhaled 44–255 LD₅₀ doses of aerosolized Yersinia pestis strain CO92. Continuous telemetry, arterial blood gases, chest radiography, blood culture, and clinical pathology monitored disease progression. Results Onset of fever, >39°C detected by continuous telemetry, 52–80 hours post‐exposure was the first sign of systemic disease and provides a distinct signal for treatment initiation. Secondary endpoints of disease severity include tachypnea measured by telemetry, bacteremia, extent of pneumonia imaged by chest x‐ray, and serum lactate dehydrogenase enzyme levels. Conclusions Inhaled Y. pestis in the AGM results in a rapidly progressive and uniformly fatal disease with fever and multifocal pneumonia, serving as a rigorous test model for antibiotic efficacy studies.     

The baboon as a non-human primate model of human schistosome infection

Over the past three decades, intensive studies of murine schistosomiasis have provided important clues to the understanding of the human disease, but growing evidence suggests that these results derived from highly inbred strains of mice might not have direct applicability to the human infection. Recent data based on the baboon indicate that infection in this non-human primate might mirror the human situation. In this review, Mramba Nyindo and Idle Farah demonstrate that baboons provide an excellent non-human primate model that produces pathology and disease closely resembling that observed in humans, and address how studies in baboons can provide insights into mechanisms regulating schistosomiasis mansoni pathology and immunity. They also address, in a general way, issues related to the use of non-human primates in biomedical research.     

Resveratrol suppresses body mass gain in a seasonal non-human primate model of obesity

Background  

Resveratrol, a natural polyphenolic compound, was shown to protect rodents against high-fat-diet induced diabesity by boosting energy metabolism. To the best of our knowledge, no data is yet available on the effects of resveratrol in non-human primates. Six non-human heterotherm primates (grey mouse lemurs, Microcebus murinus) were studied during four weeks of dietary supplementation with resveratrol (200 mg/kg/day) during their winter body-mass gain period. Body mass, spontaneous energy intake, resting metabolic rate, spontaneous locomotor activity and daily variations in body temperature were measured. In addition, the plasma levels of several gut hormones involved in satiety control were evaluated.     

Sexually selected skin colour is heritable and related to fecundity in a non-human primate

Sexual selection promotes the prevalence of heritable traits that increase an individual''s reproductive rate. Despite theoretically strong directional selection, sexually selected traits can show inter-individual variation. Here, we investigate whether red skin ornamentation, a rare example of a male mammalian trait involved in mate attraction, influences fecundity and is heritable in rhesus macaques (Macaca mulatta), and explore the mechanisms that are involved in maintaining trait variation. Interestingly, the trait is expressed by and is attractive to both sexes. We collected facial images of 266 free-ranging individuals and modelled skin redness and darkness to rhesus macaque vision. We used 20 years of genetic parentage data to calculate selection gradients on the trait and perform heritability analyses. Results show that males who were both darkly coloured and high-ranking enjoyed higher fecundity. Female skin redness was positively linked to fecundity, although it remains unclear whether this influences male selectiveness. Heritability explained 10–15% of the variation in redness and darkness, and up to 30% for skin darkness when sexes are considered separately, suggesting sex-influenced inheritance. Our results suggest that inter-individual variation is maintained through condition-dependence, with an added effect of balancing selection on male skin darkness, providing rare evidence for a mammalian trait selected through inter-sexual selection.     

Characterization of alveolar macrophage eicosanoid production in a non-human primate model of mineral dust exposure

The relative activation of eicosanoid production which results from the exposure of the alveolar macrophage (AM) to mineral dusts is thought to be a key factor in the pathophysiology of occupational lung disease. We compared in vitro basal and silica-stimulated production of prostaglandin E₂ (PGE₂) and thromboxane A₂ (TXA₂) by AM from normal humans and non-human primates (Macaca nemistrina). In addition, we instilled mineral dusts directly into one lung of the non-human primate and evaluated AM eicosanoid production at two week intervals following dust instillation. Unstimulated AM from humans produce more PGE₂ and TXA₂ than do AM from M. nemistrina. However, in vitro exposure of AM from both species to silica dust produced a qualitatively similar increase in TXA₂ production accompanied by no change in PGE₂ production. Sequential analysis of AM eicosanoid production following a single bolus exposure to bituminous or anthracite coal dusts, titanium dioxide (TiO₂) dust or crystalline silica showed marked variability among individual non-human primates in qualitative and quantitative aspects of dust-induced eicosanoid production. However, the rank order of potency of the different dusts (silica > anthracite > bituminous) correlated with epidemiological evidence relating the type of dust mined to the incidence of pneumoconiosis. These studies suggest that the non-human primate may serve as a model for the study of both the role of eicosanoids in the etiology of dust-induced occupational lung disease and the biochemical basis for individual variability in the response of lung cells to mineral dust exposure.     

Cultural evolution of systematically structured behaviour in a non-human primate

Culture pervades human life and is at the origin of the success of our species. A wide range of other animals have culture too, but often in a limited form that does not complexify through the gradual accumulation of innovations. We developed a new paradigm to study cultural evolution in primates in order to better evaluate our closest relatives'' cultural capacities. Previous studies using transmission chain experimental paradigms, in which the behavioural output of one individual becomes the target behaviour for the next individual in the chain, show that cultural transmission can lead to the progressive emergence of systematically structured behaviours in humans. Inspired by this work, we combined a pattern reproduction task on touch screens with an iterated learning procedure to develop transmission chains of baboons (Papio papio). Using this procedure, we show that baboons can exhibit three fundamental aspects of human cultural evolution: a progressive increase in performance, the emergence of systematic structure and the presence of lineage specificity. Our results shed new light on human uniqueness: we share with our closest relatives essential capacities to produce human-like cultural evolution.     

Efficient regulation of viral replication by siRNA in a non-human primate surrogate model for hepatitis C

RNA interference (RNAi) represents a new technology which could offer potential applications for the therapeutics of human diseases. RNAi-mediated therapy has recently been shown to be effective toward infectious diseases in in vitro and rodent models, however, it remains unclear whether RNAi therapy with systemic application could be effective in primates. In this study, we examined if RNAi therapy could be effective toward infectious diseases by using a non-human primate surrogate model for hepatitis C. Administration into marmosets of cationic liposome-encapsulated siRNA (CL-siRNA) for GB virus B (GBV-B), which is most closely related to hepatitis C virus, repressed GBV-B replication in a dose-dependent manner. Especially, 5 mg/kg of the CL-siRNA completely inhibited the viral replication. Since the serum interferons (IFNs) were induced by CL-siRNA in vivo, inhibition of viral regulation by anti-GBV-B CL-siRNA may include an antiviral effect of IFN. However, contribution of induced IFN may be partial, since the control CL-siRNA which induced a stronger IFN response than GBV-B CL-siRNA could only delay the viral replication. Our results suggest the feasibility of systemic administration of CL-siRNA as an antiviral strategy.     

鼠疫动物模型:经典与问题

一直以来,鼠疫菌的研究多围绕致病机制、疫苗效果评价及鼠疫治疗方案选择展开,而合理有效的鼠疫动物模型正是这些实验研究的基础与前提。研究者构建了包括小鼠、豚鼠及非人灵长类模型在内的多种动物模型,而这些模型也为人类认识、防御及治疗鼠疫菌提供了帮助,特别是一些经典动物模型沿用至今。但随着研究的深入,有些动物模型由于存在某种问题而逐渐被淘汰。本文就鼠疫菌动物模型构建以来,不同模型的优势与不足及其应用展开综述,以期为研究者提供参考。     

鼠疫的发病机制研究进展

鼠疫是由鼠疫耶尔森菌(Yersinia pestis,Y. pestis)感染引起的一种人畜共患病。鼠疫在世界范围内出现过3次大流行,均引起致命的瘟疫。由于自然疫源面积不断扩大和人口流动愈加频繁,我国的鼠疫防治形势依旧严峻。本文就鼠疫耶尔森菌的毒力因子、对宿主细胞的黏附和侵袭、胞内繁殖、宿主内播散等机制的研究进展进行总结,有助于揭示鼠疫独特的致病和传播机制,为精准防治鼠疫提供工作基础。     

Yersinia pestis YopE contains a dominant CD8 T cell epitope that confers protection in a mouse model of pneumonic plague

Septic bacterial pneumonias are a major cause of death worldwide. Several of the highest priority bioterror concerns, including anthrax, tularemia, and plague, are caused by bacteria that acutely infect the lung. Bacterial resistance to multiple antibiotics is increasingly common. Although vaccines may be our best defense against antibiotic-resistant bacteria, there has been little progress in the development of safe and effective vaccines for pulmonary bacterial pathogens. The Gram-negative bacterium Yersinia pestis causes pneumonic plague, an acutely lethal septic pneumonia. Historic pandemics of plague caused millions of deaths, and the plague bacilli's potential for weaponization sustains an ongoing quest for effective countermeasures. Subunit vaccines have failed, to date, to fully protect nonhuman primates. In mice, they induce the production of Abs that act in concert with type 1 cytokines to deliver high-level protection; however, the Y. pestis Ags recognized by cytokine-producing T cells have yet to be defined. In this study, we report that Y. pestis YopE is a dominant Ag recognized by CD8 T cells in C57BL/6 mice. After vaccinating with live attenuated Y. pestis and challenging intranasally with virulent plague, nearly 20% of pulmonary CD8 T cells recognize this single, highly conserved Ag. Moreover, immunizing mice with a single peptide, YopE(69-77), suffices to confer significant protection from lethal pulmonary challenge. These findings suggest YopE could be a valuable addition to subunit plague vaccines and provide a new animal model in which sensitive, pathogen-specific assays can be used to study CD8 T cell-mediated defense against acutely lethal bacterial infections of the lung.     

Pre-clinical evaluation of an sLe x-glycosylated complement inhibitory protein in a non-human primate model of reperfused stroke

BACKGROUND: Soluble complement receptor-1 (sCR1), a potent complement inhibitor, confers neuroprotection in a murine stroke model. Additional neuroprotective benefit is achieved by sLe x-glycosylation of sCR1. In an effort to translate sCR1-sLe x to clinical trials, we evaluated this agent in a primate stroke model. METHODS: Adult male baboons randomly received either sCR1-sLe x or vehicle. Stroke volume was assessed on day 3, and neurological examinations were conducted daily. Complement activity (CH50) was measured at 30 minute, 2, 6, 12 hour, 3, and 10 days post-ischemia. RESULTS: The experiment was terminated prematurely following an interim analysis. In a preliminary cohort (n = 3 per arm), infarct volume was greater in the treated animals. No difference in neurological score was found between groups. CH50 levels were significantly reduced in the sCR1sLe x-treated groups. A hypotensive response was also observed in animals treated with sCR1-sLe x. Conclusions Further work is necessary to explain the hypotensive response observed in primates prior to further clinical development of sCR1-sLe x.     

自然感染途径建立肺炎链球菌感染性肺炎小鼠模型

目的探索自然感染途径制备肺炎链球菌感染性肺炎小鼠模型的方法,为肺炎链球菌性肺炎的相关研究提供实验基础。方法肺炎链球菌标准菌株(CMCC 31203)经血平板培养18 h后配成2麦氏浊度。小鼠浅麻醉状态下,破损鼻黏膜,滴注一定量肺炎链球菌菌液。于3、7、14和21 d分别处死小鼠,取肺脏进行组织病理学观察,确定肺炎小鼠模型制备的最佳方式。结果鼻黏膜破损组小鼠3 d时肺泡间隔内毛细血管扩张,肺泡腔内以红细胞和纤维素渗出为主;7 d时病变以纤维素和中性粒细胞浸润为主;14 d时肺泡腔内渗出减少,炎症开始减轻;21 d时肺脏外观趋于正常,肺泡腔内渗出物质基本溶解吸收。结论以3×107CFU的肺炎链球菌菌液通过破损的鼻黏膜感染7 d时可以制备出比较典型、稳定的肺炎小鼠模型。     

Successful implementation of cooperative handling eliminates the need for restraint in a complex non-human primate disease model

Background Streptozotocin‐induced diabetic non‐human primates are used to study efficacy and safety of innovative immunosuppression after islet transplantation. We implemented a training program for medical management of a chronic disease state. Methods Cooperation with hand feeding and drinking, shifting, and limb presentation were trained utilizing predominately positive but also negative reinforcement in 52 animals compared with 28 macaques subjected to conventional physical and/or chemical restraint. The success and timing of behavior acquisition was evaluated in a representative subset of 14 animals. Results Over 90% of animals were successful in behavior acquisition. Programmatically this resulted in complete elimination of chair restraint and negligible requirement for sedation. About half of the trained animals had no‐to‐moderate thymic involution, indicative of a substantial reduction in stress. Conclusion Cooperative handling enhances animal well‐being. This contributes to validity of scientific results and eliminates model‐induced confounding that can obstruct interpretation of safety and efficacy data.