From structure to solutions: the role of basic research in developing anthrax countermeasures: Microbiology Graduate Program Seminar: Anthrax toxin

Dr. John Collier traced the discoveries that elucidated the structure and function of the anthrax toxin in his talk "Anthrax Toxin," which was part of the Microbiology Graduate Program Seminar Series at Yale School of Medicine on February 23, 2012. Dr. Collier, Professor of Microbiology and Immunobiology at Harvard University, began by noting the advantages to studying anthrax pathogenesis in a biosafety level-1 lab. This designation does not merely facilitate his research, but also reflects a larger trend of basic research being leveraged to develop translational applications. Basic research on toxin structure has led to the development of a vaccine by Dr. Collier's group. Next-generation prophylactics also may stem from recent discoveries uncovering a role for cellular cofactors that mediate toxin function. Finally, basic research into the toxin substructure has facilitated efforts to change the receptor tropism to target dysregulated cells for therapeutic purposes. The urgency around biodefense agents makes the choice of research priorities a salient issue. As such, this author submits that basic research occupies a unique and lucrative niche driving clinical applications.     

Passive transfer of maternal GnRH antibodies does not affect reproductive development in elk (Cervus elaphus nelsoni) calves

Gonadotropin-releasing hormone is intermittently released from the hypothalamus in consistent patterns from before birth to final maturation of the hypothalamic-pituitary-gonadal axis at puberty. Disruption of this signaling via GnRH vaccination during the neonatal period can alter reproduction at maturity. The objective of this study was to investigate the long-term effects of GnRH-antibody exposure on reproductive maturation and function in elk calves passively exposed to high concentrations of GnRH antibodies immediately after birth. Fifteen elk calves (eight males and seven females) born to females treated with GnRH vaccine or sham vaccine during midgestation were divided into two groups based on the concentration of serum GnRH antibodies measured during the neonatal period. Those with robust (>15 pmol ¹²⁵I-GnRH bound per mL of serum) titers (N = 10; four females and six males) were designated as the exposed group, whereas those with undetectable titers (N = 5; three females and two males) were the unexposed group. Onset of puberty, reproductive development, and endocrine function in antibody-exposed and unexposed male and female elk calves were compared. Neonatal exposure to high concentrations of GnRH antibodies had no effect on body weight (P = 0.968), endocrine profiles (P > 0.05), or gametogenesis in either sex. Likewise, there were no differences between groups in gross or histologic structure of the hypothalamus, pituitary, testes, or ovaries. Pituitary stimulation with a GnRH analog before the second potential reproductive season induced substantial LH secretion in all experimental elk. All females became pregnant during their second reproductive season and all males exhibited similar mature secondary sexual characteristics. There were no differences between exposure groups in hypothalamic GnRH content (P = 0.979), pituitary gonadotropin content (P > 0.05) or gonadal structure. We concluded that suppressing GnRH signaling through immunoneutralization during the neonatal period likely does not alter long-term reproductive function in this species.     

Cross‐reactive antibody response to the pandemic A (H1N1) 2009 influenza virus induced by vaccination with a seasonal trivalent influenza vaccine: a longitudinal study of three influenza seasons in Japan

The cross‐reactivity of antibody to the swine‐origin pandemic influenza A (H1N1) 2009 virus induced by vaccination with a seasonal trivalent influenza vaccine was studied. Paired sera from a cohort of adult volunteers vaccinated with a trivalent seasonal influenza vaccine every year from 2006 to 2008 were collected each year and tested by hemagglutination inhibition (HI) for antibody against the pandemic influenza A (H1N1) 2009 virus. There was little increase in the geometric mean titer overall; a slight increase was detected in the sera obtained in the 2007–2008 season but not in the other two seasons. The proportion of individuals with HI antibody titers ≥ 1:40 did not change significantly from year to year. These results indicate that cross‐reactivity of the antibodies induced by a trivalent seasonal vaccine to the pandemic influenza A (H1N1) 2009 virus is marginal.     

A chloroplast‐derived Toxoplasma gondii GRA4 antigen used as an oral vaccine protects against toxoplasmosis in mice

The parasitic protozoan Toxoplasma gondii, the causal agent of toxoplasmosis, can infect most mammals and birds. In human medicine, T. gondii can cause complications in pregnant women and immunodeficient individuals, while in veterinary medicine, T. gondii infection has economic importance due to abortion and neonatal loss in livestock. Thus, the development of an effective anti‐Toxoplasma vaccine would be of great value. In this study, we analysed the expression of T. gondii GRA4 antigen by chloroplast transformation (chlGRA4) in tobacco plants and evaluated the humoral and cellular responses and the grade of protection after oral administration of chlGRA4 in a murine model. The Western blot analysis revealed a specific 34‐kDa band mainly present in the insoluble fractions. The chlGRA4 accumulation levels were approximately 6 μg/g of fresh weight (equivalent to 0.2% of total protein). Oral immunization with chlGRA4 resulted in a decrease of 59% in the brain cyst load of mice compared to control mice. ChlGRA4 immunization elicited both a mucosal immune response characterized by the production of specific IgA, and IFN‐γ, IL‐4 and IL‐10 secretion by mesenteric lymph node cells, and a systemic response in terms of GRA4‐specific serum antibodies and secretion of IFN‐γ, IL‐4 and IL‐10 by splenocytes. Our results indicate that oral administration of chlGRA4 promotes the elicitation of both mucosal and systemic balanced Th1/Th2 responses that control Toxoplasma infection, reducing parasite loads.     

Robust production of virus-like particles and monoclonal antibodies with geminiviral replicon vectors in lettuce

Pharmaceutical protein production in plants has been greatly promoted by the development of viral-based vectors and transient expression systems. Tobacco and related Nicotiana species are currently the most common host plants for the generation of plant-made pharmaceutical proteins (PMPs). Downstream processing of target PMPs from these plants, however, is hindered by potential technical and regulatory difficulties owing to the presence of high levels of phenolics and toxic alkaloids. Here, we explored the use of lettuce, which grows quickly yet produces low levels of secondary metabolites and viral vector-based transient expression systems to develop a robust PMP production platform. Our results showed that a geminiviral replicon system based on the bean yellow dwarf virus permits high-level expression in lettuce of virus-like particles (VLP) derived from the Norwalk virus capsid protein and therapeutic monoclonal antibodies (mAbs) against Ebola and West Nile viruses. These vaccine and therapeutic candidates can be readily purified from lettuce leaves with scalable processing methods while fully retaining functional activity. Furthermore, this study also demonstrated the feasibility of using commercially produced lettuce for high-level PMP production. This allows our production system to have access to unlimited quantities of inexpensive plant material for large-scale production. These results establish a new production platform for biological pharmaceutical agents that are effective, safe, low cost, and amenable to large-scale manufacturing.     

新型抗结核蛋白亚单位疫苗Ag85A-γ干扰素的免疫效果评价

本研究旨在评价新型抗结核融合蛋白亚单位疫苗Ag85A-γ干扰素(Ag85A-IFN-γ)的免疫效果.在成功表达并纯化了去除Ag85A信号肽的融合蛋白Ag85A-IFN-γ后,将其与免疫佐剂二甲基三十六烷基铵(DDA)混合,皮下免疫C57BL/6小鼠3次,每次间隔2周,末次免疫2周后进行效果评价.采用酶联免疫吸附试验(ELISA)检测血清中IgG、IgG1和IgG2c水平.结果显示,融合蛋白Ag85A-IFN-γ组的IgG水平均高于单独抗原组,且融合蛋白组IgG2c/IgG1 比值显著高于对照组,表明融合蛋白能刺激宿主产生更强烈的体液免疫反应,更倾向于激发T辅助细胞1型(Th1型)免疫反应.流式细胞术检测特异性CD4+和CD8+ T细胞比例,发现Ag85A-IFN-γ组CD8+/CD4+最高,显示该融合蛋白能显著增强CD8+ T细胞增殖.上述结果表明,融合蛋白Ag85A-IFN-γ有望成为有效的新型抗结核融合蛋白亚单位疫苗.     

Molecular diversity of live-attenuated prototypic vaccine strains and clinical isolates of Staphylococcus aureus

Bovine mastitis Staphylococcus aureus isolates and prototypic live-attenuated vaccine strains were analyzed by SmaI pulsed-field gel electrophoresis (PFGE) typing and automated ribotyping. The discriminatory index of these methods was 0.91 and 0.69, respectively. SmaI PFGE typing assigned all laboratory strains into cluster Q, which shared 49% similarity with clusters A and B, and 35% similarity with cluster C. Automated ribotyping placed laboratory strains within ribogroups different from those of bovine isolates. These methods have 70% concordance and permitted identification of the prototypic vaccine background from those of clinical isolates. This information is required before conducting field trials with the vaccine.     

Immunization with cytomegalovirus envelope glycoprotein M and glycoprotein N DNA vaccines can provide mice with complete protection against a lethal murine cytomegalovirus challenge

Human cytomegalovirus virions contain three major glycoprotein complexes (gC I, II, III), all of which are required for CMV infectivity. These complexes also represent major antigenic targets for anti-viral immune responses. The gC II complex consists of two glycoproteins, gM and gN. In the current study, DNA vaccines expressing the murine cytomegalovirus (MCMV) homologs of the gM and gN proteins were evaluated for protection against lethal MCMV infection in a mouse model. Humoral and cellular immune responses, spleen viral titers, and mice survival and body-weight changes were examined. The results showed that immunization with gM or gN DNA vaccine alone was not able to offer good protection, whereas co-immunization with both gM and gN induced an effective neutralizing antibody response and cellular immune response, and provided mice with complete protection against a lethal MCMV challenge. This study provides the first in vivo evidence that the gC II (gM-gN) complex may be able to serve as a protective subunit antigen for future HCMV vaccine development.     

细菌芽胞—— 一种新型的疫苗载体

杆菌属的芽胞作为益生菌已经应用于人和动物的食品生产和细菌疗法.目前,芽胞作为一种新型的疫苗载体,开始用于破伤风、炭疽等疫苗的研究.与目前的第二代疫苗相比,细菌芽胞热稳定性好,遗传操作方便,是一种理想的疫苗载体.本文就其作为疫苗载体的相关研究进行综述.