Noroviruses (NoV) are the leading cause of epidemic acute gastroenteritis in humans worldwide. A safe and effective vaccine that prevents NoV infection or minimizes NoV disease burden is needed, especially for children and the elderly who are particularly susceptible to NoV disease. A plant-based expression system (magnICON®) was used to manufacture two different virus-like particle (VLP) immunogens derived from human NoV genogroups I and II, genotype 4 (GI.4 and GII.4), which were subsequently blended 1:1 (w/w) into a bivalent vaccine composition (rNV-2v).
Here, we report on the safety and immunogenicity of rNV-2v from one pilot and two GLP-compliant toxicity studies in New Zealand White rabbits administered the vaccine subcutaneously (SC) or intramuscularly (IM). Strong genogroup-specific immune responses were induced by vaccination without adjuvant at various doses (200 to 400 μg VLP/administration) and administration schedules (Days 1 and 7; or Days 1, 15 and 29). The results showed sporadic local irritation at the injection site, which resolved over time, and was non-adverse and consistent with expected reactogenicity.
There were no signs of systemic toxicity related to vaccine administration relative to vehicle-treated controls with respect to clinical chemistry, haematology, organ weights, macroscopic examinations, or histopathology. In a 3-administration regimen (n + 1 the clinical regimen), the NOAEL for rNV-2v via the SC or IM route was initially determined to be 200 μg. An improved GI.4 VLP variant mixed 1:1 (w/w) with the wild-type GII.4 VLP was subsequently evaluated via the IM route at a higher dose in the same 3-administration model, and the NOAEL was raised to 300 µg.
Serology performed in samples of both toxicity studies showed significant and substantial anti-VLP-specific antibody titers for rNV-2v vaccines administered via the IM or SC route www.joplink.net/rabbit-antibodies, as well as relevant NoV blocking antibody responses. These results support initiation of clinical development of the plant-made NoV vaccine.
Seasonality of Coxiella burnetii among Wild Rabbits ( Oryctolagus cuniculus) and the Hyalomma lusitanicum (Acari: Ixodidae) in a Meso-Mediterranean Ecosystem
(1) Background: Q fever is a worldwide zoonosis caused by Coxiella burnetii that have cases reported in humans and animals almost everywhere. The aim of this study was to describe the seasonality of Coxiella burnetii in the wild rabbit (Oryctolagus cuniculus) and the tick Hyalomma lusitanicum in a meso-Mediterranean ecosystem. (2) Methods: two populations of wild rabbits that differ in whether or not they share habitat with ungulates, mainly red deer (Cervus elaphus) were sampled for a year to collect ticks, blood and vaginal or anal swabs.
The presence of C. burnetii DNA in swabs and the tick H. lusitanicum was determined by PCR and serum antibodies by ELISA. (3) Results: C. burnetii DNA was detected in 47.2% of 583 rabbits, in 65.5% of sera, and in more than half of the H. lusitanicum. There were small variations according to sex and age of the rabbits but significant according to the habitat (4) Conclusions: The results indicate that C. burnetii circulates freely between wild rabbits and H. lusitanicum and the sylvatic cycle in meso-Mediterranean environments relies in the presence of wild rabbits and H. lusitanicum above all if sharing habitat with red deer.
GroEL Chaperonin-Based Assay for Early Diagnosis of Scrub Typhus
A point-of-care diagnostic for early and rapid diagnosis of scrub typhus caused by Orientia tsutsugamushi is required for prompt and proper treatment of patients presenting with undifferentiated febrile illnesses. In this study, an immunochromatographic antigen detection test kit (ICT AgTK) that targets the highly conserved O. tsutsugamushi 60 kDa GroEL chaperonin (heat shock protein 60) was developed. E. coli-derived recombinant GroEL expressed from DNA coding for the consensus sequence of 32 GroEL gene sequences extracted from the GenBank database was used to immunize rabbits and mice.
Rabbit polyclonal antibodies (pAb) were used for preparing a gold-pAb conjugate, and the rGroEL-specific mouse monoclonal antibody was used as the antigen detection reagent at the ICT test line. In-house validation revealed that the ICT AgTK gave 85, 100 and 95% diagnostic sensitivity, specificity and accuracy, respectively, compared to the combined clinical features and standard IFA when tested on 40 frozen serum samples.
The test kits correctly identified 10 scrub typhus samples out of 15 fresh plasma/buffy coat samples of patients with febrile illnesses. For independent laboratory validation, the ICT AgTK was sent to one provincial hospital. The ICT AgTK utilized by the hospital medical technologist correctly identified six scrub typhus samples out of 20 serum samples of patients with fever, as confirmed by specific IgM/IgG detection by IFA. The ICT AgTK is easy to perform with rapid turn-around time. It has the potential to be used as an important tool for on-site and early scrub typhus diagnosis by allowing testing of freshly collected samples (serum, plasma or buffy coat), especially in resource-limited healthcare settings.
Molecular characterization and antibacterial immunity functional analysis of the antimicrobial peptide hepcidin from Coregonus ussuriensis berg
Antimicrobial peptides are immune system molecules existing in different organisms including mollusks, crustaceans and vertebrates. Hepcidins are a group of cysteine rich antimicrobial peptides, which plays an important role in fish response to a variety of pathogens. In this study, we cloned and identified Hepcidin from the Coregonus ussuriensis Berg, and its functions in vivo and in vitro was investigated.
- Our results showed that, CuHepc contains a 267 bp coding sequence (CDS) region that encodes 88 putative amino acids with a molecular weight of 9.77 kD. Hepcidin transcripts were most abundant in the liver of healthy C. ussuriensis Berg.
- The synthesized Hepcidin peptide exhibited a wide range of antibacterial activity against Gram-positive and Gram-negative bacteria in vitro, and the results of in vivo bacterial attack assays showed that the CuHepc gene was differentially up-regulated in the six tissues investigated after infection with Aeromonas hydrophila.
- To analyze the changes in protein levels in C. ussuriensis, we generated Hepc polyclonal antibodies in rabbits and verified that the protein expression was increased after bacterial infection with Western blot assay.
- MIC assay results showed a geometric mean value of 5.513 μM for CuHepc peptide. In the in vivo experiment, immune-related genes IL-10, NF-κB, TLR3 were up-regulated post-infection CuHepc peptide in liver and intestine.
- Finally, CuHepc peptide reduced the tissues microbial load compared to infection with Aeromonas hydrophila. The above results indicate that Hepc plays a role in the immune response of C. ussuriensis to exogenous disturbances, indicate that CuHepc might act a candidate for modulation of the innate immune system in C. ussuriensis.
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