Gamma Irradiated Pasteurella multocida Vaccine induces strong humoral immunity and protects rabbits from disease.

Pasteurella multocida is affecting a multitude of animals and severely affects livestock production. Existing vaccines are mostly chemically inactivated and do not lead to wide protection. Irradiated vaccines are enjoying a renaissance and the concept of "replication defficient but metabolically active" vaccines was recently evaluated in several vaccine trials. P. multocida was isolated from the nasal swab, blood, and lung swab samples from infected rabbits. Gamma irradiation of P. multocida for inhibition of replication was evaluated at an optimized irradiation dose of 10 Kgy established. Four groups of rabbits were (mock) vaccinated with a commercial P. multocida vaccine and three irradiated formulations as liquid, lyophilized formulations with added Trehalose and lyophilized-Trehalose with an "activation" culturing the irradiated bacteria for 24 in broth. Evaluation of humoral immune response by ELISA showed that all three irradiated vaccines produced an effective, protective, and continued IgG serum level after vaccination and bacterial challenge. The IFN-γ expression is maintained at a normal level, within each individual group however, the lyophilized trehalose irradiated vaccine showed peak mean of IFN-γ titer at one week after booster dose (day 21) which was statistically significant. Cumulatively, the results of this study show that gamma-irradiated P. multocida vaccines are safe and protect rabbits against disease. Moreover, Rabbits' immunization with the three irradiated formulations avoided adverse side effects as compared to commercial polyvalent vaccine, the body weight gain for the irradiated vaccine groups indicates less stress compared to the commercial polyvalent vaccine.

Detection of immune effects of the Mannheimia haemolytica gamma irradiated vaccine in sheep.

Exposure to gamma rays from cobalt 60 (Co60) can induce a complete inactivation of Mannheimia haemolytica. The inactivated bacterial pathogen is a potential vaccine candidate for immunization of ruminants such as sheep. The subcutaneous administration of irradiated vaccine in a two-dose regimen (4.0 × 109 colony forming unit (CFU) per dose) results in no mortality in any of the vaccinated sheep during immunization and after subsequent challenge of the live bacteria of the same strain of M. haemolytica. A significant rise in serum IgG titer, detected through ELISA, is observed after the passage of two weeks from the inoculation of the first dose whereas, the peak of the mean serum antibody titer occurred after two weeks of booster dose. The vaccination does not bring significant change to the IFN-γ levels in serum. The bacterial challenge of the vaccinated sheep does not induce a further seroconversion relative to serum antibody titer. In conclusion, the vaccinated sheep are protected by the elevated IgG titer and increased levels of IL-4 (Th-2 response) compared to the non-vaccinated sheep. Radiation technology can provide the opportunity for mass production of immunologically safe vaccines against animal and zoonotic diseases. Ethics Approval by the National Research Center Ethics Committee (Trial Registration Number (TRN) no 13,602,023, 13/5/2023) was obtained.

Comparative assessment of homemade ELISA and lateral flow assay (LFA)in the rapid, specific and sensitive detection of SARS-CoV-2 anti-nucleocapsid protein in sera of Egyptian patients.

Several diagnostic measures have been employed to precisely detect the SARS-CoV-2 viral infection using viral antigens, nucleic acids, and other serological approaches. The sensitivity and specificity of the serological tests remain a challenging need. Here, we describe the detection of human anti-SARS-CoV-2 IgG and IgM antibodies qualitatively through two optimized in-house ELISA and lateral flow immunoassay. Both approaches are based on the prokaryotic expression of 50 kDa SARS-CoV-2 recombinant nucleocapsid protein. This SARS-CoV-2rN-6×His was used either to coat ELISA plates or to be conjugated to gold nanoparticles followed by colorimetric detection of bound human IgG or IgM. In the LFA, we show the optimization of nanoparticle size, protein-binding capacity, membrane treatment, and finally testing the potential capacity of using either the optimized ELISA or LFA in detecting antibodies raised against viral infection. Assessment of both methods was carried out using human sera-positive and negative SARS-CoV-2 antibodies. The ELISA and LFA tests showed 86%, 96.5% sensitivity, 92%, 93.75% specificity, 97%, 98.2% PPV, and 64%, 88.2% NPV, respectively. In conclusion, both approaches were able to successfully detect human antibodies against SARS-CoV-2 nucleocapsid protein. The importance of both protocols cannot be overstated in the detection and diagnosis of viral infections, especially in developing countries.

Assessment of specific human antibodies against SARS-CoV-2 receptor binding domain by rapid in-house ELISA.

BACKGROUND: The recently emerged SARS-CoV-2 caused a global pandemic since the last two years. The urgent need to control the spread of the virus and rapid application of the suitable health measures raised the importance of available, rapid, and accurate diagnostic approaches. OBJECTIVE: The purpose of this study is to describe a rapid in-house optimized ELISA based on the expression of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein in a prokaryotic system. METHODS: We show the expression of the 30 kDa recombinant SARS-CoV-2 RBD-6×His in four different E. coli strains (at 28∘C using 0.25mM IPTG) including the expression strain E. coli BL21 (DE3) Rosetta Gami. SARS-CoV-2 rRBD-6×His protein was purified, refolded, and used as an antigen coat to assess antibody response in human sera against SARS-CoV-2 infection. RESULTS: The assessment was carried out using a total of 155 human sero-positive and negative SARS-CoV-2 antibodies. The ELISA showed 69.5% sensitivity, 88% specificity, 78.5% agreement, a positive predictive value (PPV) of 92.3%, and a negative predictive value of 56.5%. Moreover, the optical density (OD) values of positive samples significantly correlated with the commercial kit titers. CONCLUSIONS: Specific human antibodies against SARS-CoV-2 spike protein were detected by rapid in-house ELISA in sera of human COVID-19-infected patients. The availability of this in-house ELISA protocol would be valuable for various diagnostic and epidemiological applications, particularly in developing countries. Future studies are planned for the use of the generated SARS-CoV-2 rRBD-6×His protein in vaccine development and other diagnostic applications.

Functional diversification of tomato HsfA1 factors is based on DNA binding domain properties.

In all eukaryotes, the response to heat stress (HS) is dependent on the activity of HS transcription factors (Hsfs). Plants contain a large number of Hsfs, however, only members of the HsfA1 subfamily are considered as master regulators of stress response and thermotolerance. In Solanum lycopersicum, among the four HsfA1 members, only HsfA1a has been proposed to possess a master regulator function. We performed a comparative analysis of HsfA1a, HsfA1b, HsfA1c and HsfA1e at different levels of regulation and function. HsfA1a is constitutively expressed under control and stress conditions, while the other members are induced in specific tissues and stages of HS response. Despite that all members are localized in the nucleus when expressed in protoplasts, only HsfA1a shows a wide range of basal activity on several HS-induced genes. In contrast, HsfA1b, HsfA1c, and HsfA1e show only high activity for specific subsets of genes. Domain swapping mutants between HsfA1a and HsfA1c revealed that the variation in that transcriptional transactivation activity is due to differences in the DNA binding domain (DBD). Specifically, we identified a conserved arginine (R107) residue in the turn of ß3 and ß4 sheet in the C-terminus of the DBD of HsfA1a that is highly conserved in plant HsfA1 proteins, but is replaced by leucine and cysteine in tomato HsfA1c and HsfA1e, respectively. Although not directly involved in DNA interaction, R107 contributes to DNA binding and consequently the activity of HsfA1a. Thus, we demonstrate that this variation in DBD in part explains the functional diversification of tomato HsfA1 members.

Identification and Characterization of a Thermotolerant TILLING Allele of Heat Shock Binding Protein 1 in Tomato.

The identification of heat stress (HS)-resilient germplasm is important to ensure food security under less favorable environmental conditions. For that, germplasm with an altered activity of factors regulating the HS response is an important genetic tool for crop improvement. Heat shock binding protein (HSBP) is one of the main negative regulators of HS response, acting as a repressor of the activity of HS transcription factors. We identified a TILLING allele of Solanum lycopersicum (tomato) HSBP1. We examined the effects of the mutation on the functionality of the protein in tomato protoplasts, and compared the thermotolerance capacity of lines carrying the wild-type and mutant alleles of HSBP1. The methionine-to-isoleucine mutation in the central heptad repeats of HSBP1 leads to a partial loss of protein function, thereby reducing the inhibitory effect on Hsf activity. Mutant seedlings show enhanced basal thermotolerance, while mature plants exhibit increased resilience in repeated HS treatments, as shown by several physiological parameters. Importantly, plants that are homozygous for the wild-type or mutant HSBP1 alleles showed no significant differences under non-stressed conditions. Altogether, these results indicate that the identified mutant HSBP1 allele can be used as a genetic tool in breeding, aiming to improve the thermotolerance of tomato varieties.

The repressor and co-activator HsfB1 regulates the major heat stress transcription factors in tomato.

Plants code for a multitude of heat stress transcription factors (Hsfs). Three of them act as central regulators of heat stress (HS) response in tomato (Solanum lycopersicum). HsfA1a regulates the initial response, and HsfA2 controls acquired thermotolerance. HsfB1 is a transcriptional repressor but can also act as co-activator of HsfA1a. Currently, the mode of action and the relevance of the dual function of HsfB1 remain elusive. We examined this in HsfB1 overexpression or suppression transgenic tomato lines. Proteome analysis revealed that HsfB1 overexpression stimulates the co-activator function of HsfB1 and consequently the accumulation of HS-related proteins under non-stress conditions. Plants with enhanced levels of HsfB1 show aberrant growth and development but enhanced thermotolerance. HsfB1 suppression has no significant effect prior to stress. Upon HS, HsfB1 suppression strongly enhances the induction of heat shock proteins due to the higher activity of other HS-induced Hsfs, resulting in increased thermotolerance compared with wild-type. Thereby, HsfB1 acts as co-activator of HsfA1a for several Hsps, but as a transcriptional repressor on other Hsfs, including HsfA1b and HsfA2. The dual function explains the activation of chaperones to enhance protection and regulate the balance between growth and stress response upon deviations from the homeostatic levels of HsfB1.