Development of a bivalent protein-based vaccine candidate against invasive pneumococcal diseases based on novel pneumococcal surface protein A in combination with pneumococcal histidine triad protein D.

Extensive efforts have been made toward improving effective strategies for pneumococcal vaccination, focusing on evaluating the potential of multivalent protein-based vaccines and overcoming the limitations of pneumococcal polysaccharide-based vaccines. In this study, we investigated the protective potential of mice co-immunization with the pneumococcal PhtD and novel rPspA proteins against pneumococcal sepsis infection. The formulations of each antigen alone or in combination were administered intraperitoneally with alum adjuvant into BALB/c mice three times at 14-day intervals. The production of antigen-specific IgG, IgG1 and IgG2a subclasses, and IL-4 and IFN-γ cytokines, were analyzed. Two in vitro complement- and opsonophagocytic-mediated killing activities of raised antibodies on day 42 were also assessed. Finally, the protection against an intraperitoneal challenge with 106 CFU/mouse of multi-drug resistance of Streptococcus pneumoniae ATCC49619 was investigated. Our findings showed a significant increase in the anti-PhtD and anti-rPspA sera IgG levels in the immunized group with the PhtD+rPspA formulation compared to each alone. Moreover, the results demonstrated a synergistic effect with a 6.7- and 1.3- fold increase in anti-PhtD and anti-rPspA IgG1, as well as a 5.59- and 1.08- fold increase in anti-PhtD and anti-rPspA IgG2a, respectively. Co-administration of rPspA+PhtD elicited a mixture of Th-2 and Th-1 immune responses, more towards Th-2. In addition, the highest complement-mediated killing activity was observed in the sera of the immunized group with PhtD+rPspA at 1/16 dilution, and the opsonophagocytic activity was increased from 74% to 86.3%. Finally, the survival rates showed that mice receiving the rPspA+PhtD formulation survived significantly longer (100%) than those receiving protein alone or PBS and exhibited the strongest clearance with a 2 log10 decrease in bacterial load in the blood 24h after challenge compared to the control group. In conclusion, the rPspA+PhtD formulation can be considered a promising bivalent serotype-independent vaccine candidate for protection against invasive pneumococcal infection in the future.

Case Report: Novel biallelic moderately damaging variants in RTTN in a patient with cerebellar dysplasia.

Rotatin, encoded by the RTTN gene, is a centrosomal protein with multiple, emerging functions, including left-right specification, ciliogenesis, and neuronal migration. Recessive variants in RTTN are associated with a neurodevelopmental disorder with microcephaly and malformations of cortical development known as "Microcephaly, short stature, and polymicrogyria with seizures" (MSSP, MIM #614833). Affected individuals show a wide spectrum of clinical manifestations like intellectual disability, poor/absent speech, short stature, microcephaly, and congenital malformations. Here, we report a subject showing a distinctive neuroradiological phenotype and harboring novel biallelic variants in RTTN: the c.5500A>G, p.(Asn1834Asp), (dbSNP: rs200169343, ClinVar ID:1438510) and c.19A>G, p.(Ile7Val), (dbSNP: rs201165599, ClinVar ID:1905275) variants. In particular brain magnetic resonance imaging (MRI) showed a peculiar pattern, with cerebellar hypo-dysplasia, and multiple arachnoid cysts in the lateral cerebello-medullary cisterns, in addition to left Meckel cave. Thus, we compare his phenotypic features with current literature, speculating a possible role of newly identified RTTN variants in his clinical picture, and supporting a relevant variability in this emerging condition.

Electrosprayed cefazolin-loaded niosomes onto electrospun chitosan nanofibrous membrane for wound healing applications.

Chronic wounds are among the most therapeutically challenging conditions, which are commonly followed by bacterial infection. The ideal approach to treat such injuries are synergistic infection therapy and skin tissue regeneration. In the recent decades, nanotechnology has played a critical role in eradicating bacterial infections by introducing several carriers developed for drug delivery. Moreover, advances in tissue engineering have resulted in new drug delivery systems that can improve the skin regeneration rate and quality. In this study, cefazolin-loaded niosomes were electrosprayed onto chitosan membrane for wound healing applications. For this purpose, niosomes were obtained by the thin-film hydration method; electrospinning was then conducted to fabricate nanofibrous mats. In vitro characterization of the scaffold was performed to evaluate the physicochemical and biological properties. Finally, in vivo studies were carried out to evaluate the potential use of the membrane for skin regeneration. In vitro results indicated the antibacterial properties of the membrane against Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) due to the gradual release of cefazolin from niosomes. The scaffolds also showed no cell toxicity. In vivo studies also confirmed the ability of the membrane to enhance skin regeneration by improving re-epithelialization, tissue remodeling, and angiogenesis. The current study could well show the promising role of the prepared scaffold for skin regeneration and bacterial infection elimination.

Oncolytic herpes simplex virus type-1 expressing IL-12 efficiently replicates and kills human colorectal cancer cells.

An increasing attitude towards oncolytic viruses (OVs) is witnessed following T-VEC's approval. In this study, we aimed to delete ICP47 and insert IL-12 in the ICP34.5 deleted HSV-1 backbone to improve the oncolytic properties and provide an immune-stimulatory effect respectively. The wild-type and recombinant viruses infected both cancerous, SW480 and HCT116, and non-cancerous, HUVEC, cell lines. Green-red Δ47/Δ34.5 was constructed by replacing ICP47 with GFP. Both ICP34.5 copies were replaced by hIL12. Cytotoxicity and growth kinetics of Δ47/Δ34.5/IL12 and Δ47/Δ34.5 were comparable to the wild virus in the cancerous cells. Δ47/Δ34.5/IL12 was able to produce IL12 in the infected cell lines. INF-γ production and PBMC proliferation were observed in the PBMCs treated with the lysate of Δ47/Δ34.5/IL12 infected cells. These results demonstrated that Δ47/Δ34.5/IL12 was competent in taking advantage of the cytotoxic effect of HSV-1 plus immune-stimulatory characteristics of IL-12.

Effects of cefazolin-containing niosome nanoparticles against methicillin-resistant Staphylococcus aureus biofilm formed on chronic wounds.

The ability of biofilm formation in methicillin-resistant Staphylococcus aureus (MRSA) causes significant mortality and morbidity in wound infections. Nanoparticles because of the drug concentration increment at the point of contact of nanoparticles and bacteria, and slower release of the drug at the desired location are considered as proper tools to overcome the therapeutic problem of antimicrobial-resistant infections. This study was aimed to evaluate the anti-biofilm activity of cefazolin-loaded nanoparticles against MRSA isolates. The 27 clinical isolates of MRSA were collected from patients with pressure sores and diabetic ulcers referred to Loghman Hospital in Tehran-Iran. MRSA isolates were detected by polymerase chain reaction (PCR) and biochemical tests. Cefazolin-loaded niosome was synthesized using the thin-film hydration method and were characterized by zeta potential measurement and transmission electron microscopy (TEM). The round-shaped cefazolin-loaded niosomes had a diameter of 100 nm and a -63 mV zeta potential. The cefazolin-containing niosomes removed 1, 3, and 5 d old biofilms at the concentration of 128 µg ml-1, 128 µg ml-1, and 256 µg ml-1, respectively. Histological results indicated that BALB/c mice receiving cefazolin-loaded niosomes were treated effectively faster than those treated by cefazolin or untreated group. In conclusion, the cefazolin-loaded niosome could be considered as a promising candidate for the treatment of biofilm-mediated infections of MRSA.

Co-administration of 2'3'-cGAMP STING activator and CpG-C adjuvants with a mutated form of HPV 16 E7 protein leads to tumor growth inhibition in the mouse model.

Persistent infection with high-risk genotypes of human papillomavirus (HPV) is the leading cause of cervical cancer. The HPV oncoprotein E7 is constitutively expressed in cervical cancer and considered as an essential target for tumor-specific immunity. The goal of this study was to develop a candidate therapeutic vaccine based on the mutated E7 protein that had possibly reduced transformation capacity while was able to elicit a robust immune response. Therefore, the mutant type of HPV 16 E7 (E7GRG) protein was recombinantly expressed in E. coli. The protein was then purified and formulated with 2'-3'cGAMP CDN and/or CpG-C ODN adjuvants and subcutaneously injected to female C57BL/6 mice. To evaluate the immunogenic response, lymphocyte proliferation, secretion levels of IFN-γ and IL-4 cytokines, granzyme B level, and total IgG and subclasses of IgG antibody were measured. The anti-tumor activity was evaluated in tumor-harboring C57BL/6 mice. The highest rate of cell proliferation, IFN-γ and granzyme B levels, and amount of IgG antibody were found in mice group that were injected by E7GRG + 2'-3'cGAMP + CpG-C. Therapeutic immunization with E7GRG + 2'-3'cGAMP + CpG-C also significantly suppressed TC-1 tumor growth in mice. In conclusion, the results demonstrated that E7GRG + 2'-3'cGAMP + CpG-C induced strong cell-mediated and humoral immune responses that resulted in inhibition of tumor in mouse model.