Strong immunogenicity & protection in mice with PlaCCine: A COVID-19 DNA vaccine formulated with a functional polymer.

DNA- based vaccines have demonstrated the potential as a safe and effective modality. PlaCCine, a DNA-based vaccine approach described subsequently relies on a synthetic DNA delivery system and is independent of virus or device. The synthetic functionalized polymer combined with DNA demonstrated stability over 12 months at 4C and for one month at 25C. Transfection efficiency compared to naked DNA increased by 5-15-fold in murine skeletal muscle. Studies of DNA vaccines expressing spike proteins from variants D614G (pVAC15), Delta (pVAC16), or a D614G + Delta combination (pVAC17) were conducted. Mice immunized intramuscular injection (IM) with pVAC15, pVAC16 or pVAC17 formulated with functionalized polymer and adjuvant resulted in induction of spike-specific humoral and cellular responses. Antibody responses were observed after one immunization. And endpoint IgG titers increased to greater than 1x 105 two weeks after the second injection. Neutralizing antibodies as determined by a pseudovirus competition assay were observed following vaccination with pVAC15, pVAC16 or pVAC17. Spike specific T cell immune responses were also observed following vaccination and flow cytometry analysis demonstrated the cellular immune responses included both CD4 and CD8 spike specific T cells. The immune responses in vaccinated mice were maintained for up to 14 months after vaccination. In an immunization and challenge study of K18 hACE2 transgenic mice pVAC15, pVAC16 and pVAC17 induced immune responses lead to decreased lung viral loads by greater than 90 % along with improved clinical score. These findings suggest that PlaCCine DNA vaccines are effective and stable and further development against emerging SARS-CoV-2 variants is warranted.

A multivalent mRNA monkeypox virus vaccine (BNT166) protects mice and macaques from orthopoxvirus disease.

In response to the 2022 outbreak of mpox driven by unprecedented human-to-human monkeypox virus (MPXV) transmission, we designed BNT166, aiming to create a highly immunogenic, safe, accessible, and scalable next-generation vaccine against MPXV and related orthopoxviruses. To address the multiple viral forms and increase the breadth of immune response, two candidate multivalent mRNA vaccines were evaluated pre-clinically: a quadrivalent vaccine (BNT166a; encoding the MPXV antigens A35, B6, M1, H3) and a trivalent vaccine (BNT166c; without H3). Both candidates induced robust T cell responses and IgG antibodies in mice, including neutralizing antibodies to both MPXV and vaccinia virus. In challenge studies, BNT166a and BNT166c provided complete protection from vaccinia, clade I, and clade IIb MPXV. Furthermore, immunization with BNT166a was 100% effective at preventing death and at suppressing lesions in a lethal clade I MPXV challenge in cynomolgus macaques. These findings support the clinical evaluation of BNT166, now underway (NCT05988203).

Disinsert, retract and rotate technique of plaque brachytherapy.

BACKGROUND: Plaque brachytherapy is commonly used in the management of choroidal melanomas. The surgical steps usually involve creating a conjunctival peritomy, fixing the recti muscles, with or without disinserting them based on the location of the lesion, and placing the plaque. The inferior oblique muscle is attached close to the macula, and in cases of perimacular or peripapillary lesions, the muscle needs to be sacrificed. PURPOSE: The authors here demonstrate a novel technique of placing radioactive plaque without disinserting the inferior oblique muscle in cases of perimacular or peripapillary choroidal melanomas. SYNOPSIS: The video demonstrates how the "disinsert, retract, and rotate technique" of brachytherapy plaque placement can be performed and what are the fundamentals behind this technique. The authors have performed this procedure multiple times and there has been no incidence of plaque tilt or migration. HIGHLIGHTS: In perimacular and peripapillary choroidal melanoma brachytherapy plaque placement, the inferior oblique muscle can be spared. The simple technique does not lead to any tilt or migration of the radioactive plaque. VIDEO LINK: https://youtu.be/YMIg3rYyp2o.

Retinoblastoma in Asia: Clinical Presentation and Treatment Outcomes in 2112 Patients from 33 Countries.

PURPOSE: To describe the clinical presentation and treatment outcomes of children who received a diagnosis of retinoblastoma in 2017 throughout Asia. DESIGN: Multinational, prospective study including treatment-naïve patients in Asia who received a diagnosis of retinoblastoma in 2017 and were followed up thereafter. PARTICIPANTS: A total of 2112 patients (2797 eyes) from 96 retinoblastoma treatment centers in 33 Asian countries. INTERVENTIONS: Chemotherapy, radiotherapy, enucleation, and orbital exenteration. MAIN OUTCOME MEASURES: Enucleation and death. RESULTS: Within the cohort, 1021 patients (48%) were from South Asia (SA), 503 patients (24%) were from East Asia (EA), 310 patients (15%) were from Southeast Asia (SEA), 218 patients (10%) were from West Asia (WA), and 60 patients (3%) were from Central Asia (CA). Mean age at presentation was 27 months (median, 23 months; range, < 1-261 months). The cohort included 1195 male patients (57%) and 917 female patients (43%). The most common presenting symptoms were leukocoria (72%) and strabismus (13%). Using the American Joint Committee on Cancer Staging Manual, Eighth Edition, classification, tumors were staged as cT1 (n = 441 [16%]), cT2 (n = 951 [34%]), cT3 (n = 1136 [41%]), cT4 (n = 267 [10%]), N1 (n = 48 [2%]), and M1 (n = 129 [6%]) at presentation. Retinoblastoma was treated with intravenous chemotherapy in 1450 eyes (52%) and 857 eyes (31%) underwent primary enucleation. Three-year Kaplan-Meier estimates for enucleation and death were 33% and 13% for CA, 18% and 4% for EA, 27% and 15% for SA, 32% and 22% for SEA, and 20% and 11% for WA (P < 0.0001 and P < 0.0001), respectively. CONCLUSIONS: At the conclusion of this study, significant heterogeneity was found in treatment outcomes of retinoblastoma among the regions of Asia. East Asia displayed better outcomes with higher rates of globe and life salvage, whereas Southeast Asia showed poorer outcomes compared with the rest of Asia. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Identification of novel genes by targeted exome sequencing in Retinoblastoma.

BACKGROUND: Retinoblastoma (RB) is initiated by mutation in both alleles of RB1 gene. However, few cases may occur even in the absence of RB1 mutation suggesting the role of genes other than RB1. METHODOLOGY: The current study was planned to utilize targeted exome sequencing in Indian RB patients affected with unilateral non-familial RB. 75 unilateral RB patients below 5 years of age were enrolled. Genomic DNA was extracted from blood and tumor tissue. From peripheral blood DNA, all coding and exon/intron regions were amplified using PCR and direct sequencing. Cases which did not harbor pathogenic variants in peripheral blood DNA were further screened for mutations in their tumor tissue DNA using targeted exome sequencing. Three pathogenicity prediction tools (Mutation Taster, SIFT, and PolyPhen-2) were used to determine the pathogenicity of non-synonymous variations. An in-house bioinformatics pipeline was devised for the mutation screening by targeted exome sequencing. Protein modeling studies were also done to predict the effect of the mutations on the protein structure and function. RESULTS: Using the mentioned approach, we found two novel variants (g.69673_69674insT and g.48373314C>A) in RB1 gene in peripheral blood DNA. We also found novel variants in eight genes (RB1, ACAD11, GPR151, KCNA1, OTOR, SOX30, ARL11, and MYCT1) that may be associated with RB pathogenesis. CONCLUSION: The present study expands our current knowledge regarding the genomic landscape of RB and also highlights the importance of NGS technologies to detect genes and novel variants that may play an important role in cancer initiation, progression, and prognosis.

Combined association of massive choroidal and optic nerve invasion as a prognostic relevance in primary retinoblastoma: A 10-year study.

PURPOSE: To determine the significance of both massive choroidal invasion and optic nerve invasion (retrolaminar [(RL]+cut end [CE]) as a criterion for classifying high metastatic potential retinoblastoma and their relationship with other known histopathological high-risk features. METHODS: A retrospective review of 650 eyes diagnosed as retinoblastoma over a 10-year period. In our study, there is male predominance and a higher percentage of the poorly differentiated tumors. The age of most of the patients ranges from 1 month to 8 years with a median age of 2 years. RESULTS: There were 24% of eyes with massive choroidal invasion and 18% of eyes with optic nerve invasion up to the cut end. On performing Cox-proportional hazard analysis, it was found that massive choroidal invasion in association with optic nerve invasion up to the cut end was an independent prognostic parameter. On Kaplan-Meier analysis, overall survival had reduced in patients having both massive choroidal invasion and an optic nerve cut end invasion along with orbital invasion (P < .05). CONCLUSION: The presence of massive choroidal invasion in association with optic nerve cut end invasion (RL+CE) could be used as a better prognostic predictor in assessing retinoblastoma patients with high metastatic potential and need to be kept for longer follow up.

Trabeculectomy in an unsuspected extramedullary iris-ciliary body plasmacytoma.

Extramedullary plasmacytoma of the iris and ciliary body is extremely rare. We present a case which was misdiagnosed as granulomatous uveitis with neovascular glaucoma, and underwent a trabeculectomy with mitomycin-c along with iris biopsy. The post-operative period showed early bleb failure and catastrophic growth of the suspected mass. Histopathological examination revealed a diagnosis iris plasmacytoma. Subsequent ultrasound biomicroscopy showed involvement of the iris and ciliary body. A prompt systemic workup was done, and an associated systemic plasma cell dyscrasia was ruled out. The affected eye was enucleated, and the patient remains disease free at the end of 3-year follow-up.

Evaluation of the role of ultrasound biomicroscopy in advanced retinoblastoma: A prospective study on Asian Indian children.

Aim: To evaluate the role of ultrasound biomicroscopy (UBM) in retinoblastoma (RB).Methods: Children with advanced unilateral RB were included. UBM was performed to look for tumour invasion into the anterior segment (AS) and for evaluation of quantitative parameters. Enucleation was done and UBM findings were correlated with histopathology. The main outcome measures were sensitivity and specificity of UBM for detecting AS invasion and comparison of quantitative parameters between the tumour affected and fellow eyes.Results: Fifty patients were evaluated. The mean age was 2.76 ± 1.63 years. Enucleation was performed in 50 eyes. The sensitivity and specificity of UBM for AS invasion were 80% (95% CI, 44-97%) and 95% (95% CI, 83-99%), respectively. UBM showed a sensitivity and specificity of 100% (95% CI, 59-100%) and 95% (95% CI, 84-99%), respectively, for iris invasion, 88% (95% CI, 47-100%) and 100% (95% CI, 92-100%), respectively, for ciliary body invasion, and 63% (95% CI, 24-91%) and 100% (95% CI, 92-100%), respectively, for anterior chamber (AC) angle invasion. Quantitative parameters were studied in 100 eyes. As compared to the fellow eyes, the AC angle was narrow (p < 0.05), posterior chamber was shallow (p = 0.004) and zonular length was increased (p = 0.001) in RB eyes.Conclusion: This clinicopathological study provides valuable insights into the role of UBM for evaluation of anterior extension of RB and for assessment of architectural changes in the AS due to the tumour.

Human papillomavirus in ocular malignant tumours: a study from a tertiary eye care centre in North India.

OBJECTIVE: The present study aims to detect the presence of human papillomavirus (HPV) in ocular malignant tumours, including retinoblastoma, eyelid squamous cell carcinoma (SCC), and sebaceous gland carcinoma (SGC), in the North Indian population. DESIGN: Prospective observational non randomized study. PARTICIPANTS: In this study, 142 prospective cases of ocular malignant tumours (retinoblastoma, SGC, and SCC) were included. METHODS: HPV was detected by multiplex PCR using PGMY09/11 primers in 142 patients with ocular malignancies. This was followed by genotyping using linear array (reverse hybridization). RESULTS: Of the 142 tumours studied, 72 were retinoblastoma, 30 SGC, and 40 SCC. The HPV genome was detected in 2.8% (4 of 142) of cases by multiplex PCR; all positive cases (4 of 40) were SCC. Genotyping revealed that all positives belonged to the high-risk HPV16 genotype. HPV-positive SCC patients had better disease-free survival. Retinoblastoma and SGC cases were negative for HPV. CONCLUSIONS: Low prevalence of HPV in ocular malignancies was observed in this study. The HPV genome was detected only in ocular squamous cell carcinoma cases and these patients were associated with better prognosis. HPV may not have a role in retinoblastoma and SGC in the North Indian population.

To compare intralesional and oral propranolol for treating periorbital and eyelid capillary hemangiomas.

Purpose: A pilot randomized control trial to compare the efficacy and side effects of intralesional and oral propranolol in periorbital and eyelid capillary hemangiomas. Methods: Twenty patients were prospectively randomized to two groups of ten each. Group 1 was initiated on oral propranolol 1 mg/kg/day titrated to final dose of 3 mg/kg/day over 1 week which was continued for 6 months and then tapered over 1 week; Group 2 received 3 doses of direct intralesional propranolol hydrochloride 1 mg/ml; 0.2 ml/cm 4-6 weeks apart. Hemangioma area and corneal astigmatism were measured. Results: Within each group at 6 months there was a significant reduction in area (group 1: 83.48 ± 11.67%,P= 0.0019; group 2: 67.78 ± 21.71%,P= 0.0019) and improvement in astigmatism (pre, post: group 1: 2.98D @ 179.8°, 1.13D @ 179.8°,P= 0.0045; group 2: 1.62D @ 90.16°, 0.75D @ 179.9°,P= 0.0001). There was no difference in area reduction (P = 0.056), change in appearance (P = 0.085), ptosis (P = 0.23) and side effects (lethargy, poor feeding;P= 0.171) between the two groups. Conclusion: Efficacy and side effects with intralesional propranolol are comparable to oral propranolol for periorbital and eyelid lesions.

Comparison of ocular pharmacokinetics of etoposide and its nanoemulsion after subtenon administration in rabbits.

Background Subtenon anticancer drugs are given as an adjunct to systemic chemotherapy for conditions like retinoblatoma. This study evaluated the ocular kinetics of nano-emulsion formulation of etoposide (NanoEt) and compared it with an equal dose of commercially available alcohol-based etoposide formulation in healthy rabbits. Methods A nanoemulsion formulation of NanoEt was developed and then evaluated for its ocular kinetics by subtenon administration in healthy rabbits. After the sterile subtenon administration of the drug, the eyes were enucleated after CO2 euthanasia at time intervals of 2 h, 6 h, 12 h, and 24 h, and ocular tissues, blood, and plasma were separated. The concentration of etoposide in the ocular tissues and blood was quantified using liquid chromatography tandem mass spectrometry (LC MS/MS). Results This study found that subtenon injection of NanoEt showed 24 times higher concentration in rabbit retina compared to an equal dose of conventional marketed formulation. Based on the ocular tissue bioavailability calculations (AUC0-24), the present study revealed that the formulation enhanced 90% ocular bioavailability of etoposide, when it was injected in the form of nano-emulsion in most of the tissues. Conclusions NanoEt has better bioavailability compared to the commercial alcohol-based formulation for subtenon injection. Low systemic exposure showed further advantage for its projected use in retinoblastoma (Rb) as an adjunct therapy. Further studies in Rb animal models are required to evaluate its safety and efficacy, for its clinical utility.

Antibody-Dependent, Gamma Interferon-Independent Sterilizing Immunity Induced by a Subunit Malaria Vaccine.

The development of effective malaria vaccines is hampered by incomplete understanding of the immunological correlates of protective immunity. Recently, the moderate clinical efficacy of the Plasmodium falciparum circumsporozoite protein (CSP)-based RTS,S/AS01E vaccine in phase 3 studies highlighted the urgency to design and test more efficacious next-generation malaria vaccines. In this study, we report that immunization with recombinant CSP from Plasmodium yoelii (rPyCSP), when delivered in Montanide ISA 51, induced sterilizing immunity against sporozoite challenge in C57BL/6 and BALB/c strains of mice. This immunity was antibody dependent, as evidenced by the complete loss of immunity in B-cell-knockout (KO) mice and by the ability of immune sera to neutralize sporozoite infectivity in mice. Th2-type isotype IgG1 antibody levels were associated with protective immunity. The fact that immunized gamma interferon (IFN-γ)-KO mice and wild-type (WT) mice have similar levels of protective immunity and the absence of IFN-γ-producing CD4+ and CD8+ T cells in protected mice, as shown by flow cytometry, indicate that the immunity is IFN-γ independent. Protection against sporozoite challenge correlated with higher frequencies of CD4+ T cells that express interleukin-2 (IL-2), IL-4, and tumor necrosis factor alpha (TNF-α). In the RTS,S study, clinical immunity was associated with higher IgG levels and frequencies of IL-2- and TNF-α-producing CD4+ T cells. The other hallmarks of immunity in our study included an increased number of follicular B cells but a loss in follicular T helper cells. These results provide an excellent model system to evaluate the efficacy of novel adjuvants and vaccine dosage and determine the correlates of immunity in the search for superior malaria vaccine candidates.

Identification of canonical NFκB (C-NFκB) pathway in uveal melanoma and their relation with patient outcome.

Inflammation in uveal melanoma (UM) is linked to a bad prognosis. It is rare type of cancer, of which the metastases are usually fatal within a year. Infiltration with an inflammatory infiltrate increases with disease progression but does not seem to inhibit metastasis. The Canonical NFκB (C-NFκB) pathway is known to play a crucial role in tumor inflammation. We therefore, studied the expression of canonical NFκB proteins and their prognostic relevance in UM. Our study evaluated the expression of C-NFκB proteins (p65, p50, and c-Rel) by using immunohistochemistry on sections from 75 formalin-fixed UM. Activation of the NFκB subunit was determined on fresh tumor specimens by measuring the DNA-binding activity in nuclei using an NFκB ELISA assay. Real-time PCR was performed on frozen material on 58 tumors. The presence of native C-NFκB heterodimers (p65/p50 and c-Rel/p50) was confirmed by co-immunoprecipitation followed by Western blotting. We observed a high nuclear immunoreactivity of p65, p50, and c-Rel proteins in 54, 60 and 41% UM cases, respectively. Expression of C-NFκB proteins significantly correlated with parameters which are related to the inflammatory environment of UM. Nuclear immunoreactivity of p65 and p50 was associated with lower patient survival (p = 0.041; p = 0.048) while c-Rel was not. Our finding reveals that C-NFκB proteins expressed are more often in UM with inflammation than those without inflammation. Activation of the canonical NFκB pathway is more frequent in high risk UM patients. These observations might help to understand the behaviour of high risk tumors, with upregulation of C-NFκB proteins contributing to tumor aggressiveness.

Retinoblastoma in Asia.

Asia-Pacific region bears a significant global burden of retinoblastoma (RB), therefore understanding RB in Asia-Pacific region is important. Based on the year 2013 population estimates, 43% (3452 of 8099 children) of the global burden of RB lives in 6 countries of Asia-Pacific region: 1486 children in India, 1103 children in China, 277 children in Indonesia, 260 children in Pakistan, 184 children in Bangladesh, 142 children in Philippines. There exists a wide disparity, technological and socio-economical, within countries in this region resulting in a varied pattern of clinical presentation and survival varies. Challenges in developing nations are not just technological, but also social. Opportunities emerge for research to study and understand the socio-economical aspects of the disease to develop interventions that are relevant culturally and feasible economically. Possible steps include disease education and counselling, universal screening, highly subsidized/free of cost treatment for low socioeconomic strata, raising funds through the government and non-governmental organizations, sensitization and training of man-power in screening, diagnosis and treatment, and developing new specialized centers with tele-ophthalmology services.

Impact of yoga based lifestyle intervention on psychological stress and quality of life in the parents of children with retinoblastoma.

BACKGROUND: Childhood cancers are associated with a psychological burden to the parents and hence, decline their mental and physical health and overall quality of life. PURPOSE: The purpose of the present study is to investigate the impact of 12-weeks yoga based lifestyle intervention on psychological stress and quality of life in the parents of children affected with retinoblastoma. METHOD: Single arm prospective clinical trial conducted from October 2015 to October 2017 at the Laboratory for Molecular Reproduction and Genetics, Department of Anatomy, All India Institute of Medical Sciences, New Delhi, India. A pre-tested 12-weeks yoga based lifestyle intervention included asanas (physical postures), pranayama (breathing exercises), dhyana (meditation), relaxation techniques, lectures and films on yoga, interactive sessions and individualized advice was administered to the participants. RESULTS: 12-weeks of yoga based lifestyle intervention programme leads to a significant improvement in psychological stress and overall quality of life in the parents of retinoblastoma patients. There was a significant improvement in all the domains (physical health, psychological health, social relationships, and environment) of WHOQOL-BREF from baseline (day 0) to 12-weeks of yoga based lifestyle intervention. Yoga based lifestyle intervention also led to a significant increase in the levels of brain derived neurotrophic factor, dehydroepiandrosterone sulphate, sirtuin 1 and decreased the cortisol and IL-6 levels. CONCLUSION: Yoga based lifestyle intervention reduced the severity of psychological stress and resulted in improvement in overall quality of life and upregulation in levels of systemic biomarkers of neuroplasticity. YBLI may serve as a beneficial therapy and may also act as an effective medium for better stress management to develop better coping strategies in the parents of retinoblastoma patients.

Magnetic resonance imaging for tumor restaging after chemotherapy in retinoblastoma with optic nerve invasion.

PURPOSE: Extraocular retinoblastoma with optic nerve invasion is treated by a multimodal protocol consisting of neoadjuvant chemotherapy, enucleation, and adjuvant therapy. This study was conducted to evaluate the performance of magnetic resonance imaging (MRI) used for tumor restaging in these children after systemic chemotherapy administration. METHODS: Contrast-enhanced MRI scan of orbits and brain was performed at diagnosis and patients were treated with neoadjuvant chemotherapy. After chemotherapy, MRI scan was repeated for tumor restaging and residual post-laminar thickening and/or enhancement of the affected optic nerve, if any, was recorded. MRI findings were correlated with histopathology in enucleated specimens. The main outcome measures were specificity, sensitivity, and accuracy of MRI in predicting post-laminar invasion after neoadjuvant chemotherapy. RESULTS: A total of 46 eyes (46 patients) were studied. Optic nerve thickening on MRI had a sensitivity, specificity, and accuracy of 100% (95% Confidence Interval (CI): 64.6-100%), 76.9% (95% CI: 61.7-87.4%), and 80.4% (95% CI: 66.8-89.4%), respectively. Optic nerve enhancement had a sensitivity, specificity, and accuracy of 85.7% (95% CI: 48.7-97.4%), 79.5 % (95% CI: 64.5-89.2%), and 80.4% (95% CI: 66.8-89.4%), respectively. Combined thickening and enhancement of the optic nerve had a sensitivity, specificity, and accuracy of 100% (95% CI: 60.9-100%), 82.4% (95% CI: 66.5-91.7%), and 85% (95% CI: 70.9-92.9%), respectively. CONCLUSION: MRI is a valuable tool for restaging of retinoblastoma and predicting residual optic nerve disease after neoadjuvant chemotherapy. Combined thickening and enhancement on MRI appeared to be a more reliable indicator of post-laminar invasion as compared to thickening or enhancement alone.

Oxidative Stress and Polymorphism in MTHFR SNPs (677 and 1298) in Paternal Sperm DNA is Associated with an Increased Risk of Retinoblastoma in Their Children: A Case-Control Study.

Sperm DNA is considered as the most vulnerable to oxidative stress-induced damage that also impairs global sperm DNA methylation leading to sperm-associated pathologies. C677T and A1298C polymorphisms of the methylene tetrahydrofolate reductase (MTHFR) gene affect MTHFR enzyme activity. This study was planned as a case-control study to determine the MTHFR gene polymorphisms in the fathers of children affected with sporadic nonfamilial heritable retinoblastoma in an Indian population. MTHFR polymorphisms for single nucleotide polymorphisms 677 and 1298 were also determined in sporadic nonfamilial heritable retinoblastoma patients to estimate the risk for retinoblastoma development and to evaluate the role of MTHFR in retinoblastoma pathogenesis.