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The ongoing COVID-19 pandemic highlights the need to tackle viral variants, expand the number of antigens, and assess diverse delivery systems for vaccines against emerging viruses. In the present study, a DNA vaccine candidate was generated by combining in tandem envelope protein domain III (EDIII) of dengue virus serotypes 1–4 and a dengue virus (DENV)-2 non-structural protein 1 (NS1) protein-coding region. Each domain was designed as a serotype-specific consensus coding sequence derived from different genotypes based on the whole genome sequencing of clinical isolates in India and complemented with data from Africa. This sequence was further optimized for protein expression. In silico structural analysis of the EDIII consensus sequence revealed that epitopes are structurally conserved and immunogenic. The vaccination of mice with this construct induced pan-serotype neutralizing antibodies and antigen-specific T cell responses. Assaying intracellular interferon (IFN)-γ staining, immunoglobulin IgG2(a/c)/IgG1 ratios, and immune gene profiling suggests a strong Th1-dominant immune response. Finally, the passive transfer of immune sera protected AG129 mice challenged with a virulent, non-mouse-adapted DENV-2 strain. Our findings collectively suggest an alternative strategy for dengue vaccine design by offering a novel vaccine candidate with a possible broad-spectrum protection and a successful clinical translation either as a stand alone or in a mix and match strategy., Graphical Abstract, Developing a successful vaccine against dengue has been challenging. Arun Sankaradoss and colleagues developed a genotype-specific DNA DENV vaccine candidate that elicits robust dengue immune responses. The low cost and thermostability of DNA vaccines should allow countries to implement large-scale vaccination programs, which could greatly reduce dengue cases.

Following the recent clinical clearance of an Indian DNA COVID-19 vaccine, India and Africa are potential regions where DNA vaccines may become a major delivery system subject to a range of immunological and regulatory scrutiny. The ongoing COVID pandemic highlights the need to tackle viral variants and expand the number of antigens and assess diverse delivery systems. To address some of these key issues, we have created a Dengue DNA vaccine candidate with the EDIII region as the key antigen given the promise of this segment in not causing ADE, a challenge with this disease. In addition, we have added the NS1 region to broaden the immune response. Following a large Dengue viral sequencing exercise in India, complemented with data from east Africa, our approach was to generate a consensus of four serotypes ED3-NS1 vaccine to explore tackling the issue of diversity. OurIn silicostructural analysis of EDIII consensus vaccine sequence revealed that epitopes are structurally conserved and immunogenic across HLA diversity. Vaccination of mice with this construct induced pan-serotype neutralizing antibodies and antigen-specific T cell responses. Furthermore, the DNA vaccination confers protection against DENV challenge in AG129 mice. Finally, assaying of intracellular staining for IFN-γ, immunoglobulin IgG2(a/c) /IgG1 ratios as well as immune gene profiling suggested a strong Th1-dominant immune response. Our Dengue DNA platform with a focus on Indo-African sequences offers an approach for assessing cross reactive immunity in animal models and lays the foundation for human vaccine roll out either as a stand-alone or mix and match strategy.

BackgroundAfrica has one of the highest incidences of gonorrhoea, but not much information is available on the relatedness with strains from other geographical locations. Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a major public health threat, with the bacteria gaining resistance to most of the available antibiotics, compromising treatment across the world. Whole-genome sequencing is an efficient way of predicting AMR determinants and their spread in the human population. Previous studies on Kenyan gonococcal samples have focused on plasmid-mediated drug resistance and fluoroquinolone resistance using Illumina sequencing.Recent advances in next-generation sequencing technologies like Oxford Nanopore Technology (ONT) have helped in the generation of longer reads of DNA in a shorter duration with lower cost. However, long-reads are error-prone. The increasing accuracy ofbase-calling algorithms, high throughput, error-correction strategies, and ease of using the mobile sequencer in remote areas is leading to the adoption of the MinION sequencer (ONT), for routine microbial genome sequencing.MethodsTo investigate whether MinION-only sequencing is sufficient for diagnosis, genome sequencing and downstream analysis like inferring phylogenetic relationships and detection of AMR in resource-limited settings, we sequenced the genomes of fourteen clinical isolates suspected to be N. gonorrhoeae from Nairobi, Kenya. The isolates were tested using standard bacteriological methods for identification, interpreted using analytical profile index and antibiotic susceptibility tests had indicated ciprofloxacin and gentamycin resistance. Using whole-genome sequencing, the isolates were confirmed to be cases of N. gonorrhoeae (n=12), Additionally, we identified reads from N. meningitidis (n=2) and both of N. gonorrhoeae and Moraxella osloensis (n=3) in the sample (co-infections) respectively, which have been implicated in sexually transmitted infections in the recent years. The near-complete N. gonorrhoeae genomes (n=10) were analysed further for mutations/factors causing AMR using an in-house database of mutations curated from the literature. We attempted to understand the basis of drug resistance using homology modelling of AMR proteins, using known structures from other bacteria.ResultsWe observe that Ciprofloxacin resistance is associated with multiple mutations in both gyrA and parC. We identified mutations conferring tetracycline (rpsJ) and Sulfonamide (folA) resistance in all the isolates and plasmids encoding beta-lactamase and tet(M) were identified in almost all of the strains. Phylogenetic analysis clustered the nine isolates into clades containing previously sequenced genomes from Kenya and countries across the world.ConclusionHere, we demonstrate the utility of mobile DNA sequencing technology supplemented with reference-based assembly in sequence typing and elucidating the basis of AMR. Bioinformatics profiling to predict AMR can be used along with routine AMR susceptibility tests in clinics. The workflow followed in the study, including AMR mutation dataset creation and the genome identification, assembly and analysis, can be used for the genome assembly and analysis of any clinical isolate. Further studies are required to determine the utility of real-time sequencing in the outbreak investigations, diagnosis and management of infections, especially in resource-limited settings.Data availabilityThe raw reads generated for this study have been deposited in BioProject Accession: PRJNA660404 (https://www.ncbi.nlm.nih.gov). The biosample details are available under the ids SAMN15960547, SAMN15960548, SAMN15960549, SAMN15960550, SAMN15960551, SAMN15960552, SAMN15960553, SAMN15960554, SAMN15960555. The genomes and annotation files are available under the bioproject.

Anjana Sadanand,1 Anjali Janardhanan,1 Arun Sankaradoss,1 Arambakkam J Vanisree,1 Thamilpavai Arulnambi,2 Kesavamurthy Bhanu2 1Department of Biochemistry, University of Madras, 2Department of Neurology and Neurosurgery, Madras Medical College, Chennai, India Abstract: Neurodegenerative diseases have many similar pathological conditions, and very few studies exist which detail their molecular features. Proteins like Na+/K+-ATPase (NKA), α-spectrin (SPTA) and drebrin have been reported to be involved in the integrity of neuronal cell membrane and their functions. Furthermore, recent studies have highlighted their implication in neurodegeneration. In the current study, we wanted to identify the role of NKA, SPTA and drebrin in the erythrocyte membranes obtained from the blood of patients with neurodegenerative disorders (NDs) subjected to motor impairment such as Parkinson’s disease, amyotrophic lateral sclerosis, ataxia and dementia. We have studied the activity of NKA and the expression of NKA, SPTA and drebrin in the erythrocyte membrane by quantitative real-time PCR and Western blot obtained from the blood samples of patients with NDs culminating in movement and memory dysfunction. We observed a significant reduction in the expressions of NKA, SPTA and drebrin when compared to control and significant variations among the recruited ND samples. On correlating, we found a significant relationship between the expressions and the clinical features such as bradykinesia. Thus, we suggest that the reduction in the expressions of NKA, SPTA and drebrin could function as tools of assessment and speculate the particular neurodegenerative condition. Keywords: Na+/K+-ATPase, spectrin, drebrin, neurodegeneration and movement disorder