Immunogenicity of SARS-CoV-2 vaccines BBV152 (COVAXIN®) and ChAdOx1 nCoV-19 (COVISHIELD™) in seronegative and seropositive individuals in India: a multicentre, nonrandomised observational study.

Background: There are limited global data on head-to-head comparisons of vaccine platforms assessing both humoral and cellular immune responses, stratified by pre-vaccination serostatus. The COVID-19 vaccination drive for the Indian population in the age group 18-45 years began in April 2021 when seropositivity rates in the general population were rising due to the delta wave of COVID-19 pandemic during April-May 2021. Methods: Between June 30, 2021, and Jan 28, 2022, we enrolled 691 participants in the age group 18-45 years across four clinical sites in India. In this non-randomised and laboratory blinded study, participants received either two doses of Covaxin® (4 weeks apart) or two doses of Covishield™ (12 weeks apart) as per the national vaccination policy. The primary outcome was the seroconversion rate and the geometric mean titre (GMT) of antibodies against the SARS-CoV-2 spike and nucleocapsid proteins post two doses. The secondary outcome was the frequency of cellular immune responses pre- and post-vaccination. Findings: When compared to pre-vaccination baseline, both vaccines elicited statistically significant seroconversion and binding antibody levels in both seronegative and seropositive individuals. In the per-protocol cohort, Covishield™ elicited higher antibody responses than Covaxin® as measured by seroconversion rate (98.3% vs 74.4%, p < 0.0001 in seronegative individuals; 91.7% vs 66.9%, p < 0.0001 in seropositive individuals) as well as by anti-spike antibody levels against the ancestral strain (GMT 1272.1 vs 75.4 binding antibody units/ml [BAU/ml], p < 0.0001 in seronegative individuals; 2089.07 vs 585.7 BAU/ml, p < 0.0001 in seropositive individuals). As participants at all clinical sites were not recruited at the same time, site-specific immunogenicity was impacted by the timing of vaccination relative to the delta and omicron waves. Surrogate neutralising antibody responses against variants-of-concern including delta and omicron was higher in Covishield™ recipients than in Covaxin® recipients; and in seropositive than in seronegative individuals after both vaccination and asymptomatic infection (omicron variant). T cell responses are reported from only one of the four site cohorts where the vaccination schedule preceded the omicron wave. In seronegative individuals, Covishield™ elicited both CD4+ and CD8+ spike-specific cytokine-producing T cells whereas Covaxin® elicited mainly CD4+ spike-specific T cells. Neither vaccine showed significant post-vaccination expansion of spike-specific T cells in seropositive individuals. Interpretation: Covishield™ elicited immune responses of higher magnitude and breadth than Covaxin® in both seronegative individuals and seropositive individuals, across cohorts representing the pre-vaccination immune history of most of the vaccinated Indian population. Funding: Corporate social responsibility (CSR) funding from Hindustan Unilever Limited (HUL) and Unilever India Pvt. Ltd. (UIPL).

IMPA1 dependent regulation of phosphatidylinositol 4,5-bisphosphate and calcium signalling by lithium.

Lithium (Li) is widely used as a mood stabilizer to treat bipolar affective disorder. However, the molecular targets of Li that underpin its therapeutic effect remain unresolved. Inositol monophosphatase (IMPA1) is an enzyme involved in phosphatidylinositol 4,5-bisphosphate (PIP2) resynthesis after PLC signaling. In vitro, Li inhibits IMPA1, but the relevance of this inhibition within neural cells remains unknown. Here, we report that treatment with therapeutic concentrations of Li reduces receptor-activated calcium release from intracellular stores and delays PIP2 resynthesis. These effects of Li are abrogated in IMPA1 deleted cells. We also observed that in human forebrain cortical neurons, treatment with Li reduced neuronal excitability and calcium signals. After Li treatment of human cortical neurons, transcriptome analyses revealed down-regulation of signaling by glutamate, a key excitatory neurotransmitter in the human brain. Collectively, our findings suggest that inhibition of IMPA1 by Li reduces receptor-activated PLC signaling and neuronal excitability.

PI3P-dependent regulation of cell size and autophagy by phosphatidylinositol 5-phosphate 4-kinase.

Phosphatidylinositol 3-phosphate (PI3P) and phosphatidylinositol 5-phosphate (PI5P) are low-abundance phosphoinositides crucial for key cellular events such as endosomal trafficking and autophagy. Phosphatidylinositol 5-phosphate 4-kinase (PIP4K) is an enzyme that regulates PI5P in vivo but can act on both PI5P and PI3P in vitro. In this study, we report a role for PIP4K in regulating PI3P levels in Drosophila Loss-of-function mutants of the only Drosophila PIP4K gene show reduced cell size in salivary glands. PI3P levels are elevated in dPIP4K 29 and reverting PI3P levels back towards WT, without changes in PI5P levels, can rescue the reduced cell size. dPIP4K 29 mutants also show up-regulation in autophagy and the reduced cell size can be reverted by depleting Atg8a that is required for autophagy. Lastly, increasing PI3P levels in WT can phenocopy the reduction in cell size and associated autophagy up-regulation seen in dPIP4K 29 Thus, our study reports a role for a PIP4K-regulated PI3P pool in the control of autophagy and cell size.

In vitro human stem cell derived cultures to monitor calcium signaling in neuronal development and function.

The development of the human brain involves multiple cellular processes including cell division, migration, and dendritic growth. These processes are triggered by developmental cues and lead to interactions of neurons and glial cells to derive the final complex organization of the brain. Developmental cues are transduced into cellular processes through the action of multiple intracellular second messengers including calcium. Calcium signals in cells are shaped by large number of proteins and mutations in several of these have been reported in human patients with brain disorders.  However, the manner in which such mutations impact human brain development in vivo remains poorly understood. A key limitation in this regard is the need for a model system in which calcium signaling can be studied in neurons of patients with specific brain disorders. Here we describe a protocol to differentiate human neural stem cells into cortical neuronal networks that can be maintained as live cultures up to 120 days in a dish. Our protocol generates a 2D in vitro culture that exhibits molecular features of several layers of the human cerebral cortex. Using fluorescence imaging of intracellular calcium levels, we describe the development of neuronal activity as measured by intracellular calcium transients during development in vitro. These transients were dependent on the activity of voltage gated calcium channels and were abolished by blocking sodium channel activity. Using transcriptome analysis, we describe the full molecular composition of such cultures following differentiation in vitro thus offering an insight into the molecular basis of activity. Our approach will facilitate the understanding of calcium signaling defects during cortical neuron development in patients with specific brain disorders and a mechanistic analysis of these defects using genetic manipulations coupled with cell biological and physiological analysis.

Phosphoinositides: Regulators of Nervous System Function in Health and Disease.

Phosphoinositides, the seven phosphorylated derivatives of phosphatidylinositol have emerged as regulators of key sub-cellular processes such as membrane transport, cytoskeletal function and plasma membrane signaling in eukaryotic cells. All of these processes are also present in the cells that constitute the nervous system of animals and in this setting too, these are likely to tune key aspects of cell biology in relation to the unique structure and function of neurons. Phosphoinositides metabolism and function are mediated by enzymes and proteins that are conserved in evolution, and analysis of knockouts of these in animal models implicate this signaling system in neural function. Most recently, with the advent of human genome analysis, mutations in genes encoding components of the phosphoinositide signaling pathway have been implicated in human diseases although the cell biological basis of disease phenotypes in many cases remains unclear. In this review we evaluate existing evidence for the involvement of phosphoinositide signaling in human nervous system diseases and discuss ways of enhancing our understanding of the role of this pathway in the human nervous system's function in health and disease.

Rewiring of one carbon metabolism in Salmonella serves as an excellent live vaccine against systemic salmonellosis.

Live attenuated vaccines are superior to the killed or subunit vaccines. We designed a Salmonella Typhimurium strain by deleting folD gene (encoding methylenetetrahydrofolate dehydrogenase-cyclohydrolase) in the presence of a heterologous fhs gene (encoding formyltetrahydrofolate synthetase) and tested its vaccine potential under stringent conditions of lethal and sub-lethal challenges with virulent Salmonella in the murine model. The efficacy of the vaccine in conferring protection against Salmonella infection was determined in a wide range of host conditions of systemic infection, corresponding to human young adults, neonates, geriatric age and, importantly, to the immune compromised state of pregnancy. The standardized vaccination regime comprised a primary dose of 104 CFU/animal followed by a booster dose of 102 CFU/animal on day 7. Challenge with the virulent pathogen was done at day 7 post-administration of the booster. Subsequently, the mortality, morbidity, systemic colonization, antibody response and cytokine profiling were determined. The vaccinated cohort showed a strong protection against virulent pathogen in all models tested. The serum anti-Salmonella antibody titers and cytokine levels were significantly higher in the vaccinated cohort compared to the mock vaccinated cohort. Thus, we report the development and validation of a live attenuated vaccine candidate conferring excellent protection against Salmonellosis and typhoid fever.