Aging reshapes the CD8⁺ T cell compartment through contraction of the naïve pool and expansion of memory-phenotype populations, including the progressive accumulation of antigen-inexperienced virtual memory (VM) CD8⁺ T cells. VM CD8⁺ T cells arise in the absence of foreign antigen priming and acquire a memory-like phenotype. In aged hosts, VM CD8⁺ T cells constitute a substantial fraction of the antigen-inexperienced memory-phenotype CD8⁺ T cell pool, and their differentiation is shaped by self-reactivity, homeostatic cytokines, and transcriptional programs linked to IL-15 and EOMES. During aging, VM CD8⁺ T cells accumulate numerically, display altered proliferative responses, retain sensitivity to homeostatic and inflammatory cytokines, and can preserve effector function during infection. These features suggest that VM CD8⁺ T cells may partially compensate for the age-associated loss of naïve CD8⁺ T cells by providing rapid effector-like responses. At the same time, aged VM CD8⁺ T cells appear to comprise heterogeneous subsets, including populations with cytotoxic and inflammatory potential. Emerging evidence suggests that inhibitory receptors may mark distinct VM states during aging, although their functional significance and potential regulatory role remain to be established.

Clostridioides difficile is a leading cause of hospital-acquired enteric infections, with outcomes ranging from mild diarrhea to life-threatening pseudomembranous colitis and death. Dietary modulations can influence the course of antibiotic-induced C. difficile infection (CDI), and high-fat diets are associated with increased mortality. Here, we profiled host innate immune cells, cecal microbial composition, and the host transcriptome of normal diet (ND) and high-fat diet (HFD)-fed C57BL/6 male and female mice following experimental CDI during the acute and recovery phases. HFD-fed male mice exhibited delayed disease onset but increased mortality following CDI. During acute infection, HFD-fed males showed increased monocyte infiltration and expansion of CD103+CD11b- and CD103-CD11b+ dendritic cell subsets; transcriptomic modules were enriched for antigen presentation and signatures consistent with T cell activation, coincident with enrichment of Escherichia and Lactococcus. In contrast, during recovery, HFD-fed males and females displayed sustained neutrophil and monocyte infiltration, depletion of eosinophils (prominent in males), tissue-resident Tim4+/CD206+ macrophage subsets and ILC3s, increased expression of Tlr4 and endothelial activation markers, downregulation of metabolic pathways, and failure to restore cecal microbial diversity. Collectively, these data indicate that high-fat feeding disrupts resolution programs after CDI and promotes progression to severe disease, with the most pronounced susceptibility in males.

Aging and obesity are associated with pro-inflammatory changes in adipose tissue. Overlapping mechanisms, such as the infiltration of inflammatory macrophages and T cells into visceral adipose tissue, have been implicated in contributing to inflammation. However, a comparative analysis of both states is needed to identify distinct regulatory targets. Here, we performed single-cell RNA sequencing of stromal vascular fractions (SVF) isolated from gonadal white adipose tissue (gWAT) of young mice fed either a normal or a high-fat diet, and aged mice fed a normal diet. Our analysis revealed that physiological aging, compared to high-fat diet-induced obesity, was associated with an accumulation of phenotypically distinct CD8 T cells resembling virtual memory (VM) CD8 T cells. These cells expressed high levels of Cd44, Sell, Il7r, Il2rb, lacked Itga4, and exhibited elevated Fcgr2b expression which was associated with pseudotime differentiation trajectories. Flow cytometry confirmed an age-associated increase in Fcgr2b + CD49d- VM-like CD8 T cells in gWAT. Notably, these Fcgr2b-expressing cells exhibited a cytotoxic profile and expressed granzyme M. Functional analysis using recombinant granzyme M revealed its potential in inducing inflammation in mouse fibroblasts and macrophages. Together, our study has identified Fcgr2b + CD49d- VM-like CD8 T cells in the adipose tissue of aged mice with regulatory, cytotoxic, and inflammatory potential.

Aging and obesity have been linked to driving inflammatory changes in adipose tissue. Similar mechanisms, such as the infiltration of inflammatory macrophages and T cells into visceral adipose tissue, have been suggested to contribute to these inflammatory changes. Recent studies have identified exhausted PD1+ CD8 T cells in the visceral adipose tissue of both aged and obese mice suggested to be involved in the inflammation. In our study, we used single-cell sequencing to explore the heterogeneity of visceral adipose tissue during aging compared to obesity. We isolated stromal vascular fractions (SVF) from the gonadal white adipose tissue of young (4–5 months) mice fed either a normal or a high-fat diet, and from aged (21–22 months) mice fed a normal diet. Our observations revealed accumulation of B cells and T cells in the SVF of aged mice. Specifically, CD8 T cells from the SVF exhibited increased incoming signaling from various cells (high number of Ligand-receptor pairs), which was more pronounced in aged mice compared to those fed a high-fat or normal diet. Further investigation of CD8 T cells showed the accumulation of previously defined age-associated PD1+ Gzmk+ CD8 T cells, along with CD44+CD62L+CD122+CD49d- central/virtual memory phenotype in the adipose tissue of aged mice. The expression profiles of these cells revealed the presence of different inhibitory receptors among various memory CD8 T cells, potentially affecting the functionality of the CD8 T memory cell. Supported by NIAID R01AI162787 Cellular Adhesion, Migration, and Inflammation (CAM)

Abstract Background Clostridioides difficile (C. difficile) infection (CDI) is a leading cause of nosocomial infection that can range from mild diarrhea to life threatening pseudomembranous colitis and death. Aging as well as defects in the host immune response including failure to develop antibody response against C. difficile antigens increases the risk of bacterial colonization, hospitalization, mortality and developing recurrent CDI. The present study aimed to investigate the differences in the B cell response between young and aged C. difficile infected mice. Methods C57BL/6 mice were rendered susceptible to CDI via the administration of oral cefoperazone for 10 days and then the mice were challenged with 103 spores of C. difficile R20291 strain (Figure 1). The young (3-4 months) and aged (22-24 months) mice were euthanized at 14 days post infection (dpi). The germinal center response and IgA+ antibody-secreting cells (ASCs) were studied using multicolor flow cytometry. Results C. difficile infected aged mice exhibited greater weight loss at 4 dpi compared to young C. difficile infected mice. The gut germinal center response studied at 14 dpi showed mild changes in T follicular helper cells (CXCR5+PD-1+ of CD4+) and germinal center B cells (GL7+CD95+ of CD19+B200+) of young C. difficile infected mice, whereas the response was dampened in the aged C. difficile infected mice (Figure 2). Further, at 14 dpi ASCs (CD138+CD98+) were seen in the lamina propria of the cecum and colon of the C. difficile infected mice, but the frequencies of CD138+CD98+ ASCs as well as IgA+ ASCs were significantly lower in the C. difficile infected aged mice compare to young mice (Figure 3). Conclusion The defect in B cell response during CDI may be responsible for more severe infection and recurrent CDI among aged individual compared to young individuals. It remains to be seen if age-related changes impact antibody production and whether the antibody production during primary CDI provides long-term immunity. Disclosures Vincent B. Young, MD, PhD, ASM: Senior Editor for mSphere Journal|Debiopharm: Consultant|mSphere: Senior Editor|Vendanta Biosciences: Consultant

Dectin-1 is an innate immune receptor that recognizes and binds β-1,3/1,6 glucans on fungi. We evaluated Dectin-1 function in myeloid cells in a cohort of HIV-positive and HIV-negative young and older adults. Stimulation of monocytes with β-D-glucans induced a pro-inflammatory phenotype in monocytes of HIV-infected individuals that was characterized by increased levels of IL-12, TNF-α, and IL-6, with some age-associated cytokine increases also noted. Dendritic cells showed a striking HIV-associated increase in IFN-α production. These increases in cytokine production paralleled increases in Dectin-1 surface expression in both monocytes and dendritic cells that were noted with both HIV and aging. Differential gene expression analysis showed that HIV-positive older adults had a distinct gene signature compared to other cohorts characterized by a robust TNF-α and coagulation response (increased at baseline), a persistent IFN-α and IFN-γ response, and an activated dendritic cell signature/M1 macrophage signature upon Dectin-1 stimulation. Dectin-1 stimulation induced a strong upregulation of MTORC1 signaling in all cohorts, although increased in the HIV-Older cohort (stimulation and baseline). Overall, our study demonstrates that the HIV Aging population has a distinct immune signature in response to Dectin-1 stimulation. This signature may contribute to the pro-inflammatory environment that is associated with HIV and Aging.

Abstract Background Dectin-1 is an innate immune pattern recognition receptor that recognizes and binds b-1,3/1,6 glucans on fungal pathogens. Methods We evaluated Dectin-1 function in innate immune cells (Monocytes and Dendritic cells) in a cohort of HIV-positive and HIV-negative young and older adults (n= 81). PBMCs were stimulated with whole glucan particles (1,3/1,6-b-glucan, WGP), a specific Dectin-1 agonist and analyzed via intracellular cytokine staining (ICS) and multicolor flow cytometry of monocytes and dendritic cells. Sorted CD14+ CD16+ monocytes stimulated with WGP and subjected to RNA-seq analysis. Results Stimulation of monocytes with β-D-glucans induced a pro-inflammatory phenotype in monocytes of HIV-infected individuals that was characterized by increased levels of IL-12, TNF-a, and IL-6, with some age-associated cytokine increases also noted. Dendritic cells showed a striking HIV-associated increase in IFN-a production. This increase in cytokine production paralleled increases in Dectin-1 surface expression in both monocytes and dendritic cells that were noted with both HIV and aging. Differential gene expression analysis showed that HIV-positive older adults had a distinct gene signature compared to other cohorts characterized by a robust TNF-a and coagulation response that was already increased at baseline, and a persistent IFN-α and IFN-γ response. HIV-older adults also demonstrated an activated dendritic cell signature and M1 macrophage signature upon stimulation that was not seen in young individuals. Pathways upregulated in all cohorts were the signaling pathways MTORC-1, hypoxia pathway (pathways involved in trained immunity) and the unfolded protein response. Of note MTORC-1 signaling was upregulated at baseline in HIV-Older adults. Conclusion Overall, our study demonstrates that increased age and HIV-infection can affect the function of Dectin-1 and lead to a distinct inflammatory signature. Dectin-1 stimulation in HIV-Older adults led to a more pro-inflammatory phenotype that was demonstrated through both protein and gene expression. Dectin-1 induced inflammation in HIV-infected individuals may be contributing to the pro-inflammatory environment that is seen in the setting of both aging and HIV-infection. Disclosures All Authors: No reported disclosures.

Nipah virus (NiV) is an emerging and priority pathogen from the Paramyxoviridae family with a high fatality rate. It causes various diseases such as respiratory ailments and encephalitis and poses a great threat to humans and livestock. Despite various efforts, there is no approved antiviral treatment available. Therefore, to expedite and assist the research, we have developed an integrative resource NipahVR (http://bioinfo.imtech.res.in/manojk/nipahvr/) for the multi-targeted putative therapeutics and epitopes for NiV. It is structured into different sections, i.e. genomes, codon usage, phylogenomics, molecular diagnostic primers, therapeutics (siRNAs, sgRNAs, miRNAs) and vaccine epitopes (Bcell, CTL, MHC-I and -II binders). Most decisively, potentially efficient therapeutic regimens targeting different NiV proteins and genes were anticipated and projected. We hope this computational resource would be helpful in developing combating strategies against this deadly pathogen. Database URL: http://bioinfo.imtech.res.in/manojk/nipahvr/

Viruses are responsible for causing various epidemics and pandemics with a high mortality rate e.g. ongoing SARS-CoronaVirus-2 crisis. The discovery of novel antivirals remains a challenge but drug repurposing is emerging as a potential solution to develop antivirals in a cost-effective manner. In this regard, we collated the information of repurposed drugs tested for antiviral activity from literature and presented it in the form of a user-friendly web server named 'DrugRepV'. The database contains 8485 entries (3448 unique) with biological, chemical, clinical and structural information of 23 viruses responsible to cause epidemics/pandemics. The database harbors browse and search options to explore the repurposed drug entries. The data can be explored by some important fields like drugs, viruses, drug targets, clinical trials, assays, etc. For summarizing the data, we provide overall statistics of the repurposed candidates. To make the database more informative, it is hyperlinked to various external repositories like DrugBank, PubChem, NCBI-Taxonomy, Clinicaltrials.gov, World Health Organization and many more. 'DrugRepV' database (https://bioinfo.imtech.res.in/manojk/drugrepv/) would be highly useful to the research community working to develop antivirals.

Background The increasing antimalarial drug resistance is a significant hindrance to malaria control and elimination programs. For the last six decades, chloroquine (CQ) plus pyrimethamine remains the first-line treatment for P. vivax malaria. Regions where both P. falciparum and P. vivax co-exist, P. vivax is exposed to antifolate drugs due to either misdiagnosis or improper treatment that causes selective drug pressure to evolve. Therefore, the present study aims to estimate antimalarial drug resistance among the complicated and uncomplicated P. vivax patients. Methods A total of 143 P. vivax malaria positive patients were enrolled in this study, and DNA was isolated from their blood samples. Pvcrt-o, Pvmdr-1, Pvdhps, and Pvdhfr genes were PCRs amplified, and drug resistance-associated gene mutations were analyzed. Statistical analysis of the drug resistance genes and population diversity was performed using MEGA vs. 7.0.21 and DnaSP v software. Results Among the CQ resistance marker gene Pvcrt-o, the prevalence of K10 insertion was 17.5% (7/40) and 9.5% (7/73) of complicated and uncomplicated P vivax group isolates respectively. In Pvmdr-1, double mutant haplotype ( M 958 /L 1076 ) was found in 99% of the clinical isolates. Among the pyrimethamine resistance-associated gene Pvdhfr, the double mutant haplotype I 13 P 33 F 57 R 58 T 61 N 117 I 173 was detected in 23% (11/48) in complicated and 20% (17/85) in uncomplicated group isolates. In the sulphadoxine resistance-associated Pvdhps gene, limited polymorphism was observed with the presence of a single mutant (D459A) among 16 and 5% of the clinical isolates in the complicated and uncomplicated group respectively. Conclusion The study presents the situations of polymorphism in the antimalarial drug resistance-associated genes and emphasizes the need for regular surveillance. It is imperative for the development of suitable antimalarial drug policy in India.

•Scrub typhus shows a great diversity in its strains over a large geographical area of north India.•Most common strain in north India is Karp, followed by Gilliam.•Kato-like strains found in South of India were not found in the present study.

Introduction: Varicella outbreaks are known to occur in developing nations as vaccine coverage is still low. Material and Methods: In the present study, an institutional outbreak from Chandigarh, India, is reported wherein the utility of non-invasive samples such as saliva and urine was studied for the molecular diagnosis of varicella by conventional polymerase chain reaction (PCR), real-time PCR and real-time loop-mediated isothermal amplification (real-time LAMP). Results: The results of the present study showed that saliva and urine samples can be used for outbreak investigation of varicella compared to varicella-zoster virus DNA in vesicular swab samples with reasonable sensitivity. Conclusion: Thus, molecular techniques may be useful in the early identification of the outbreak and timely isolation, and the treatment of cases can further prevent its spread.

Nipah virus (NiV) caused several outbreaks in Asian countries including the latest one from Kerala state of India. There is no drug available against NiV till now, despite its urgent requirement. In the current study, we have provided a computational one-stop solution for NiV inhibitors. We have developed the first “anti-Nipah” web resource, which comprising of a data repository, prediction method, and data visualization module. The database contains of 313 (181 unique) chemicals extracted from research articles and patents, which were tested for different strains of NiV isolated from various outbreaks. Moreover, the quantitative structure–activity relationship (QSAR) based regression predictors were developed using chemicals having half maximal inhibitory concentration (IC50). Predictive models were accomplished using support vector machine employing 10-fold cross validation technique. The overall predictor showed the Pearson's correlation coefficient of 0.82 on training/testing dataset. Likewise, it also performed equally well on the independent validation dataset. The robustness of the predictive model was confirmed by applicability domain (William's plot) and scatter plot between actual and predicted efficiencies. Further, the data visualization module from chemical clustering analysis displayed the diversity in the NiV inhibitors. Therefore, this web platform would be of immense help to the researchers working in developing effective inhibitors against NiV. The user-friendly web server is freely available on URL: http://bioinfo.imtech.res.in/manojk/antinipah/.

BACKGROUND:Malaria is one of the important vector-borne diseases with high fatality rates in tropical countries. The pattern of emergence and spread of novel antigenic variants, leading to escape of vaccine-induced immunity might be factors responsible for severe malaria. A high level of polymorphism has been reported among malarial antigens which are under selection pressure imposed by host immunity. There are limited reports available on comparative stage-specific genetic diversity among Plasmodium vivax candidate genes in complicated vivax malaria. The present study was planned to study genetic diversity (Pvcsp and Pvs25) among complicated and uncomplicated P. vivax isolates.METHODS:Pvcsp and Pvs2-specific PCRs and DNA sequencing were performed on P. vivax PCR positive samples. Genetic diversity was analysed using appropriate software.RESULTS:The present study was carried out on 143 P. vivax clinical isolates, collected from Postgraduate Institute of Medical Education and Research, Chandigarh. Among the classic and variant types of Pvcsp, the VK210 (99%; 115/116) was found to be predominant in both complicated and uncomplicated group isolates. Out of the various peptide repeat motifs (PRMs) observed, GDRADGQPA (PRM1) and GDRAAGQPA (PRM2) was the most widely distributed among the P. vivax isolates. Whereas among the Pvs25 isolates, 100% of double mutants (E97Q/I130T) in both the complicated (45/45) as well as in the uncomplicated (81/81) group was observed.CONCLUSION:An analysis of genetic variability enables an understanding of the role of genetic variants in severe vivax malaria.

Nipah virus (NiV) is responsible to cause various outbreaks in Asian countries, with latest from Kerala state of India. Till date there is no drug available despite its urgent requirement. In the current study, we have provided a computational one-stop solution for NiV inhibitors. We have developed “anti-Nipah” web resource, which comprised of a data repository, prediction method, and data visualization modules. The database comprised of 313 (181 unique) inhibitors from different strains and outbreaks of NiV extracted from research articles and patents. However, the quantitative structure–activity relationship (QSAR) based predictors were accomplished using classification approach employing 10-fold cross validation through support vector machine with 120 (68p + 52n) inhibitors. The overall predictor showed the accuracy and Matthew’s correlation coefficient of 88.89% and 0.77 on training/testing dataset respectively. The independent validation dataset also performed equally well. The data visualization modules from chemical clustering and principal component analyses displayed the diversity in the NiV inhibitors. Therefore, our web platform would be of immense help to the researchers working in developing effective inhibitors against NiV. The user-friendly webserver is freely available on URL: http://bioinfo.imtech.res.in/manojk/antinipah/

Human adenovirus (HAdV) is a major cause of viral conjunctivitis. The various serotypes implicated in the causation are 3, 4, 8, 19 and 37. The present study aimed to know the circulating types of HAdV causing acute conjunctivitis in North India. A total of 23 conjunctival swabs were collected from patients with clinically suspected acute viral conjunctivitis during 2014-2015. The HAdV was implicated in the etiology in 65.2% of cases. The sequencing of representative samples using hexon gene suggests the presence of serotype 8 and 4. The serotype eight sequences showed 99%-100% similarity with other Indian strains. The phylogenetic analysis showed that the current circulating serotypes, responsible for conjunctivitis, belonged to epidemic keratoconjunctivitis strains.

The protective role of vitamin D supplementation has recently been shown to be present in various ocular inflammatory diseases. The oral supplementation of vitamin D may take time to achieve adequate levels in intraocular fluids. Therefore, the present study was performed to understand the ocular pharmacokinetics of 25-hydroxyvitamin D3 (25D3) in aqueous humor after weekly supplementation of 25D3 in rabbits.

In the recent years Plasmodium vivax has been reported to cause severe infections associated with mortality. Clinical evaluation has limited accuracy for the early identification of the patients progressing towards the fatal condition. Researchers have tried to identify the serum and the plasma-based indicators of the severe malaria. Discovery of MicroRNA (miRNA) has opened up an era of identification of early biomarkers for various infectious and non-infectious diseases. MicroRNAs (miRNA) are the small non-coding RNA molecules of length 19–24 nts and are responsible for the regulation of the majority of human gene expressions at post transcriptional level.

The antiviral and immunomodulatory role of vitamin D has been shown in various viral infections. However, there is scanty literature available about the effect of vitamin D supplementation in herpes simplex virus-1 (HSV-1) infection. Therefore, the present study aimed to evaluate the role of two different forms of vitamin D: 25-hydroxyvitamin D3 (25D3) and 1,25-dihydroxyvitamin D3 (1,25D3) against HSV-1 in HeLa cells. The HeLa cells were supplemented with either 25D3 or 1,25D3 before HSV-1 infection and were studied after 6, 12, and 24 h postinfection (p.i.). The mRNA levels of toll-like receptors (TLRs), (2, 3, 4, 7, and 9), vitamin D signaling genes, and HSV-1 were studied using real-time PCR. The HSV-1 DNA load was estimated in culture supernatant. The supplementation of 25D3 and 1,25D3 significantly downregulated the mRNA levels of TLR2 (p < 0.0001) at 12 h p.i. The mRNA levels of TLR9 were found to be significantly downregulated in 1,25D3-supplemented cells at 12 h p.i. Furthermore, the significant downregulation was observed in HSV-1 titer in both 25D3- and 1,25D3-supplemented cells at 24 h p.i.(p < 0.0001). However, the effect of 25D3 supplementation persisted till 24 h p.i. with significant downregulation of TLR2 (p < 0.05) mRNA levels. The supplementation of both 25D3 and 1,25D3 before HSV-1 infection was found to downregulate the viral titer and TLR2 mRNA during the intial phase of infection. However, the effect of 25D3 supplementation was found to last for a longer duration compared with 1,25D3.