Equine immunoglobulin fragment F(ab')2 displays high neutralizing capability against multiple SARS-CoV-2 variants.

Neutralizing antibody-based passive immunotherapy could be an important therapeutic option against COVID-19. Herein, we demonstrate that equines hyper-immunized with chemically inactivated SARS-CoV-2 elicited high antibody titers with a strong virus-neutralizing potential, and F(ab')2 fragments purified from them displayed strong neutralization potential against five different SARS-CoV-2 variants. F(ab')2 fragments purified from the plasma of hyperimmunized horses showed high antigen-specific affinity. Experiments in rabbits suggested that the F(ab')2 displays a linear pharmacokinetics with approximate plasma half-life of 47 h. In vitro microneutralization assays using the purified F(ab')2 displayed high neutralization titers against five different variants of SARS-CoV-2 including the Delta variant, demonstrating its potential efficacy against the emerging viral variants. In conclusion, this study demonstrates that F(ab')2 generated against SARS-CoV-2 in equines have high neutralization titers and have broad target-range against the evolving variants, making passive immunotherapy a potential regimen against the existing and evolving SARS-CoV-2 variants in combating COVID-19.

Milk exosomes elicit a potent anti-viral activity against dengue virus.

BACKGROUND: Exosomes are nano-sized vesicles secreted by various cells into the intra and extracellular space and hence is an integral part of biological fluids including milk. In the last few decades, many research groups have proved the potential of milk exosomes as a sustainable, economical and non-immunogenic drug delivery and therapeutic agent against different pathological conditions. However, its anti-viral properties still remain to be unearthed. METHODS: Here, we have been able to isolate, purify and characterize the milk derived exosomes from Cow (CME) and Goat (GME) and further studied its antiviral properties against Dengue virus (DENV), Newcastle Disease Virus strain Komarov (NDV-K) and Human Immunodeficiency Virus (HIV-1) using an in-vitro infection system. RESULTS: TEM, NTA and DLS analysis validated the appropriate size of the isolated cow and goat milk exosomes (30-150 nm). Real-time PCR and immunoblotting results confirmed the presence of several milk exosomal miRNAs and protein markers. Our findings suggest that GME significantly decreased the infectivity of DENV. In addition, we confirmed that GME significantly reduces DENV replication and reduced the secretion of mature virions. Furthermore, heat inactivation of GME did not show any inhibition on DENV infection, replication, and secretion of mature virions. RNase treatment of GME abrogates the anti-viral properties indicating direct role of exosomes in DENV inhibition. In addition GME inhibited the infectivity of NDV-K, but not HIV-1, suggesting that the GME mediated antiviral activity might be virus specific. CONCLUSION: This study demonstrates the anti-viral properties of milk exosomes and opens new avenues for the development of exosome-based therapies to treat viral diseases.

Amino acid starvation sensing dampens IL-1ß production by activating riboclustering and autophagy.

Activation of the amino acid starvation response (AAR) increases lifespan and acute stress resistance as well as regulates inflammation. However, the underlying mechanisms remain unclear. Here, we show that activation of AAR pharmacologically by Halofuginone (HF) significantly inhibits production of the proinflammatory cytokine interleukin 1ß (IL-1ß) and provides protection from intestinal inflammation in mice. HF inhibits IL-1ß through general control nonderepressible 2 kinase (GCN2)-dependent activation of the cytoprotective integrated stress response (ISR) pathway, resulting in rerouting of IL-1ß mRNA from translationally active polysomes to inactive ribocluster complexes-such as stress granules (SGs)-via recruitment of RNA-binding proteins (RBPs) T cell-restricted intracellular antigen-1(TIA-1)/TIA-1-related (TIAR), which are further cleared through induction of autophagy. GCN2 ablation resulted in reduced autophagy and SG formation, which is inversely correlated with IL-1ß production. Furthermore, HF diminishes inflammasome activation through suppression of reactive oxygen species (ROS) production. Our study unveils a novel mechanism by which IL-1ß is regulated by AAR and further suggests that administration of HF might offer an effective therapeutic intervention against inflammatory diseases.

Activation of integrated stress response pathway regulates IL-1ß production through posttranscriptional and translational reprogramming in macrophages.

Immune cells sense and programme its cellular machinery appropriately to the environmental changes through the activation of cytoprotective adaptive pathway so-called the "integrated stress response (ISR)". However, the mechanisms implicated in ISR-induced protective responses are poorly understood. Here, we show that ISR activation by arsenite (Ar) results in suppression of IL-1ß production in macrophages and inhibition of DSS-induced colitis in a murine model through a novel posttranscriptional and translation regulatory (PTR) mechanism. Ar triggers PTR events through eIF2α-phosphorylation, which results in the attenuation of active polysome formation leading to the accumulation of translationally stalled IL-1ß mRNAs. Translationally stalled IL-1ß mRNAs recruit RNA-binding proteins (TIA-1/TIAR), resulting in the formation of RBP-RNA complexes known as stress granules (SGs). The SGs bound IL-1ß mRNAs might undergo degradation through induction of autophagy. Also, we show that Ar posttranslationally impairs processing and secretion of IL-1ß by diminishing inflammasome activation. Altogether, this study unveils a novel mechanism of IL-1ß regulation and further suggests that pharmacological activation of cytoprotective ISR pathway might provide an effective therapeutic intervention against inflammatory diseases.

Dengue virus envelope protein domain III induces pro-inflammatory signature and triggers activation of inflammasome.

Dengue virus poses a considerable clinical problem, with the four closely related serotypes of dengue virus (DENV) infecting around 50-100 million people per year world-wide. The drastic increase in the dengue infection could be partly attributed to geographic expansion of the vector due to increasing urbanization, unavailability of specific antiviral therapies, licensed dengue vaccine, and poor understanding of the host immune responses. It has been reported that the immune-dominant envelope protein (E protein) domain III region (EDIII) of DENV is one of the most potent vaccine candidates because of its ability to trigger host immunity by inducing production of protective neutralizing antibodies. However, its role in the modulation of innate inflammatory responses hitherto remains unexplored. Herein, we demonstrate that EDIII protein of DENV induces pro-inflammatory signature by inducing production of inflammatory cytokines such as IL-1ß and TNF-α in THP-1 cells through NF-κB pathway. Also, we observed increase in the maturation of IL-1ß, which was found to be associated with increased ROS production and potassium efflux. Further, our findings reveal that the IL-1ß production by EDIII protein is mediated through caspase-1 and NLRP3 inflammasome activation. In conclusion this study unearths the role of DENV EDIII protein in modulating innate inflammatory responses, which might provide possible mechanism of pathogenesis and open-up new avenues for the development of therapeutics against DENV.

Transcriptome meta-analysis identifies immune signature comprising of RNA binding proteins in ulcerative colitis patients.

Ulcerative colitis (UC) is a persistent inflammatory illness, which is clinically categorised as Inflammatory bowel disease (IBD), affecting millions of people worldwide. The precise cause behind the pathology of the disease remains unknown. However, the involvement of multiple factors including genetic predisposition, immunological deregulations, microbiota imbalance, and environmental triggers has been suggested. Amongst all these factors, the over-active immunological response reported in UC patients seems to be a promising target for therapy. Moreover, identification of gene signatures associated with disease onset and progression would help in better understanding of the molecular mechanisms involved in the disease pathogenesis. Here, we have conducted meta-analysis of gene expression profiles of UC patient microarray datasets accessible in public databases and further validated the in-silico findings in UC patients' blood samples. Our study reveals that UC pathogenesis perturbs expression of several inflammatory genes. In addition, we report a novel gene signature comprising of TIA1 (T cell restricted intracellular antigen) and TIAR (TIA1 related protein; also known as TIAL1), which were found to be significantly downregulated in UC patients. TIA1 and TIAR are RNA-binding proteins (RBPs), which function as a translational represser by binding to ARE sequences in the 3' UTR of mRNAs encoding inflammatory mediators including cytokines. Our findings demonstrate that deletion of TIAR using gene specific siRNAs in-vitro results in enhanced production of inflammatory cytokine IL-1ß. In conclusion, the findings of this study reveal that down regulation of TIA1/TIAR genes could be responsible for UC associated inflammation. This study highlights the usefulness of the meta-analysis approach in the identification of unique gene signatures that might deliver mechanistic insights into UC pathogenesis and possibly assist in discovery of prognostic markers and therapeutic interventions.

Inadequate structural constraint on Fab approach rather than paratope elicitation limits HIV-1 MPER vaccine utility.

Broadly neutralizing antibodies (bnAbs) against HIV-1 target conserved epitopes, thereby inhibiting viral entry. Yet surprisingly, those recognizing linear epitopes in the HIV-1 gp41 membrane proximal external region (MPER) are elicited neither by peptide nor protein scaffold vaccines. Here, we observe that while Abs generated by MPER/liposome vaccines may exhibit human bnAb-like paratopes, B-cell programming without constraints imposed by the gp160 ectodomain selects Abs unable to access the MPER within its native "crawlspace". During natural infection, the flexible hinge of IgG3 partially mitigates steric occlusion of less pliable IgG1 subclass Abs with identical MPER specificity, until affinity maturation refines entry mechanisms. The IgG3 subclass maintains B-cell competitiveness, exploiting bivalent ligation resulting from greater intramolecular Fab arm length, offsetting weak antibody affinity. These findings suggest future immunization strategies.

Inadequate structural constraint on Fab approach rather than paratope elicitation limits HIV-1 MPER vaccine utility.

Broadly neutralizing antibodies (bnAbs) against HIV-1 target conserved envelope (Env) epitopes to block viral replication. Here, using structural analyses, we provide evidence to explain why a vaccine targeting the membrane-proximal external region (MPER) of HIV-1 elicits antibodies with human bnAb-like paratopes paradoxically unable to bind HIV-1. Unlike in natural infection, vaccination with MPER/liposomes lacks a necessary structure-based constraint to select for antibodies with an adequate approach angle. Consequently, the resulting Abs cannot physically access the MPER crawlspace on the virion surface. By studying naturally arising Abs, we further reveal that flexibility of the human IgG3 hinge mitigates the epitope inaccessibility and additionally facilitates Env spike protein crosslinking. Our results suggest that generation of IgG3 subtype class-switched B cells is a strategy for anti-MPER bnAb induction. Moreover, the findings illustrate the need to incorporate topological features of the target epitope in immunogen design.

Tetravalent formulation of polymeric nanoparticle-based vaccine induces a potent immune response against dengue virus.

Dengue is a mosquito-borne disease caused by the four serotypes of the dengue virus (DENV 1-4). It is growing at an alarming rate globally, which could be partly attributed to the lack of an effective therapeutic regimen. Therefore, strategies for developing an effective vaccine have gained more significance in the given scenario. Failure of the existing live attenuated vaccine candidates to mount effective and broader protection against all the four serotypes of DENV has generated a new interest in exploring novel strategies for augmenting the efficacy of non-infectious, non-replicating subunit vaccines. In the current study, we employed a new strategy of encapsulating the immunodominant EDIII domain of Envelop protein of all the serotypes of DENV (1-4) into PLGA nanoparticles separately. All four nano formulations were physically mixed to develop a tetravalent nano formulation in combination with TLR agonists. Further, we examined its immunological efficacy using a mouse and in vitro infection model system. Interestingly, our results demonstrate that majority of EDIII protein loaded PLGA nanoparticles were polydispersed and less than 1 µm in size with optimal encapsulation efficacy. Tetravalent nanoformulation along with TLR agonists (MPLA + R837) enhanced the magnitude of antigen-specific polyfunctional T cell response. It triggered robust antibody responses in mice concurrent with the increased level of genes involved in the programming of memory B-cell formation and the maintenance and maturation of GCs, leading to the formation of long-lived plasma cells secreting antigen-specific antibodies. Further assessment revealed that tetravalent nanoformulation in combination with TLR ligands upon immunization in mice aids in the enhanced production of serotype-specific neutralizing antibodies, which can effectively neutralize all the four serotypes of DENV (DENV 1-4). The findings of this study reveal a new strategy for enhancing the immunogenicity of vaccine candidates and might pave the way for the development of a tetravalent vaccine against all the serotypes of Dengue Virus.

Immunogenic profiling of Mycobacterium tuberculosis DosR protein Rv0569 reveals its ability to switch on Th1 based immunity.

Mycobacterium tuberculosis (M.tb) is a multifaceted bacterial pathogen known to infect more than 2 billion people globally. However, a majority of the individuals (>90%) show no overt clinical symptoms of active Tuberculosis (TB) and, it is reported that M.tb in these individuals resides in the latent form. Therefore, a huge burden of latently infected population poses serious threat to the human health. Inconsistent efficacy of BCG vaccine and poor understanding of latency-associated determinants contribute to the failure of combating M.tb. The discovery of DosR as the master regulator of dormancy, opened new avenues to understand the pathophysiology of the bacterium. Though the specific functions of various DosR genes are yet to be discovered, they have been reported as potent T-cell activators and could elicit strong protective immune responses. Rv0569 is a DosR-encoded conserved hypothetical protein overexpressed during dormancy. However, it is not clearly understood how this protein modulates the host immune response. In the present study, we have demonstrated that Rv0569 has a high antigenic index and induces enhanced secretion of Th1 cytokines IL-12p40 and TNF-α as compared to Th2 cytokine IL-10 in macrophages. Mechanistically, Rv0569 induced the transcription of these pro-inflammatory signatures through the activation of NF-κB pathway. Further, immunization of mice with DosR protein Rv0569 switched the immune response towards Th1-biased cytokine pattern, characterized by the enhanced production of IFN-γ, IL-12p40, and TNF-α. Rv0569 augmented the expansion of antigen-specific IFN-γ and IL-2 producing effector CD4+and CD8+ T-cells which are hallmarks of Th1 biased protective immunity. Additionally, IgG2a/IgG1 and IgG2b/IgG1 ratio in the serum of immunized mice further confirmed the ability of Rv0569 to skew Th1 biased immune response. In conclusion, we emphasize that Rv0569 has the ability to generate signals to switch on Th1-dominated responses and further suggest that it could be a potential vaccine candidate against latent M.tb infection.

Light-triggered selective ROS-dependent autophagy by bioactive nanoliposomes for efficient cancer theranostics.

Light-responsive nanoliposomes are being reported to induce cancer cell death through heat and reactive oxygen species (ROS). Nanoliposomes (CIR NLPs) encapsulating a near-infrared (NIR) light-sensitive dye, IR780, and a bioactive chlorophyll-rich fraction of Anthocephalus cadamba (CfAc) were synthesized and characterized. These CIR NLPs, when activated by NIR light, displayed localized synergistic cancer cell death under in vitro and in vivo conditions. We demonstrated a NIR light-mediated release of CfAc in cancer cells. The bioactive CfAc was selective in causing ROS generation (leading to autophagic cell death) in cancer cells, while normal healthy cells were unaffected. An increase in the intracellular ROS leading to enhanced lipidation of microtubule-associated protein light chain 3 (LC3-II) was observed only in cancer cells, while normal cells showed no increase in either ROS or LC3-II. In vivo analysis of CIR NLPs in an orthotopic mouse model showed better anti-tumorigenic potential through a combined effect (i.e. via heat and CfAc). We reported for the first time induction of selective and localized, bioactive phyto fraction-mediated autophagic cancer cell death through an NIR light trigger. The synergistic activation of ROS-mediated autophagy by light-triggered nanoliposomes can be a useful strategy for enhancing the anticancer potential of combinational therapies.

Detection of atherosclerosis by ankle brachial index: evaluation of palpatory method versus ultrasound Doppler technique.

BACKGROUND: Peripheral Arterial Disease [PAD], a manifestation of systemic atherosclerosis, is highly prevalent both in community studies and in primary care practice. Estimation of ankle brachial index [ABI] by ultrasound Doppler is the standard screening method for the detection of atherosclerosis in PAD patients. A low ABI is associated with increased risk of stroke or transient ischemic attack, ischemic heart disease and lower extremity gangrene. Though prevalence is high, physician and patient awareness of the condition is low. Primary care physicians are not well versed with the use of Doppler. Initial cost of the equipment is another adverse factor in low income countries. Detection of ankle systolic pressure by palpatory method may offer a cheap, simple and useful alternative approach in office care settings for early detection of disease. This may lead to the use of risk reduction strategies to avoid significant future morbidity and mortality. METHODS: The sample size of 230 participants was identified. Patients were divided into control (100 cases), high risk asymptomatic (100 cases) and symptomatic groups (30 cases). Ankle systolic pressure was measured by digital palpation of foot arteries and by the gold standard Doppler technique in all patients in the three groups. PAD was defined by an ABI of < 0.9. RESULTS: In control group, mean +/- SD value of ABI was 1.0115 +/- 0.08167 by Doppler method versus 0.9923 +/- 0.08609 by palpatory method (p = 0.1), in high risk asymptomatic group, 0.9838 +/- 0.08878 versus 0.9608 +/- 0.10377 (p = 0.13) and in symptomatic group, 0.9302 +/- 0.14064 versus 0.9088 +/- 0.13274 (p = 0.12). Against the more precise Doppler method, palpatory method was equally good in detecting the PAD positive cases. CONCLUSION: Early diagnosis of PAD in primary care practice by manual palpation of foot arteries is a reliable method to identitfy the population at risk and may lead to aggressive preventive therapies.