Studies on the sequential pathology of Kyasanur Forest Disease (KFD) in Mouse brain: KFD virus induces apoptosis of neurons in cerebrum and hippocampus.

The sequential pathology of Kyasanur forest disease (KFD) in mouse brain was assessed in this study. Kyasanur forest disease virus (KFDV) strain P9605 used in this study was confirmed by real-time reverse transcriptase-polymerase chain reaction targeting the NS5 gene. Mouse Lethal Dose 50 (MLD50) of the virus was determined by in-vivo mice inoculation test. One MLD50 of the KFDV was inoculated intra-cerebrally into 36 mice aged 2-3 weeks. Another group of 36 age-matched mice that served as control group were inoculated with plain media. Six mice each from infected and control groups were euthanized every 24 hrs intervals for six days. Brain tissues were collected in 10% NBF. The collected brain tissues were processed and subjected to histopathological studies by Hematoxylin and Eosin staining. Grossly, the infected mice showed symptoms of dullness, hunched back appearance, weakness, sluggish movements with indication of hind quarter paralysis on day four post-infection. These symptoms got aggravated with complete paralysis of the hind quarters, inability to move and death on 5th and 6th day post-infection. Microscopically, the brain showed apoptosis of neurons, perivascular cuffing, gliosis, congestion, neuropil vacuolation, meningitis, degeneration, and necrotic neurons. The real-time RT-PCR on hippocampus of the KFDV-infected mouse brain showed three-fold higher expression levels of Caspase 3, a crucial mediator of apoptosis. The cerebral cortex, cerebellum and hippocampus that control the motor neuron activities and muscle tone were primarily affected, possibly correlating with the gross symptoms of hind quarter paralysis, ataxia, and other motor neuron dysfunctions noticed. Taken together, these findings reveal that KFDV induces apoptosis of neurons in the cerebrum and hippocampus of KFDV infected mice. Further studies are needed to confirm if the lesions noticed in mice brain simulate the brain lesions in humans since gross motor-neuron symptoms are similar in mice as well as humans.

Evaluation of Safety and Potency of Kyasanur Forest Disease (KFD) Vaccine Inactivated with Different Concentrations of Formalin and Comparative Evaluation of In Vitro and In Vivo Methods of Virus Titration in KFD Vaccine.

We evaluated the safety and potency of the Kyasanur Forest disease (KFD) vaccine inactivated with different formalin concentrations in mice, since the side effects due to higher formalin concentrations have been a major reason for vaccine refusal. Furthermore, with an objective to reduce the use of mice in vaccine testing, we performed quantification of the KFD virus by real-time PCR and compared it with in vivo titration in mice. The KFD vaccine prepared in chicken embryo fibroblast cells was inactivated with 0.04%, 0.06%, and 0.08% concentrations of formalin. The vaccine inactivated with 0.04% and 0.06% formalin failed the safety test, whereas the KFD vaccine inactivated with 0.08% formalin was safe and potent with a log protective index of 5678 in mice. This reduced formalin content may induce no/lesser side-effects of pain/swelling which may increase the vaccine acceptance. The real-time PCR on individual KFD vaccine harvests interpreted that when the CT value of each harvest is <20, the vaccine will have sufficient viral particles to pass the potency test. Comparison of the real-time PCR on tenfold dilutions of the pooled harvests with in vivo mice inoculation test revealed that the 1MLD50 of the vaccine lies in the tenfold dilution that yields CT values between 31 and 34.

Isolation, Characterization, and Drug Sensitivity of Mycobacterium tuberculosis in Captive Sloth Bears (Melursus ursinus): Unnatural Habitat With Human Environment May Predispose Sloth Bears to Tuberculosis.

We describe the isolation, molecular characterization, and drug sensitivity of Mycobacterium tuberculosis recovered from lung tissues of four rescued captive sloth bears (Melursus ursinus) at Bannerghatta Biological Park (BBP), Bangalore, India. These bears had lived most of their life with humans in circus companies. They were rescued and housed in the Bear Rescue Center (BRC) of BBP. Upon rescue, they showed signs of unthriftiness, chronic debility, and failed to respond to symptomatic treatments. Over the period of the next 12-14 months, the four sloth bears died and the post-mortem examination revealed nodular lesions in the lungs that showed the presence of acid-fast bacilli. Polymerase chain reaction (PCR), culture, and nucleotide sequencing confirmed the bacilli as Mycobacterium tuberculosis. Histopathology of the lungs revealed characteristic granulomatous reaction with caseation. We determined the sensitivity of these isolates to rifampicin and isoniazid drugs by a WHO approved test, Line Probe Assay (LPA) using Genotype MTBDRplus VER 2.0. We discuss the role of unnatural habitat with the human environment in predisposing captive sloth bears for tuberculosis (TB). In the absence of any other reliable ante-mortem diagnostic test, this study recommends the use of LPA for early detection of TB in captive wild animals, which will help in taking necessary steps to prevent its further spread to animal caretakers and other susceptible animals in captivity.

Combination Adjuvants Affect the Magnitude of Effector-Like Memory CD8 T Cells and Protection against Listeriosis.

The development of T cell-based subunit protein vaccines against diseases such as tuberculosis and malaria remains a challenge for immunologists. Here, we have identified a nanoemulsion adjuvant, Adjuplex (ADJ), which enhanced dendritic cell (DC) cross-presentation and elicited effective memory T cell-based immunity to Listeria monocytogenes. We further evaluated whether cross-presentation induced by ADJ can be combined with the immunomodulatory effects of Toll-like receptor (TLR) agonists (CpG or glucopyranosyl lipid adjuvant [GLA]) to evoke systemic CD8 T cell-based immunity to L. monocytogenes. Mechanistically, vaccination with ADJ, alone or in combination with CpG or GLA, augmented activation and antigen uptake by CD103+ migratory and CD8α+ resident DCs and upregulated CD69 expression on B and T lymphocytes in vaccine-draining lymph nodes. By engaging basic leucine zipper ATF-like transcription factor 3-dependent cross-presenting DCs, ADJ potently elicited effector CD8 T cells that differentiated into granzyme B-expressing CD27LO effector-like memory CD8 T cells, which provided effective immunity to L. monocytogenes in the spleen and liver. CpG or GLA alone did not elicit effector-like memory CD8 T cells and induced moderate protection in the spleen but not in the liver. Surprisingly, combining CpG or GLA with ADJ reduced the number of ADJ-induced memory CD8 T cells and compromised protective immunity to L. monocytogenes, especially in the liver. Taken together, the data presented in this study provide a glimpse of protective CD8 T cell memory differentiation induced by a nanoemulsion adjuvant and demonstrate the unexpected negative effects of TLR signaling on the magnitude of CD8 T cell memory and protective immunity to L. monocytogenes, a model intracellular pathogen.

Carbomer-based adjuvant elicits CD8 T-cell immunity by inducing a distinct metabolic state in cross-presenting dendritic cells.

There is a critical need for adjuvants that can safely elicit potent and durable T cell-based immunity to intracellular pathogens. Here, we report that parenteral vaccination with a carbomer-based adjuvant, Adjuplex (ADJ), stimulated robust CD8 T-cell responses to subunit antigens and afforded effective immunity against respiratory challenge with a virus and a systemic intracellular bacterial infection. Studies to understand the metabolic and molecular basis for ADJ's effect on antigen cross-presentation by dendritic cells (DCs) revealed several unique and distinctive mechanisms. ADJ-stimulated DCs produced IL-1ß and IL-18, suggestive of inflammasome activation, but in vivo activation of CD8 T cells was unaffected in caspase 1-deficient mice. Cross-presentation induced by TLR agonists requires a critical switch to anabolic metabolism, but ADJ enhanced cross presentation without this metabolic switch in DCs. Instead, ADJ induced in DCs, an unique metabolic state, typified by dampened oxidative phosphorylation and basal levels of glycolysis. In the absence of increased glycolytic flux, ADJ modulated multiple steps in the cytosolic pathway of cross-presentation by enabling accumulation of degraded antigen, reducing endosomal acidity and promoting antigen localization to early endosomes. Further, by increasing ROS production and lipid peroxidation, ADJ promoted antigen escape from endosomes to the cytosol for degradation by proteasomes into peptides for MHC I loading by TAP-dependent pathways. Furthermore, we found that induction of lipid bodies (LBs) and alterations in LB composition mediated by ADJ were also critical for DC cross-presentation. Collectively, our model challenges the prevailing metabolic paradigm by suggesting that DCs can perform effective DC cross-presentation, independent of glycolysis to induce robust T cell-dependent protective immunity to intracellular pathogens. These findings have strong implications in the rational development of safe and effective immune adjuvants to potentiate robust T-cell based immunity.

Programming Multifaceted Pulmonary T Cell Immunity by Combination Adjuvants.

Induction of protective mucosal T cell memory remains a formidable challenge to vaccinologists. Using a combination adjuvant strategy that elicits potent CD8 and CD4 T cell responses, we define the tenets of vaccine-induced pulmonary T cell immunity. An acrylic-acid-based adjuvant (ADJ), in combination with Toll-like receptor (TLR) agonists glucopyranosyl lipid adjuvant (GLA) or CpG, promotes mucosal imprinting but engages distinct transcription programs to drive different degrees of terminal differentiation and disparate polarization of TH1/TC1/TH17/TC17 effector/memory T cells. Combination of ADJ with GLA, but not CpG, dampens T cell receptor (TCR) signaling, mitigates terminal differentiation of effectors, and enhances the development of CD4 and CD8 TRM cells that protect against H1N1 and H5N1 influenza viruses. Mechanistically, vaccine-elicited CD4 T cells play a vital role in optimal programming of CD8 TRM and viral control. Taken together, these findings provide further insights into vaccine-induced multifaceted mucosal T cell immunity with implications in the development of vaccines against respiratorypathogens, including influenza virus and SARS-CoV-2.

Polymeric Pathogen-Like Particles-Based Combination Adjuvants Elicit Potent Mucosal T Cell Immunity to Influenza A Virus.

Eliciting durable and protective T cell-mediated immunity in the respiratory mucosa remains a significant challenge. Polylactic-co-glycolic acid (PLGA)-based cationic pathogen-like particles (PLPs) loaded with TLR agonists mimic biophysical properties of microbes and hence, simulate pathogen-pattern recognition receptor interactions to safely and effectively stimulate innate immune responses. We generated micro particle PLPs loaded with TLR4 (glucopyranosyl lipid adjuvant, GLA) or TLR9 (CpG) agonists, and formulated them with and without a mucosal delivery enhancing carbomer-based nanoemulsion adjuvant (ADJ). These adjuvants delivered intranasally to mice elicited high numbers of influenza nucleoprotein (NP)-specific CD8+ and CD4+ effector and tissue-resident memory T cells (TRMs) in lungs and airways. PLPs delivering TLR4 versus TLR9 agonists drove phenotypically and functionally distinct populations of effector and memory T cells. While PLPs loaded with CpG or GLA provided immunity, combining the adjuvanticity of PLP-GLA and ADJ markedly enhanced the development of airway and lung TRMs and CD4 and CD8 T cell-dependent immunity to influenza virus. Further, balanced CD8 (Tc1/Tc17) and CD4 (Th1/Th17) recall responses were linked to effective influenza virus control. These studies provide mechanistic insights into vaccine-induced pulmonary T cell immunity and pave the way for the development of a universal influenza and SARS-CoV-2 vaccines.