Harnessing the Potential of MAIT Cells as Cellular Adjuvants in Mucosal Vaccines

<p>The development of vaccines is considered one of the most successful medical interventions to date, preventing millions of deaths every year. However, the majority of vaccines are administered peritoneally, despite the vast majority of pathogens invade the human host at mucosal sites. By vaccinating at distal sites, little to no protection is developed at the mucosa where the initial invasion occurs. There are however, a handful of licenced mucosally administered vaccines against infections such as poliovirus, influenza and Salmonella Typhi that are able to induce both a systemic and mucosal protective immune response. All but one of the current licenced mucosal vaccines are live attenuated due in part to the difficulty of developing new mucosal adjuvants. Recombinant cholera toxin subunit B is the only adjuvant used in the current licenced mucosal vaccines. While inactivated and subunit vaccines are considered safer as they are unable to revert back to virulent pathogens, adjuvants are required to boost their immunogenicity. This thesis therefore explores whether mucosal-associated invariant T (MAIT) cells which are found in mucosal tissues, are invariant in nature and have rapid activation, could be exploited as cellular adjuvants in mucosal vaccines. This thesis was able to show that intranasally administered MAIT cell agonist components, 5-A-RU and methylglyoxal (MG), are able to induce both MAIT cell and conventional dendritic cell (cDC) activation in the lung tissue and mediastinal lymph node (mLN). In this model CD40L and RANKL co-stimulatory interactions are involved in ICOSL expression on cDCs in the lung and associated with cDC activation. The MAIT cells within this model also maintained a RORyT and GATA3 phenotype after both one and three doses of the 5-A-RU + MG vaccine. Furthermore, a prime-boost intranasal vaccine scheme of 5-A-RU + MG and the model antigen OVA, was able to induce MR1-dependent accumulation of TFH cells and antigen-specific germinal center B cells in the mLN along with systemic antigen-specific IgG antibody production. This humoral response was also dependent on the presence of both cDC1 and cDC2 populations. Together, this thesis suggests MAIT cells have the potential to be utilised as cellular adjuvants in mucosal vaccines.</p>

Harnessing the Potential of MAIT Cells as Cellular Adjuvants in Mucosal Vaccines

<p>The development of vaccines is considered one of the most successful medical interventions to date, preventing millions of deaths every year. However, the majority of vaccines are administered peritoneally, despite the vast majority of pathogens invade the human host at mucosal sites. By vaccinating at distal sites, little to no protection is developed at the mucosa where the initial invasion occurs. There are however, a handful of licenced mucosally administered vaccines against infections such as poliovirus, influenza and Salmonella Typhi that are able to induce both a systemic and mucosal protective immune response. All but one of the current licenced mucosal vaccines are live attenuated due in part to the difficulty of developing new mucosal adjuvants. Recombinant cholera toxin subunit B is the only adjuvant used in the current licenced mucosal vaccines. While inactivated and subunit vaccines are considered safer as they are unable to revert back to virulent pathogens, adjuvants are required to boost their immunogenicity. This thesis therefore explores whether mucosal-associated invariant T (MAIT) cells which are found in mucosal tissues, are invariant in nature and have rapid activation, could be exploited as cellular adjuvants in mucosal vaccines. This thesis was able to show that intranasally administered MAIT cell agonist components, 5-A-RU and methylglyoxal (MG), are able to induce both MAIT cell and conventional dendritic cell (cDC) activation in the lung tissue and mediastinal lymph node (mLN). In this model CD40L and RANKL co-stimulatory interactions are involved in ICOSL expression on cDCs in the lung and associated with cDC activation. The MAIT cells within this model also maintained a RORyT and GATA3 phenotype after both one and three doses of the 5-A-RU + MG vaccine. Furthermore, a prime-boost intranasal vaccine scheme of 5-A-RU + MG and the model antigen OVA, was able to induce MR1-dependent accumulation of TFH cells and antigen-specific germinal center B cells in the mLN along with systemic antigen-specific IgG antibody production. This humoral response was also dependent on the presence of both cDC1 and cDC2 populations. Together, this thesis suggests MAIT cells have the potential to be utilised as cellular adjuvants in mucosal vaccines.</p>

Use of cholera toxin subunit B to label neural projections to lower urinary tract organs v1

This protocol is used to visualise sensory and autonomic neurons innervating organs of the lower urinary tract in an experimental adult male or female rat. The protocol is performed under anesthesia and should incorporate all local requirements for standards of animal experimentation, including methods of anesthesia, surgical environment, and post-operative monitoring and care.

Immunohistochemical labelling of lower urinary tract afferents in spinal cord v1

This protocol is used for immunohistochemical visualisation of cholera toxin subunit B within afferents innervating the lower urinary tract in cryosections of rat lumbosacral spinal cord. Free-floating sections are processed in a double labelling protocol to distinguish regions of innervation by these afferents. Cholera toxin B antibody [lower urinary tract afferents] Choline acetyltransferase antibody [preganglionic autonomic neurons and motoneurons]

Immunolabelling and clearing of intact spinal cord for visualization of lower urinary tract afferents v1

The whole-mount immunolabeling and clearing method (iDISCO) was used to visualize cholera toxin subunit B-labelled lower urinary tract afferents in the lumbosacral spinal cord of the rat. Imaging of spinal cord was performed on a light sheet microscope with a 12x lens. Concurrently, choline acetyltransferase identified preganglionic autonomic neurons and motoneurons within the spinal cord, which were used to confirm the rostrocaudal location of afferents.

Comparison of Sensory and Motor Innervation Between the Acupoints LR3 and LR8 in the Rat With Regional Anatomy and Neural Tract Tracing

ObjectiveThis study aimed to investigate the sensory and motor innervation of “Taichong” (LR3) and “Ququan” (LR8) in the rat and provide an insight into the neural relationship between the different acupoints in the same meridian.MethodsThe LR3 and LR8 were selected as the representative acupoints from the Liver Meridian and examined by using the techniques of regional anatomy and neural tract tracing in this study. For both acupoints, their local nerves were observed with regional anatomy, and their sensory and motor pathways were traced using neural tract tracing with single cholera toxin subunit B (CTB) and dual Alexa Fluor 594/488 conjugates with CTB (AF594/488-CTB).ResultsUsing the regional anatomy, the branches of the deep peroneal nerve and saphenous nerve were separately found under the LR3 and LR8. Using single CTB, the sensory neurons, transganglionic axon terminals, and motor neurons associated with both LR3 and LR8 were demonstrated on the dorsal root ganglia (DRG), spinal dorsal horn, Clarke’s nucleus, gracile nucleus, and spinal ventral horn corresponding to their own spinal segments and target regions, respectively. Using dual AF594/488-CTB tracing, it was shown that the sensory and motor neurons associated with LR3 were separated from that of LR8.ConclusionThis study demonstrates that LR3 and LR8 are innervated by different peripheral nerves, which originated from or terminated in their corresponding spinal segments and target regions independently through the sensory and motor pathways. These results provide an example for understanding the differential innervation between the different acupoints in the same meridian.

Axon regeneration after optic nerve injury in rats can be improved via PirB knockdown in the retina

Background In the central nervous system (CNS), three types of myelin-associated inhibitors (MAIs) exert major inhibitory effects on nerve regeneration: Nogo-A, myelin-associated glycoprotein (MAG), and oligodendrocyte-myelin glycoprotein (OMgp). MAIs have two co-receptors, Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PirB). Existing studies confirm that inhibiting NgR only exerted a modest disinhibitory effect in CNS. However, the inhibitory effects of PirB on nerve regeneration after binding to MAIs are controversial too. We aimed to further investigate the effect of PirB knockdown on the neuroprotection and axonal regeneration of retinal ganglion cells (RGCs) after optic nerve injury in rats. Methods The differential expression of PirB in the retina was observed via immunofluorescence and western blotting after 1, 3, and 7 days of optic nerve injury (ONI). The retina was locally transfected with adeno-associated virus (AAV) PirB shRNA, then, the distribution of virus in tissues and cells was observed 21 days after AAV transfection to confirm the efficiency of PirB knockdown. Level of P-Stat3 and expressions of ciliary neurotrophic factor (CNTF) were detected via western blotting. RGCs were directly labeled with cholera toxin subunit B (CTB). The new axons of the optic nerve were specifically labeled with growth associated protein-43 (GAP43) via immunofluorescence. Flash visual evoked potential (FVEP) was used to detect the P1 and N1 latency, as well as N1-P1, P1-N2 amplitude to confirm visual function. Results PirB expression in the retina was significantly increased after ONI. PirB knockdown was successful and significantly promoted P-Stat3 level and CNTF expression in the retina. PirB knockdown promoted the regeneration of optic nerve axons and improved the visual function indexes such as N1-P1 and P1-N2 amplitude. Conclusions PirB is one of the key molecules that inhibit the regeneration of the optic nerve, and inhibition of PirB has an excellent effect on promoting nerve regeneration, which allows the use of PirB as a target molecule to promote functional recovery after ONI.

Dual projecting cells linking thalamic and cortical communication routes between the medial prefrontal cortex and hippocampus

The interactions between the medial prefrontal cortex (mPFC) and hippocampus (HC) are critical for memory and decision making and have been specifically implicated in several neurological disorders including schizophrenia, epilepsy, frontotemporal dementia, and Alzheimers disease. The ventral midline thalamus (vmThal), and lateral entorhinal cortex and perirhinal cortex (LEC/PER) constitute major communication pathways that facilitate mPFC-HC interactions in memory. Although vmThal and LEC/PER circuits have been delineated separately we sought to determine whether these two regions share cell-specific inputs that could influence both routes simultaneously. To do this we used a dual fluorescent retrograde tracing approach using cholera toxin subunit-B (CTB-488 and CTB-594) with injections targeting vmThal and the LEC/PER in rats. Retrograde cell body labeling was examined in key regions of interest within the mPFC-HC system including: (1) mPFC, specifically anterior cingulate cortex (ACC), dorsal and ventral prelimbic cortex (dPL, vPL), and infralimbic cortex (IL); (2) medial and lateral septum (MS, LS); (3) subiculum (Sub) along the dorsal-ventral and proximal-distal axes; and (4) LEC and medial entorhinal cortex (MEC). Results showed that dual vmThal-LEC/PER-projecting cell populations are found in MS, vSub, and the shallow layers II/III of LEC and MEC. We did not find any dual projecting cells in mPFC or in the cornu ammonis (CA) subfields of the HC. Thus, mPFC and HC activity is sent to vmThal and LEC/PER via non-overlapping projection cell populations. Importantly, the dual projecting cell populations in MS, vSub, and LEC are in a unique position to simultaneously influence both cortical and thalamic mPFC-HC pathways critical to memory.

CD171 multi‑epitope peptide design based on immunoinformatics approach as a cancer vaccine candidate for glioblastoma

Glioblastoma (GB) is a common primary malignancy of the central nervous system, and one of the highly lethal brain tumors. GB cells can promote therapeutic resistance and tumor angiogenesis. The CD171 is an adhesion molecule in neuronal cells that is expressed in glioma cells as a regulator of brain development during the embryonic period. CD171 is one of the immunoglobulin-like CAMs (cell adhesion molecules) families that can be associated with prognosis in a variety of human tumors. The multi-epitope peptide vaccines are based on synthetic peptides with a combination of both B-cell epitopes and T-cell epitopes, which can induce specific humoral or cellular immune responses. Moreover Cholera toxin subunit B (CTB), a novel TLR agonist was utilized in the final construct to polarize CD4+ T cells toward T-helper 1 to induce strong cytotoxic T lymphocytes (CTL) responses. In the present study, several immune-informatics tools were used for analyzing the CD171 sequence and studying the important characteristics of a designed vaccine. The results included the molecular docking, prediction and validation of the secondary and tertiary structure, physicochemical properties, solubility, conservancy, toxicity as well as antigenicity and allergenicity of the promising candidate for a vaccine against CD171. The immuno-informatic analyze suggested 12 predicted multi-epitope peptides, whose construction consists of 582 residues long. Therewith, cloning adaptation of designed vaccine was performed and eventually sequence was inserted into pET30a (+) vector for the application of the anti-glioblastoma vaccine development.

CD171 multi‑epitope peptide design based on immunoinformatics approach as a cancer vaccine candidate for glioblastoma

Glioblastoma (GB) is a common primary malignancy of the central nervous system, and one of the highly lethal brain tumors. GB cells can promote therapeutic resistance and tumor angiogenesis. The CD171 is an adhesion molecule in neuronal cells that is expressed in glioma cells as a regulator of brain development during the embryonic period. CD171 is one of the immunoglobulin-like CAMs (cell adhesion molecules) families that can be associated with prognosis in a variety of human tumors. The multi-epitope peptide vaccines are based on synthetic peptides with a combination of both B-cell epitopes and T-cell epitopes, which can induce specific humoral or cellular immune responses. Moreover Cholera toxin subunit B (CTB), a novel TLR agonist was utilized in the final construct to polarize CD4+ T cells toward T-helper 1 to induce strong cytotoxic T lymphocytes (CTL) responses. In the present study, several immune-informatics tools were used for analyzing the CD171 sequence and studying the important characteristics of a designed vaccine. The results included the molecular docking, prediction and validation of the secondary and tertiary structure, physicochemical properties, solubility, conservancy, toxicity as well as antigenicity and allergenicity of the promising candidate for a vaccine against CD171. The immuno-informatic analyze suggested 12 predicted multi-epitope peptides, whose construction consists of 582 residues long. Therewith, cloning adaptation of designed vaccine was performed and eventually sequence was inserted into pET30a (+) vector for the application of the anti-glioblastoma vaccine development.