Background:Nervous system injuries in mammals often involve transection or segmental loss of peripheral nerves. Such injuries result in functional (behavioral) deficits poorly restored by naturally occurring 1-2mm/d axonal outgrowths aided by primary repair or reconstruction. "Neurorrhaphy" or nerve repair joins severed connective tissues, but not severed cytoplasmic/ plasmalemmal extensions (axons) within the tissue.New Method: PEG-fusion consists of neurorrhaphy combined with a well-defined sequence of four pharmaceutical agents in solution, one containing polyethylene glycol (PEG), applied directly to closely apposed viable ends of severed axons.Results: PEG-fusion of rat sciatic nerves: (1) restores axonal continuity across coaptation site(s) within minutes, (2) prevents Wallerian degeneration of many distal severed axons, (3) preserves neuromuscular junctions, (4) prevents target muscle atrophy, (5) produces rapid and improved recovery of voluntary behaviors compared with neurorrhaphy alone, and (6) PEG-fused allografts are not rejected, despite no tissue-matching nor immunosuppression.Comparison with existing methods: If PEG-fusion protocols are not correctly executed, the results are similar to that of neurorrhaphy alone: (1) axonal continuity across coaptation site(s) is not re-established, (2) Wallerian degeneration of all distal severed axons rapidly occurs, (3) neuromuscular junctions are non-functional, (4) target muscle atrophy begins within weeks, (5) recovery of voluntary behavior occurs, if ever, after months to levels well-below that observed in unoperated animals, and (6) allografts are either rejected or not well-accepted.Conclusion: PEG-fusion produces rapid and dramatic recovery of function following rat peripheral nerve injuries.
Lassa virus (LASV) is a hemorrhagic fever virus of the Arenaviridae family with high rates of mortality and co-morbidities, including chronic seizures and permanent bilateral or unilateral deafness. LASV is endemic in West Africa and Lassa fever accounts for 10-16% of hospitalizations annually in parts of Sierra Leone and Liberia according to the CDC. An ongoing outbreak in Nigeria has resulted in 144 deaths in 568 cases confirmed as LASV as of November 2018, with many more suspected, highlighting the urgent need for a vaccine to prevent this severe disease. We previously reported on a DNA vaccine encoding a codon-optimized LASV glycoprotein precursor gene, pLASV-GPC, which completely protects Guinea pigs and nonhuman primates (NHPs) against viremia, clinical disease, and death following lethal LASV challenge. Herein we report on the immunogenicity profile of the LASV DNA vaccine in protected NHPs. Antigen-specific binding antibodies were generated in 100% (6/6) NHPs after two immunizations with pLASV-GPC. These antibodies bound predominantly to the assembled LASV glycoprotein complex and had robust neutralizing activity in a pseudovirus assay. pLASV-GPC DNA-immunized NHPs (5/6) also developed T cell responses as measured by IFNγ ELISpot assay. These results revealed that the pLASV-GPC DNA vaccine is capable of generating functional, LASV-specific T cell and antibody responses, and the assays developed in this study will provide a framework to identify correlates of protection and characterize immune responses in future clinical trials.
Plasmid DNA (pDNA) gene delivery is a highly versatile technology that has the potential to address a multitude of unmet medical needs. Advances in pDNA delivery to host tissue with the employment of in vivo electroporation (EP) have led to significantly enhanced gene expression and the recent demonstration of clinical efficacy with the platform. Building upon this platform, this study reports that enzyme-mediated modification of the muscle tissue extracellular matrix structure at the site of pDNA delivery operates in a synergistic manner with EP to enhance both local and systemic gene expression further. Specifically, administration of chondroitinase ABC (Cho ABC) to the site of intramuscular delivery of pDNA led to transient disruption of chondroitin sulfate scaffolding barrier, permitting enhanced gene distribution and expression across the tissue. The employment of Cho ABC in combination with CELLECTRA ® intramuscular EP resulted in increased gene expression by 5.5-fold in mice and 17.98-fold in rabbits. The study demonstrates how this protocol can be universally applied to an active prophylaxis platform to increase the in vivo production of functional immunoglobulin G, and to DNA vaccine protocols to permit drug dose sparing. The data indicate the Cho ABC formulation to be of significant value upon combination with EP to drive enhanced gene expression levels in pDNA delivery protocols.
Acute viral infection generates lineage-committed Th1 and T follicular helper (Tfh) memory cells that recall their lineage-specific functions following secondary challenge with virus. However, the lineage commitment of effector and memory Th cells in vivo following protein vaccination is poorly understood. In this study, we analyzed effector and memory CD4+ T cell differentiation in mice (Mus musculus) following adjuvanted glycoprotein immunization compared with acute lymphocytic choriomeningitis virus infection. Glycoprotein immunization induced CXCR5− non-Tfh effector and memory CD4+ T cells that surprisingly had not undergone polarization toward any particular Th cell lineage but had undergone memory differentiation. However, upon challenge with virus, these Th lineage–nonpolarized memory CD4+ T cells were able to generate Th1 secondary effector cells, demonstrating their lineage plasticity. In addition, Tfh and memory Tfh cells were generated in response to protein immunization, and these cells differed from infection-induced Tfh cells by their lack of the transcription factor Tbet. Rechallenge experiments demonstrated that viral infection, but not protein immunization, during either the primary or secondary immune response, restricts the recall of Bcl6 expression and the generation of germinal center Tfh cells. Together, these data demonstrate that protein immunization generates a combination of nonpolarized memory cells that are highly plastic and memory Tfh cells that can undergo further Th1-like modulation during a secondary response to viral infection.
The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.
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