Nearly four decades after HIV (Human Immunodeficiency Virus) was identified as the causal agent of the AIDS (Acquired Immunodeficiency Syndrome) pandemic, it remains a top global health concern impacting millions of people around the world particularly in Sub-Saharan Africa. Despite significant scientific, governmental and nongovernmental organizational efforts, most HIV-infected patients do not have access to prevention and treatment. Since cure is not available yet, developing a vaccine to prevent HIV from spreading is a priority. Previous studies have worked on an HIV vaccine platform using attenuated Vaccinia vector and plant-produced HIV virus-like particles (VLPs) to deliver Gag and dgp41 antigens as a heterologous prime-boost strategy. To further study this work, I conducted immunogenicity studies in rabbits which exhibited high IgG responses against Gag (p < 0.002) and less to dgp41. To increase the immunogenicity to dgp41, focusing on MPER, a combination of IgG fusions with VLPs as a vaccine platform was studied in mice. Both IgG fusion constructs showed similar serum results, though Gag-specific serum IgG responses were significantly higher (p < 0.007) for the recombinant immune complex (RIC) group than hexamer forming complexes (Hex). In an effort to expand the use of HIV VLPs, RSV (Respiratory Syncytial Virus) pre-fusion stabilized F (pre-F) protein was presented by self-assembling HIV-1 Gag as a potential vaccine strategy for RSV infections. Multiple constructs were designed to assemble into chimeric VLPs and tested for recombinant plant expression. Mouse immunogenicity study using these chimeric VLPs showed significantly high F-specific IgG (p < 0.001) in serum and superior IgA in mucosal samples for the group that received one of the pre-F stabilized VLP constructs. Moreover, when the same antigen was administered with cholera toxin intranasally, it generated IgA response in nasal flush higher than when it was administered subcutaneously. To summarize, this study showed the efficiency of a plant-produced VLP-based system as an adaptable chimeric vaccine platform for potential use with various viral antigens in pursuit of a vaccine strategy that is immunogenic in animal studies.

HIV continues to remain a global health issue, in particular in many low and middle-income countries. The World Health Organization (WHO) estimates that of the nearly 38 million HIV-1 positive individuals, 25% are unaware they are infected. Despite decades of research, a safe and effective preventative vaccine has yet to be produced. The HIV-1 envelope glycoprotein41 and the Gag structural protein have been identified to be particularly important in HIV-1 transcytosis and cytotoxic lymphocyte response, respectively. Enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of glycoprotein (dgp41) comprising the membrane proximal external region (MPER), transmembrane domain and cytoplasmic tail may present a unique and safe way of presenting these proteins in a state mimicking their natural formation. Another form of presenting the immunogenic glycoprotein41, particularly the MPER component, is by presenting it onto the N-terminal of an IgG molecule, thereby creating an IgG fusion molecule. In our lab, both VLPs and IgG fusion molecules are highly expressed and purified within GnGn Nicotiana benthamiana. The results indicated that these recombinant proteins can be assembled properly within plants and can elicit an immune response in mice. This provides a preliminary step in using such Gag/dpg41 VLPs and RIC as present a safe, effective, and inexpensive HIV vaccine.

Succinylcholine-induced apnea is a common problem in pre-hospital medicine that affects 1/1800 patients who undergo rapid sequence intubation. Succinylcholine is an anesthetic that mimics the neurotransmitter, acetylcholine. It binds to cholinergic receptors, blocking acetylcholine access, and causes paralysis for (normally) only a short time. Butyrylcholinesterase, which is responsible for succinylcholine hydrolysis, is deficient in a small percentage of the population. Previous studies have shown that wild-type butyrylcholinesterase (BChE) can be produced in transient-expression Nicotiana benthamiana, and can reverse the effects of succinylcholine induced apnea through enzyme replacement therapy. The wild type enzyme is also capable of irreversibly binding and inactivating organophosphorus nerve agents and pesticides, and has also exhibited cocaine hydrolase activity. Super cocaine-hydrolyzing BChE mutants, which exceed 2000 times the catalytic capability of the wild-type, have been optimized and expressed in N. benthamiana. The purpose of this study was to determine whether these mutants also hydrolyze succinylcholine with improved efficiency. Variant 3 and Variant 4 exhibited catalytic efficiencies of 2.08 x 106 M-1 min-1 and 3.48 x 106 M-1 min-1, respectively, against their preferred substrate, butyrylthiocholine, in the Ellman assay. The wild-type plant-expressed BChE did exhibit hydrolysis of succinylcholine, as we had previously determined; however, neither Variant 3 nor Variant 4 demonstrated the ability to hydrolyze succinylcholine in our particular assay. Therefore, N. benthamiana-expressed Variant 3 and Variant 4 may not succeed as a dual treatment against cocaine toxicity and prolonged succinylcholine-induces paralysis.