The DENV and the Zika (ZIKV) FVs frequently co-circulate and generally cause mild self-liming febrile illnesses. However, a secondary infection with a heterologous DENV serotype may lead to life threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). DHF/DSS have been linked to antibody dependent enhancement of infection (ADE), a phenomenon that occurs when antibodies (Abs) formed against an initial infection with one serotype of DENV cross-reacts but does not neutralize a heterologous DENV serotype in a secondary infection. Furthermore, Abs raised against the ZIKV have been observed to cross-react with the DENV and vice versa, which can potentially cause ADE and lead to severe DENV disease. The ZIKV can be transmitted vertically and has been linked to devastating congenital defects such as microcephaly in newborns. FDA approved treatments do not exist for DENV and ZIKV illnesses. Thus, there is a need for safe and effective treatments for these co-circulating viruses. Here, a tetravalent bispecific antibody (bsAb) targeting the ZIKV and all four serotypes of the DENV was expressed in the Nicotiana benthamiana (N. benthamiana) plant. Functional assays of the DENV/ZIKV bsAb demonstrated binding, neutralization, and a significant reduction in ADE activity against both the DENV and the ZIKV.
A single chain variable fragment (scFv) and a diabody based on an antibody directed against the immune checkpoint inhibitor PD-L1, were also expressed in N. benthamiana leaves. The smaller sizes of the scFv and diabody confers them with the ability to penetrate deeper tissues making them beneficial in diagnostics, imaging, and possibly cancer therapy. The past few decades has seen long strives in recombinant protein production in plants with significant improvements in production, safety, and efficacy. These characteristics make plants an attractive platform for the production of recombinant proteins, biologics, and therapeutics.
Plant-made virus-like particles (VLPs), composed of HIV-1 Gag and deconstructed gp41 proteins, have been shown to be safe and immunogenic in mice. Here, we report the successful production of HIV-1 Gag/dgp41 VLPs in Nicotiana benthamiana, using an enhanced geminivirus-based expression vector. This novel vector results in unique expression kinetics, with peak protein accumulation and minimal necrosis achieved on day 4 post-infiltration. In comparing various purification strategies, it was determined that a 20% ammonium sulfate precipitation is an effective and efficient method for removing plant proteins and purifying the recombinant VLPs of interest. If further purification is required, this may be achieved through ultracentrifugation. VLPs are a useful platform for a variety of biomedical applications and developing the technology to efficiently produce VLPs in the plant expression system is of critical importance.
Influenza virus A (IVA) poses a serious threat to human health, killing over 25,000 Americans in the 2022 flu season alone. In the past 10 years, vaccine efficacy has varied significantly, ranging from 20-60% each season. Because IVA is subject to high antigenic shift and strain cocirculation, more effective IVA vaccines are needed to reduce the incidence of disease. Herein we report the production of a recombinant immune complex (RIC) vaccine “4xM2e” in Nicotiana benthamiana plants using agroinfiltration for use as a potential universal IVA vaccine candidate. RICs fuse antigen to the C-terminus of an immunoglobulin heavy chain with an epitope tag cognate to the antibody, promoting immune complex formation to increase immunogenicity. IVA matrix protein 2 ectodomain (M2e) is selected to serve as vaccine antigen for its high sequence conservation, as only a small number of minor mutations have occurred since its discovery in 1981 in the human sequence. However, there is some divergence in zoonotic IVA strains, and to account for this, we designed a combination of human consensus, swine, and avian M2e variants, 4xM2e. This was fused to the C terminus of the RIC platform to improve M2e immunogenicity and IVA strain coverage. The 4xM2e RIC was produced in N. benthamiana and verified with SDS-PAGE and Western blot assays, along with an analysis of complex formation and the potential for complement activation via complement C1q ELISA. With this work, we demonstrate the potential of RICs and plant-expression systems to generate universal IVA vaccine candidates.