Production of tetravalent dengue virus envelope protein domain III based antigens in lettuce chloroplasts and immunologic analysis for future oral vaccine development.

André van Eerde,Henry Daniell,Kristine Eraker Aasland Hansen,Hetron Mweemba Munang'andu,Johanna Gottschamel,Jihong Liu Clarke,Ralph Bock

doi:10.1111/pbi.13065

Abstract

SummaryDengue fever is a mosquito (Aedes aegypti) ‐transmitted viral disease that is endemic in more than 125 countries around the world. There are four serotypes of the dengue virus (DENV 1‐4) and a safe and effective dengue vaccine must provide protection against all four serotypes. To date, the first vaccine, Dengvaxia (CYD‐TDV), is available after many decades’ efforts, but only has moderate efficacy. More effective and affordable vaccines are hence required. Plants offer promising vaccine production platforms and food crops offer additional advantages for the production of edible human and animal vaccines, thus eliminating the need for expensive fermentation, purification, cold storage and sterile delivery. Oral vaccines can elicit humoural and cellular immunity via both the mucosal and humoral immune systems. Here, we report the production of tetravalent EDIII antigen (EDIII‐1‐4) in stably transformed lettuce chloroplasts. Transplastomic EDIII‐1‐4‐expressing lettuce lines were obtained and homoplasmy was verified by Southern blot analysis. Expression of EDIII‐1‐4 antigens was demonstrated by immunoblotting, with the EDIII‐1‐4 antigen accumulating to 3.45% of the total protein content. Immunological assays in rabbits showed immunogenicity of EDIII‐1‐4. Our in vitro gastrointestinal digestion analysis revealed that EDIII‐1‐4 antigens are well protected when passing through the oral and gastric digestion phases but underwent degradation during the intestinal phase. Our results demonstrate that lettuce chloroplast engineering is a promising approach for future production of an affordable oral dengue vaccine.

Highlights

Dengue fever is caused by four antigenically distinct dengue virus serotypes (DENV-1, DENV-2, DENV-3 and DENV-4)
Secondary infections with a heterologous serotype result in more life-threatening and potentially deadly forms of the disease (WHO, 2009) due to antibody dependent enhancement. This phenomenon is associated with cross-reactive but non-neutralizing antibodies produced during the first infection (Halstead, 1988), which enhance the uptake of viruses into Fc-receptor bearing cells during secondary infection (Dejnirattisai et al, 2010; Halstead, 2003)
Clinical studies with the licensed Dengvaxia® (CYD-TDV, a live attenuated, recombinant tetravalent vaccine employing the attenuated YF virus 17D vaccine strain as the replication backbone) has been further investigated clinically since 2015

Summary

Introduction

Dengue fever is caused by four antigenically distinct dengue virus serotypes (DENV-1, DENV-2, DENV-3 and DENV-4). Secondary infections with a heterologous serotype result in more life-threatening and potentially deadly forms of the disease (WHO, 2009) due to antibody dependent enhancement This phenomenon is associated with cross-reactive but non-neutralizing antibodies produced during the first infection (Halstead, 1988), which enhance the uptake of viruses into Fc-receptor bearing cells during secondary infection (Dejnirattisai et al, 2010; Halstead, 2003). This increases the initial virus load in the cells, promotes virus replication and leads to overwhelming of the immune system, causing symptoms like fluid accumulation, plasma leaking, respiratory distress, severe bleeding and organ impairment (WHO, 2015). A safe, effective and affordable dengue vaccine against the four strains is urgently needed to control the disease and meet the WHO goal of reducing dengue morbidity by at least 25% and mortality by at least 50% by 2020 (www.who.int)

Methods

Results

Conclusion