Safety and Immunogenicity of an AS03B-Adjuvanted Inactivated Tetravalent Dengue Virus Vaccine Administered on Varying Schedules to Healthy U.S. Adults: A Phase 1/2 Randomized Study.

Dengue disease and its causative agents, the dengue viruses (DENV-1-4), cause high morbidity in tropical and subtropical regions. We evaluated three dosing regimens of the investigational tetravalent AS03B-adjuvanted dengue-purified inactivated vaccine (DPIV+AS03B). In this phase 1/2, observer-blind, placebo-controlled study (NCT02421367), 140 healthy adults were randomized 1:1:2 to receive DPIV+AS03B according to the following regimens: 0-1 month (M), 0-1-6 M, or 0-3 M. Participants received DPIV+AS03B or placebo at M0, M1, M3, and M6 according to their dosing schedule. Primary objectives were 1) to evaluate the safety of DPIV+AS03B for 28 days (D) after each dose; 2) to demonstrate the added value of a booster dose (0-1-6 M versus 0-1 M) based on neutralizing antibody titers to each DENV type (DENV-1-4) at 28 D after the last dose; and, if this objective was met, 3) to demonstrate the benefit of a longer interval between the first and second doses (0-1 M versus 0-3 M). Adverse events (AEs) within 7 D after vaccination tended to be more frequent after DPIV+AS03B doses than placebo; the number of grade 3 AEs was low (≤ 4.5% after DPIV+AS03B; ≤ 2.9% after placebo), with no obvious differences across groups. Within 28 D following each dose, the frequency of unsolicited AEs after DPIV+AS03B appeared higher for three-dose (0-1-6 M) than two-dose (0-1 M and 0-3 M) regimens. No serious AEs were considered related to vaccination, and no potential immune-mediated diseases were reported during the study. All three schedules were well tolerated. Both primary immunogenicity objectives were demonstrated. The 0-3 M and 0-1-6 M regimens were more immunogenic than the 0-1 M regimen.

E. coli production process yields stable dengue 1 virus-sized particles (VSPs).

Dengue fever is one of the most wide-spread vector-borne diseases in the world. Although dengue-associated mortality is low, morbidity and economic impact are high. Current licensed vaccines are limited and mediate only partial protection, thus a cost-effective vaccine with improved efficacy is strongly needed. In this work, recombinant dengue serotype 1 E protein was produced in E. coli, inclusion bodies were isolated and the E protein solubilized in urea and purified using an immobilized metal chelate affinity column. The protein was refolded by dialysis in order to obtain virus-like particles (VLPs). Particle assembly was confirmed using size-exclusion chromatography, dynamic light scattering (DLS), transmission electron microscopy (TEM), atomic force microscopy and stimulated emission depletion fluorescence (STED) microscopy. Particle diameter was strongly dependent on temperature, pH, buffer salt composition, and addition of L-arginine. Particles were stable in carbonate buffer at pH 9.5 and higher at 4 °C and did not aggregate during short-term temperature increase up to 55 °C. However, on basis of the above analyses, especially the results of DLS, TEM and STED, it was concluded that the particles obtained did not have an optimal virus-like structure and were therefore designated "virus-sized particles" (VSPs) rather than VLPs. Immunization of rabbits with the particles did not induce neutralizing antibodies, despite the recognition of the native virus by rabbit antibodies. As the titers against the immunogen were much higher than against the (heat-inactivated) virus, it is assumed that the conformation of the particles at the time of immunization was not optimal. Studies are currently underway to improve the quality of the E protein virus-sized particles towards true virus-like particles in order to optimize its potential as a dengue vaccine candidate.

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

Dengue 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.

Structural and Functional Characterization of a Cross-Reactive Dengue Virus Neutralizing Antibody that Recognizes a Cryptic Epitope.

Understanding the molecular basis of the neutralizing antibody response to dengue virus (DENV) is an essential component in the design and development of effective vaccines and immunotherapeutics. Here we present the structure of a cross-reactive, neutralizing antibody, 3E31, in complex with domain III (DIII) of the DENV envelope (E) protein and reveal a conserved, temperature-sensitive, cryptic epitope on DIII that is not available in any of the known conformations of E on the dengue virion. We observed that 3E31 inhibits E-mediated membrane fusion, suggesting that the antibody is able to neutralize virus through binding an as-yet uncharacterized intermediate conformation of DENV E and sterically block trimer formation. Finally, we show that, unlike cross-reactive fusion peptide-specific antibodies, 3E31 does not promote antibody-dependent enhancement of infection at sub-neutralizing concentrations. Our results highlight the 3E31 epitope on the E protein DIII as a promising target for immunotherapeutics or vaccine design.

Guiding dengue vaccine development using knowledge gained from the success of the yellow fever vaccine.

Flaviviruses comprise approximately 70 closely related RNA viruses. These include several mosquito-borne pathogens, such as yellow fever virus (YFV), dengue virus (DENV), and Japanese encephalitis virus (JEV), which can cause significant human diseases and thus are of great medical importance. Vaccines against both YFV and JEV have been used successfully in humans for decades; however, the development of a DENV vaccine has encountered considerable obstacles. Here, we review the protective immune responses elicited by the vaccine against YFV to provide some insights into the development of a protective DENV vaccine.

A novel tetravalent formulation combining the four aggregated domain III-capsid proteins from dengue viruses induces a functional immune response in mice and monkeys.

Our group developed a subunit vaccine candidate against dengue virus based on two different viral regions: the domain III of the envelope protein and the capsid protein. The novel chimeric protein from dengue-2 virus [domain III-capsid (DIIIC-2)], when presented as aggregated incorporating oligodeoxynucleotides, induced anti-viral and neutralizing antibodies, a cellular immune response and conferred significant protection to mice and monkeys. The remaining constructs were already obtained and properly characterized. Based on this evidence, this work was aimed at assessing the immune response in mice of the chimeric proteins DIIIC of each serotype, as monovalent and tetravalent formulations. Here, we demonstrated the immunogenicity of each protein in terms of humoral and cell-mediated immunity, without antigen competition on the mixture forming the formulation tetra DIIIC. Accordingly, significant protection was afforded as measured by the limited viral load in the mouse encephalitis model. The assessment of the tetravalent formulation in non-human primates was also conducted. In this animal model, it was demonstrated that the formulation induced neutralizing antibodies and memory cell-mediated immune response with IFN-γ-secreting and cytotoxic capacity, regardless the route of immunization used. Taken together, we can assert that the tetravalent formulation of DIIIC proteins constitutes a promising vaccine candidate against dengue virus, and propose it for further efficacy experiments in monkeys or in the dengue human infection model, as it has been recently proposed.

Highly efficient production of a dengue pseudoinfectious virus.

Dengue is a major infectious disease that affects people living in tropical and subtropical regions around the world. The causative agents are dengue virus serotype 1, 2, 3, and 4 (DENV1, 2, 3, and 4). Developing a vaccine for dengue is a high priority for public health, but traditional methods have faced numerous obstacles due to the unique immunopathogenesis of dengue virus infection. Here, we report a novel dengue vaccine candidate based on dengue pseudoinfectious virus (PIV) produced by the incorporation of a dengue subgenomic replicon into viral particles in highly efficient packaging cells. The subgenomic replicon was constructed by deleting the capsid protein (C) gene from the dengue viral genome and optimizing the signal peptide sequence of pre-membrane protein (prM) to facilitate the formation of viral particles. Packaging cells were developed for inducible expression of a bi-protein Cpr, where the protein pr is the "pr" segment of viral protein prM that holds the protein C on the endoplasmic reticulum (ER). When the replicon was introduced into the packaging cells, protein C was released from the bi-protein Cpr by a replicon-encoded viral protease. Coordinate expression of viral structural proteins by the replicon and packaging cells led to the incorporation of the replicon into viral particle to produce PIVs. Animal tests showed that the dengue PIV vaccine was highly immunogenic and the immune response protected mice challenged with a hundred-fold LD50 inoculation of dengue virus. The method described here has the potential to be applied to vaccine development for other flaviviruses.

Expression, purification and evaluation of diagnostic potential and immunogenicity of dengue virus type 3 domain III protein.

Dengue hemorrhagic fever and dengue shock syndrome are the severe manifestations of dengue infection. The quest for reliable dengue diagnostics and a dengue vaccine remained elusive for decades. Domain III of dengue virus envelope contains multiple conformation dependant neutralizing epitopes, thus making it an attractive diagnostic and vaccine candidate. In this report we show the expression of dengue virus type 3 envelope domain III protein (D3EDIII) and demonstrate its potential as a diagnostic and vaccine candidate. Accordingly, D3EDIII was expressed to high levels in Escherichia coli and purified by Ni-NTA affinity chromatography. The purified protein was used to develop an in-house plate ELISA and was further tested with a panel of 40 dengue infected serum samples previously characterized by commercially available serological tests. The in-house results were in excellent agreement with the commercial kits. D3EDIII was refolded by rapid dilution method and the refolded monomer protein was purified by Ion exchange chromatography. Further, the recombinant protein was biologically functional and found to inhibit dengue virus type 3 plaque formation on LLC-MK2 cells demonstrating its function of receptor interaction. Furthermore, D3EDIII in combination with Freund's complete adjuvant induced high antibody titers in BALB/c mice and these antibodies efficiently neutralized dengue 3 virus. Additionally, D3EDIII induced expression of Th1 cytokines that can inhibit the intracellular viral infections. Thus, our results demonstrate that D3EDIII protein has tremendous potential both in diagnosis of dengue infections and in vaccine development.

Cost of production of live attenuated dengue vaccines: a case study of the Instituto Butantan, Sao Paulo, Brazil.

BACKGROUND: A vaccine to prevent dengue disease is urgently needed. Fortunately, a few tetravalent candidate vaccines are in the later stages of development and show promise. But, if the cost of these candidates is too high, their beneficial potential will not be realized. The price of a vaccine is one of the most important factors affecting its ultimate application in developing countries. In recent years, new vaccines such as those for human papilloma virus and pneumococcal disease (conjugate vaccine) have been introduced with prices in developed countries exceeding $50 per dose. These prices are above the level affordable by developing countries. In contrast, other vaccines such as those against Japanese encephalitis (SA14-14-2 strain vaccine) and meningitis type A have prices in developing countries below one dollar per dose, and it is expected that their introduction and use will proceed more rapidly. Because dengue disease is caused by four related viruses, vaccines must be able to protect against all four. Although there are several live attenuated dengue vaccine candidates under clinical evaluation, there remains uncertainty about the cost of production of these tetravalent vaccines, and this uncertainty is an impediment to rapid progress in planning for the introduction and distribution of dengue vaccines once they are licensed. METHOD: We have undertaken a detailed economic analysis, using standard industrial methodologies and applying generally accepted accounting practices, of the cost of production of a live attenuated vaccine, originally developed at the US National Institutes of Health (National Institute of Allergy and Infectious Diseases), to be produced at the Instituto Butantan in Sao Paulo, Brazil. We determined direct costs of materials, direct costs of personnel and labor, indirect costs, and depreciation. These were analyzed assuming a steady-state production of 60 million doses per year. RESULTS: Although this study does not seek to compute the price of the final licensed vaccine, the cost of production estimate produced here leads to the conclusion that the vaccine can be made available at a price that most ministries of health in developing countries could afford. This conclusion provides strong encouragement for supporting the development of the vaccine so that, if it proves to be safe and effective, licensure can be achieved soon and the burden of dengue disease can be reduced.

Overview of plant-derived vaccine antigens: Dengue virus.

This review highlights the advantages and current status of plant-derived vaccine development with special reference to the dengue virus. There are numerous problems involved in dengue vaccine development, and there is no vaccine against all four dengue serotypes. Dengue vaccine development using traditional approaches has not been satisfactory in terms of inducing neutralizing antibodies. Recently, these issues were addressed by showing a very good response to inducing neutralizing antibodies by plant-derived dengue vaccine antigens. This indicates the feasibility of using plant-derived vaccine antigens as a low-cost method to combat dengue and other infectious diseases. The application of new methods and strategies such as dendritic cell targeting in cancer therapy, severe acute respiratory syndrome, tuberculosis, human immune deficiency virus, and malaria might play an important role. These new methods are more efficient than traditional protocols. It is expected that in the near future, plant-derived vaccine antigens or antibodies will play an important role in the control of human infectious diseases.

Strategies for the plant-based expression of dengue subunit vaccines.

Despite significant efforts in many countries, there is still no commercially viable dengue vaccine. Currently, attention is focused on the development of either live attenuated vaccines or live attenuated chimaeric vaccines using a variety of backbones. Alternate vaccine approaches, such as whole inactivated virus and subunit vaccines are in the early stages of development, and are each associated with different problems. Subunit vaccines offer the advantage of providing a uniform antigen of well-defined nature, without the added risk of introducing any genetic material into the person being inoculated. Preliminary trials of subunit vaccines (using dengue E protein) in rhesus monkeys have shown promising results. However, the primary disadvantages of dengue subunit vaccines are the low levels of expression of dengue proteins in mammalian or insect cells, as well as the added unknown risks of antigens produced from mammalian cells containing other potential sources of contamination. In the past two decades, plants have emerged as an alternative platform for expression of biopharmaceutical products, including antigens of bacterial, fungal or viral origin. In the present minireview, we highlight the current plant expression technologies used for expression of biopharmaceutical products, with an emphasis on plants as a production system for dengue subunit vaccines.

Development of a novel DNA SynCon tetravalent dengue vaccine that elicits immune responses against four serotypes.

The increased transmission and geographic spread of dengue fever (DF) and its most severe presentations, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), make it one of the most important mosquito-borne viral disease of humans. Four distinct serotypes of dengue viruses are transmitted to humans through the bites of the mosquitoes. Currently there is no vaccine or antiviral drug against DV infections. Cross-protection between dengue virus serotypes is limited and antibody dependent enhancement (ADE) contributes significantly to the severity of the disease. The major challenge is to induce a broad durable immune response against all four serotypes of dengue virus simultaneously while avoiding the possible exacerbation of risk of developing the severe forms of disease through incomplete or modified responses. In order to address this worldwide concern, we present a synthetic consensus (SynCon) human codon optimized DNA vaccine that elicits immunity against all four dengue serotypes. We cloned consensus DIII domain of E protein from all serotypes and expressed them as a single open reading frame in a mammalian expression vector, called pDV-U-DIII (dengue-vaccine universal). In mice, this dengue-universal construct elicits significant level of anti-DIII antibody that neutralizes all four dengue subtypes and prevents cell death induced by dengue infection. This is the first SynCon DNA vaccine that provides tetravalent immunity against all four serotypes of dengue virus.

A fusogenic peptide expressed on the surface of Salmonella enterica elicits CTL responses to a dengue virus epitope.

Attenuated Salmonella strains are used widely as live carriers of antigens because they elicit both mucosal and systemic immunity against passenger antigens. However, they generally evoke poor cytotoxic T cell (CTL) responses because Salmonella resides within vacuolar compartments and the passenger antigens must travel to the cytosol and be processed through the MHC class I-dependent pathway to simulate CTLs. To address this problem, we designed a fusion protein to destabilize the phagosome membrane and allow a dengue epitope to reach the cytosol. The fusion protein was displayed on the bacterial surface of Salmonella enterica serovar Typhimurium SL3261 through the beta domain of the autotransporter MisL. The passenger alpha domain contained, from the N-terminus, a fusogenic sequence, the NS3 protein 298-306-amino acid CTL epitope from the dengue virus type 2, a molecular tag, and a recognition site for the protease OmpT to release it to the milieu. Display of the fusion protein on the bacterial surface was demonstrated by IFA and flow cytometry using antibodies against the molecular tag. Cleavage of the fusogenic protein-dengue peptide was demonstrated by flow cytometry using OmpT+ Escherichia coli strains. The recombinant Salmonella strains displaying the fusogenic-dengue peptide were able to lyse erythrocytes, induced specific proliferative responses, and elicited CTL responses. These results suggest that the recombinant fusion proteins containing fusogenic sequences provide a promising system to induce CTLs by live vector vaccines.