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We conducted an open label, dose escalation Phase 1 clinical trial of a tetravalent dengue DNA vaccine (TVDV) formulated in Vaxfectin ® to assess safety and immunogenicity. A total of 40 dengue- and flavivirus-naive volunteers received either low-dose (1 mg) TVDV alone ( N = 10, group 1), low-dose TVDV (1 mg) formulated in Vaxfectin ( N = 10, group 2), or high-dose TVDV (2 mg, group 3) formulated in Vaxfectin ® ( N = 20). Subjects were immunized intramuscularly with three doses on a 0-, 30-, 90-day schedule and monitored. Blood samples were obtained after each immunization and various time points thereafter to assess anti-dengue antibody and interferon gamma (IFNγ) T-cell immune responses. The most common adverse events (AEs) across all groups included mild to moderate pain and tenderness at the injection site, which typically resolved within 7 days. Common solicited signs and symptoms included fatigue (42.5%), headache (45%), and myalgias (47.5%). There were no serious AEs related to the vaccine or study procedures. No anti-dengue antibody responses were detected in group 1 subjects who received all three immunizations. There were minimal enzyme-linked immunosorbent assay and neutralizing antibody responses among groups 2 and 3 subjects who completed the immunization schedule. By contrast, IFNγ T-cell responses, regardless of serotype specificity, occurred in 70%, 50%, and 79% of subjects in groups 1, 2, and 3, respectively. The largest IFNγ T-cell responses were among group 3 subjects. We conclude that TVDV was safe and well-tolerated and elicited predominately anti-dengue T-cell IFNγ responses in a dose-related fashion.

Two novel methods of dengue virus inactivation using iodonaphthyl azide (INA) and aminomethyl trioxsalen (AMT) were compared with traditional virus inactivation by formaldehyde. The AMT inactivated dengue-2 virus retained its binding to a panel of 5 monoclonal antibodies specific for dengue-2 envelope protein, whereas inactivation by formaldehyde and INA led to 30-50% decrease in binding. All three inactivated viruses elicited high level virus neutralizing antibodies in vaccinated mice. However, only mice vaccinated with AMT inactivated virus mounted T cell responses similar to live, uninactivated virus.

Dengue viruses are the most important arboviruses causing human disease. Expansion of the disease in recent decades to include more geographical areas of the world, an appreciation of the disease burden and market potentials have spurred a flurry of activity in the development of vaccines to combat dengue viruses. Recent progress in this area and some of the obstacles associated with this development are discussed.

Nearly a third of the human population is at risk of infection with the four serotypes of dengue viruses, and it is estimated that more than 100 million infections occur each year. A licensed vaccine for dengue viruses has become a global health priority. A major challenge to developing a dengue vaccine is the necessity to produce fairly uniform protective immune responses to all four dengue virus serotypes. We have developed two bivalent dengue virus vaccines, using a complex adenovirus vector, by incorporating the genes expressing premembrane (prM) and envelope (E) proteins of dengue virus types 1 and 2 (dengue-1 and -2, respectively) (CAdVax-Den12) or dengue-3 and -4 (CAdVax-Den34). Rhesus macaques were vaccinated by intramuscular inoculation of a tetravalent dengue vaccine formulated by combining the two bivalent vaccine constructs. Vaccinated animals produced high-titer antibodies that neutralized all four serotypes of dengue viruses in vitro. The ability of the vaccine to induce rapid, as well as sustained, protective immune responses was examined with two separate live-virus challenges administered at 4 and 24 weeks after the final vaccination. For both of these virus challenge studies, significant protection from viremia was demonstrated for all four dengue virus serotypes in vaccinated animals. Viremia from dengue-1 and dengue-3 challenges was completely blocked, whereas viremia from dengue-2 and dengue-4 was significantly reduced, as well as delayed, compared to that of control-vaccinated animals. These results demonstrate that the tetravalent dengue vaccine formulation provides significant protection in rhesus macaques against challenge with all four dengue virus serotypes.

There are approximately 100 million new cases of dengue (DEN) virus infection each year. Infection can result in illness ranging from a mild fever to hemorrhaging, shock, or even death. There are four serotypes of dengue virus (DEN1-4), and immunity to one serotype does not cross protect from infection with other serotypes. Currently there are no approved vaccines for dengue fever. In this report, we describe the construction of a bivalent dengue virus vaccine using a complex recombinant adenovirus approach to express multiple genes of DEN1 and DEN2 serotypes. In vaccinated mice, this vector induced humoral immune responses against all four dengue serotypes as measured by enzyme-linked immunosorbent assay. However, the neutralizing antibody responses were specific for DEN1 and DEN2 serotypes. Expansion of this vaccine development platform towards the DEN3 and DEN4 serotypes can lead towards the development of an adenovirus-based tetravalent dengue vaccine.

Dengue virus infections can cause hemorrhagic fever, shock, encephalitis, and even death. Worldwide, approximately 2.5 billion people live in dengue-infested regions with about 100 million new cases each year, although many of these infections are believed to be silent. There are four antigenically distinct serotypes of dengue virus; thus, immunity from one serotype will not cross-protect from infection with the other three. The difficulties that hamper vaccine development include requirements of the natural conformation of the envelope glycoprotein to induce neutralizing immune responses and the necessity of presenting antigens of all four serotypes. Currently, the only way to meet these requirements is to use a mixture of four serotypes of live attenuated dengue viruses, but safety remains a major problem. In this study, we have developed the basis for a tetravalent dengue vaccine using a novel complex adenovirus platform that is capable of expressing multiple antigens de novo. This dengue vaccine is constructed as a pair of vectors that each expresses the premembrane and envelope genes of two different dengue virus serotypes. Upon vaccination, the vaccine expressed high levels of the dengue virus antigens in cells to mimic a natural infection and induced both humoral and cellular immune responses against multiple serotypes of dengue virus in an animal model. Further analyses show the humoral responses were indeed neutralizing against all four serotypes. Our studies demonstrate the concept of mimicking infections to induce immune responses by synthesizing dengue virus membrane antigens de novo and the feasibility of developing an effective tetravalent dengue vaccine by vector-mediated expression of glycoproteins of the four serotypes.

DNA shuffling and screening technologies were used to produce chimeric DNA constructs expressing antigens that shared epitopes from all four dengue serotypes. Three shuffled constructs (sA, sB and sC) were evaluated in the rhesus macaque model. Constructs sA and sC expressed pre-membrane and envelope genes, whereas construct sB expressed only the ectodomain of envelope protein. Five of six, and four of six animals vaccinated with sA and sC, respectively, developed antibodies that neutralized all 4 dengue serotypes in vitro. Four of six animals vaccinated with construct sB developed neutralizing antibodies against 3 serotypes (den-1, -2 and -3). When challenged with live dengue-1 or dengue-2 virus, partial protection against dengue-1 was observed. These results demonstrate the utility of DNA shuffling as an attractive tool to create tetravalent chimeric dengue DNA vaccine constructs, as well as a need to find ways to improve the immune responses elicited by DNA vaccines in general.

We employed DNA shuffling and screening technologies to develop a single recombinant dengue envelope (E) antigen capable of inducing neutralizing antibodies against all four antigenically distinct dengue serotypes. By DNA shuffling of codon-optimized dengue 1-4 E genes, we created a panel of novel chimeric clones expressing C-terminal truncated E antigens that combined epitopes from all four dengue serotypes. DNA vaccines encoding these novel chimeras induced multivalent T cell and neutralizing antibody responses against all four dengue serotypes in mice. By contrast, a mixture of four unshuffled, parental DNA vaccines failed to produce tetravalent neutralizing antibodies in mice. The neutralizing antibody titers for some of these antigens could be further improved by extending the sequences to express full-length pre-membrane and envelope proteins. The chimeric antigens also protected mice against a lethal dengue-2 virus challenge. These data demonstrate that DNA shuffling and associated screening can lead to the selection of multi-epitope antigens against closely related dengue virus serotypes and suggest a broad utility for these technologies in optimizing vaccine antigens.

The goal of this study was to determine, for each of four dengue serotypes, whether owl monkeys (Aotus nancymae) become viremic and develop antibody responses in patterns similar to those seen in humans and whether any behavioral parameters are reliably associated with immunologic responses. A secondary goal was to investigate effects of chronic blood sampling on hematologic parameters in this genus. We inoculated 20 owl monkeys with 2 x 10(4) plaque-forming units of one of four dengue serotypes. Blood samples ranging from 0.4 to 0.7 ml per animal were taken each day for 12 consecutive days after inoculation, as well as on days 21, 28, and 60 post-inoculation. The total amount of blood taken per monkey was 8.0 ml during the first 12 days and 9.5 ml during the first 30 days of the study (i.e., up to 17% total blood volume per week and up to 20% total blood volume per month). Detailed behavioral assessments of all animals were made twice daily on every day of sample collection. The dengue-1 group were viremic for an average of 3.75 days. Dengue-2, -3, and -4 groups had average viremias of 1.00, 1.25, and 1.33 days, respectively. All animals demonstrated appropriate antibody responses as determined by enzyme-linked immunosorbency assay (ELISA). Animals tolerated repeated phlebotomy well, as all animals remained within clinically normal hematocrit (HCT) reference ranges, and no lasting effects on HCT occurred in any monkey. Final HCT for most animals was greater than 45% (mean final hematocrit, 45%). The maximum decrease in HCT ranged from 3.5 to 19 (mean, 8.9) percentage points. No consistent correlation of any behavioral disease parameters with viremia and antibody status was demonstrated, although overt illness did occur in two animals. Aotus can be an affordable and safe model for testing dengue vaccine efficacy; further testing with higher doses of dengue-2, -3 and -4 viruses is warranted.

A dengue 2 plasmid DNA vaccine (pD2) expressing the pre-membrane and envelope proteins (preM-E) was modified by replacing the dengue transmembrane and cytoplasmic sequences with those of the mouse lysosome-associated membrane protein (pD2/LAMP). Immunofluorescence and confocal microscopy of human 293, NIH 3T3, and macrophage IC21 cell lines transfected with pD2/LAMP showed that the preM-E/LAMP protein chimera was present in vesicles containing endogenous LAMP and major histocompatability complex class II (MHC II), in contrast to the non-vesicular localization of native preM-E protein lacking the LAMP targeting sequence. Mice immunized with pD2 showed an antigen-specific immunoglobulin response but the neutralizing antibodies titers (plaque reduction neutralization test, PRNT(50)) elicited by the native protein were minimal. In contrast, vaccination with pD2/LAMP resulted in PRNT(50) of 270, 320 and 160 at approximately 1, 3 and 8 months after two immunizations with 50 microg DNA, and approached 100% neutralization at 1:20 dilution. Additional immunization with pD2/LAMP, after 8 months, increased the neutralizing antibody titers to >640. Comparable neutralizing antibody responses were induced by two vector backbones, pVR1012 and pVax-1, at 5 and 50 microg of DNA. The neutralizing responses to the pD2/LAMP chimera were greatly superior to those elicited by pD2 in all conditions. These results underscore the importance of MHC class II presentation of DNA-encoded dengue-virus envelope protein for production of neutralizing antibodies.

Background The WHO estimates that there may be 50 million cases of dengue virus (DENV) infection worldwide every year. There is no safe vaccine against DENV licensed in the United States. The development of a balanced and effective anti-DENV vaccine is vital to preventing morbidity and mortality. Codagenix used its proprietary SAVE (Synthetic Attenuated Virus Engineering) platform to generate and test a live attenuated, tetravalent vaccine against DENV. Methods Codagenix used SAVE to substitute under-represented human codons and codon-pairs into the E protein sequences of contemporary strains of DENV1-4, producing either a fully human-cell-deoptimized prM-E (E-Min), or a partially deoptimized prM-E (E-W/Min) to allow for balancing of the vaccine's immunogenicity. Full genomes containing deoptimized E-Min and E-W/Min in the DENV2 backbone were transfected into cells to recover live-attenuated, human-cell-deoptimized vaccine strains. Mice were vaccinated with 106 FFU of each DENV vaccine (alone or together), boosted on day 21 and assessed for neutralizing antibodies by PRNT50 and survival after lethal challenge with mouse-adapted wild-type (WT) DENV. Cynomolgus macaques were immunized with a mixture of 106 FFU of each DENV vaccine strain. Two doses were administered on study day 1 and 57 and serum neutralizing antibodies were determined on day 57 and 85 by a microneutralization assay. Results SAVE deoptimized DENV viruses grew to wild-type (between 107 and 108 FFU/ml) levels at permissive temperatures (<37C). All vaccine strains generated neutralizing antibody levels comparable to WT. A tetravalent formulation containing all four E-Min strains protected mice from lethal challenge with DENV3. A tetravalent formulation of Codagenix DENV-E-W/Min vaccine elicited a robust and balanced neutralizing antibody response in non-human primates (NHPs) against all four DENV serotypes after a single dose. A second vaccine dose did not boost antibody titers significantly. Conclusion The ability to rationally balance the attenuation of multiple vaccine strains, thereby avoiding antibody-dependent enhancement, is a unique advantage of the Codagenix SAVE platform. Codagenix DENV vaccine viruses generated balanced, sterilizing immunity in NHPs after one dose. Disclosures All authors: No reported disclosures. [ABSTRACT FROM AUTHOR]

• Two-step purification of Dengue virus using Chromatographic methods. • Psoralen-inactivation of Dengue virus. • Evaluation of psoralen-inactivated Dengue virus vaccines in mice. • Evaluation of psoralen-inactivated Dengue virus vaccines in nonhuman primates. Dengue fever, caused by dengue viruses (DENV 1–4) is a leading cause of illness and death in the tropics and subtropics. Therefore, an effective vaccine is urgently needed. Currently, the only available licensed dengue vaccine is a chimeric live attenuated vaccine that shows varying efficacy depending on serotype, age and baseline DENV serostatus. Accordingly, a dengue vaccine that is effective in seronegative adults, children of all ages and in immunocompromised individuals is still needed. We are currently researching the use of psoralen to develop an inactivated tetravalent dengue vaccine. Unlike traditional formalin inactivation, psoralen inactivates pathogens at the nucleic acid level, potentially preserving envelope protein epitopes important for protective anti-dengue immune responses. We prepared highly purified monovalent vaccine lots of formalin- and psoralen-inactivated DENV 1–4, using Capto DeVirS and Capto Core 700 resin based column chromatography. Tetravalent psoralen-inactivated vaccines (PsIV) and formalin-inactivated vaccines (FIV) were prepared by combining the four monovalent vaccines. Mice were immunized with either a low or high dose of PsIV or FIV to evaluate the immunogenicity of monovalent as well as tetravalent formulations of each inactivation method. In general, the monovalent and tetravalent PsIVs elicited equivalent or higher titers of neutralizing antibodies to DENV than the FIV dengue vaccines and this response was dose dependent. The immunogenicity of tetravalent dengue PsIVs and FIVs were also evaluated in nonhuman primates (NHPs). Consistent with what was observed in mice, significantly higher neutralizing antibody titers for each dengue serotype were observed in the NHPs vaccinated with the tetravalent dengue PsIV compared to those vaccinated with the tetravalent dengue FIV, indicative of the importance of envelope protein epitope preservation during psoralen inactivation of DENV. [ABSTRACT FROM AUTHOR]