A tetravalent dengue vaccine based on a complex adenovirus vector provides significant protection in rhesus monkeys against all four serotypes of dengue virus

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.     

A live,attenuated dengue virus type 1 vaccine candidate with a 30-nucleotide deletion in the 3' untranslated region is highly attenuated and immunogenic in monkeys

The Delta30 deletion mutation, which was originally created in dengue virus type 4 (DEN4) by the removal of nucleotides 172 to 143 from the 3' untranslated region (3' UTR), was introduced into a homologous region of wild-type (wt) dengue virus type 1 (DEN1). The resulting virus, rDEN1Delta30, was attenuated in rhesus monkeys to a level similar to that of the rDEN4Delta30 vaccine candidate. rDEN1Delta30 was more attenuated in rhesus monkeys than the previously described vaccine candidate, rDEN1mutF, which also contains mutations in the 3' UTR, and both vaccines were highly protective against challenge with wt DEN1. Both rDEN1Delta30 and rDEN1mutF were also attenuated in HuH-7-SCID mice. However, neither rDEN1Delta30 nor rDEN1mutF showed restricted replication following intrathoracic inoculation in the mosquito Toxorhynchites splendens. The ability of the Delta30 mutation to attenuate both DEN1 and DEN4 viruses suggests that a tetravalent DEN vaccine could be generated by introduction of the Delta30 mutation into wt DEN viruses belonging to each of the four serotypes.     

Potential use of attenuated langat E5 virus as a live vaccine -- long term protection against Russian spring-summer encephalitis virus in mice

A single inoculation of Langat E5 virus provides 90-100% protection in mice against 3200-16000 lethal doses of Russian Spring-Summer Encephalitis (RSSE) virus when challenged 18 or 24 months later. The observed protection is found in the absence of neutralizing antibody (NA) against RSSE virus at the time of challenge but is associated with anamnestic NA response. Immunized mice have low titer short duration viremia. Immunized mice have late first detection and much lower multiplication in the brains, even though traces of virus could be detected in the brain up to 3 weeks following challenge. The observed protection results from sensitization due to immunization and quick host response following RSSE challenge resulting in significantly altered viral pathogenesis. Long term cross protection studies reported in this study and before coupled with the experience of the use of attenuated Langat E5 virus in humans volunteers suggests to its further testing as a live vaccine for prophylaxis against various members of Russian Spring-Summer virus complex throughout the world.     

Yellow fever virus/dengue-2 virus and yellow fever virus/dengue-4 virus chimeras: biological characterization,immunogenicity, and protection against dengue encephalitis in the mouse model

Two yellow fever virus (YFV)/dengue virus chimeras which encode the prM and E proteins of either dengue virus serotype 2 (dengue-2 virus) or dengue-4 virus within the genome of the YFV 17D strain (YF5.2iv infectious clone) were constructed and characterized for their properties in cell culture and as experimental vaccines in mice. The prM and E proteins appeared to be properly processed and glycosylated, and in plaque reduction neutralization tests and other assays of antigenic specificity, the E proteins exhibited profiles which resembled those of the homologous dengue virus serotypes. Both chimeric viruses replicated in cell lines of vertebrate and mosquito origin to levels comparable to those of homologous dengue viruses but less efficiently than the YF5.2iv parent. YFV/dengue-4 virus, but not YFV/dengue-2 virus, was neurovirulent for 3-week-old mice by intracerebral inoculation; however, both viruses were attenuated when administered by the intraperitoneal route in mice of that age. Single-dose inoculation of either chimeric virus at a dose of 10(5) PFU by the intraperitoneal route induced detectable levels of neutralizing antibodies against the homologous dengue virus strains. Mice which had been immunized in this manner were fully protected from challenge with homologous neurovirulent dengue viruses by intracerebral inoculation compared to unimmunized mice. Protection was associated with significant increases in geometric mean titers of neutralizing antibody compared to those for unimmunized mice. These data indicate that YFV/dengue virus chimeras elicit antibodies which represent protective memory responses in the mouse model of dengue encephalitis. The levels of neurovirulence and immunogenicity of the chimeric viruses in mice correlate with the degree of adaptation of the dengue virus strain to mice. This study supports ongoing investigations concerning the use of this technology for development of a live attenuated viral vaccine against dengue viruses.     

Recombinant, live-attenuated tetravalent dengue virus vaccine formulations induce a balanced, broad, and protective neutralizing antibody response against each of the four serotypes in rhesus monkeys

Three tetravalent vaccine (TV) formulations of previously described monovalent dengue (DEN) virus vaccine candidates were compared to a tetravalent formulation of wild-type DEN viruses (T-wt) for replication in SCID mice transplanted with human liver cells (SCID-HuH-7) or for replication and immunogenicity in rhesus monkeys. TV-1 consists of recombinant DEN1, -2, -3, and -4, each with a 30-nucleotide deletion in the 3' untranslated region (Delta30). TV-2 consists of rDEN1Delta30, rDEN4Delta30, and two antigenic chimeric viruses, rDEN2/4Delta30 and rDEN3/4Delta30, both also bearing the Delta30 mutation. TV-3 consists of rDEN1Delta30, rDEN2Delta30, rDEN4Delta30, and a 10-fold higher dose of rDEN3/4Delta30. TV-1 and TV-2 were attenuated in SCID-HuH-7 mice with minimal interference in replication among the virus components. TV-1, -2, and -3 were attenuated in rhesus monkeys as measured by duration and peak of viremia. Each monkey immunized with TV-1 and TV-3 seroconverted to the four DEN components by day 28 with neutralization titers ranging from 1:52 to 1:273 and 1:59 to 1:144 for TV-1 and TV-3, respectively. TV-2 induced low antibody titers to DEN2 and DEN3, but a booster immunization after 4 months increased the neutralizing antibody titers to greater than 1:100 against each serotype and elicited broad neutralizing activity against 19 of 20 DEN subtypes. A single dose of TV-2 induced protection against wild-type DEN1, DEN3, and DEN4 challenge, but not DEN2. However, two doses of TV-2 or TV-3 induced protection against DEN2 challenge. Two tetravalent formulations, TV-2 and TV-3, possess properties of a successful DEN vaccine and can be considered for evaluation in clinical trials.     

Epitope determinants of a chimpanzee dengue virus type 4 (DENV-4)-neutralizing antibody and protection against DENV-4 challenge in mice and rhesus monkeys by passively transferred humanized antibody

The chimpanzee monoclonal antibody (MAb) 5H2 is specific for dengue virus type 4 (DENV-4) and neutralizes the virus at a high titer in vitro. The epitope detected by the antibody was mapped by sequencing neutralization escape variants of the virus. One variant contained a Lys₁₇₄-Glu substitution and another contained a Pro₁₇₆-Leu substitution in domain I of the DENV-4 envelope protein (E). These mutations reduced binding affinity for the antibody 18- to >100-fold. Humanized immunoglobulin G (IgG) 5H2, originally produced from an expression vector, has been shown to be a variant containing a nine-amino-acid deletion in the Fc region which completely ablates antibody-dependent enhancement of DENV replication in vitro. The variant MAb, termed IgG 5H2 ΔD, is particularly attractive for exploring its protective capacity in vivo. Passive transfer of IgG 5H2 ΔD at 20 μg/mouse afforded 50% protection of suckling mice against challenge with 25 50% lethal doses of mouse neurovirulent DENV-4 strain H241. Passive transfer of antibody to monkeys was conducted to demonstrate proof of concept for protection against DENV challenge. Monkeys that received 2 mg/kg of body weight of IgG 5H2 ΔD were completely protected against 100 50% monkey infectious doses (MID₅₀) of DENV-4, as indicated by the absence of viremia and seroconversion. A DENV-4 escape mutant that contained a Lys₁₇₄-Glu substitution identical to that found in vitro was isolated from monkeys challenged with 10⁶ MID₅₀ of DENV-4. This substitution was also present in all naturally occurring isolates belonging to DENV-4 genotype III. These studies have important implications for possible antibody-mediated prevention of DENV infection.     

Evaluation of Francisella tularensis ΔpdpC as a candidate live attenuated vaccine against respiratory challenge by a virulent SCHU P9 strain of Francisella tularensis in a C57BL/6J mouse model

Francisella tularensis, which causes tularemia, is an intracellular gram‐negative bacterium. F. tularensis has received significant attention in recent decades because of its history as a biological weapon. Thus, development of novel vaccines against tularemia has been an important goal. The attenuated F. tularensis strain ΔpdpC, in which the pathogenicity determinant protein C gene (pdpC) has been disrupted by TargeTron mutagenesis, was investigated as a potential vaccine candidate for tularemia in the present study. C57BL/6J mice immunized s.c. with 1 × 10⁶ CFUs of ΔpdpC were challenged intranasally with 100× the median lethal dose (LD₅₀) of a virulent SCHU P9 strain 21 days post immunization. Protection against this challenge was achieved in 38% of immunized C57BL/6J mice administered 100 LD₅₀ of this strain. Conversely, all unimmunized mice succumbed to death 6 days post challenge. Survival rates were significantly higher in vaccinated than in unimmunized mice. In addition, ΔpdpC was passaged serially in mice to confirm its stable attenuation. Low bacterial loads persisted in mouse spleens during the first to tenth passages. No statistically significant changes in the number of CFUs were observed during in vivo passage of ΔpdpC. The inserted intron sequences for disrupting pdpC were completely maintained even after the tenth passage in mice. Considering the stable attenuation and intron sequences, it is suggested that ΔpdpC is a promising tularemia vaccine candidate.