Abstract
A candidate vaccine (D1ME-VRP) expressing dengue virus type 1 premembrane and envelope proteins in a Venezuelan equine encephalitis (VEE) virus replicon particle (VRP) system was constructed and tested in conjunction with a plasmid DNA vaccine (D1ME-DNA) expressing identical dengue virus sequences. Cynomolgus macaques were vaccinated with three doses of DNA (DDD), three doses of VRP (VVV group), or a heterologous DNA prime-VRP boost regimen (DDV) using two doses of DNA vaccine and a third dose of VRP vaccine. Four weeks after the final immunization, the DDV group produced the highest dengue virus type 1-specific immunoglobulin G antibody responses and virus-neutralizing antibody titers. Moderate T-cell responses were demonstrated only in DDD- and DDV-vaccinated animals. When vaccinated animals were challenged with live virus, all vaccination regimens showed significant protection from viremia. DDV-immunized animals were completely protected from viremia (mean time of viremia = 0 days), whereas DDD- and VVV-vaccinated animals had mean times of viremia of 0.66 and 0.75 day, respectively, compared to 6.33 days for the control group of animals.
Dengue viruses are members of the family Flaviviridae (4). There are four antigenically distinct serotypes with similar clinical presentations, epidemiology, and distributions, especially in tropical and subtropical regions, where 2.5 billion people are at risk of infection (9). Infection with any of the four dengue virus serotypes can cause diseases ranging from mild febrile illness to classic dengue fever or the severe and potentially fatal dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) (23). Natural infection with any of the dengue virus serotypes provides only homotypic immunity, and available epidemiologic data suggest that a secondary infection with a heterologous serotype increases the risk for DHF/DSS. Global expansion of dengue virus infections in recent decades has made the development of vaccines for dengue viruses a public health priority. Traditional approaches to vaccine development, including live attenuated viruses, inactivated viruses, and subunit vaccines, have not yet produced a licensed vaccine. Several novel technologies, such as cloned engineered viruses and chimeric viruses using the yellow fever virus backbone, are at various stages of development.
Because of the increased risk for DHF/DSS during secondary dengue virus infections (10), it is necessary that a dengue vaccine provides protective immunity to all four serotypes. Current approaches depend on developing four monovalent vaccine candidates and mixing them to produce a final tetravalent vaccine. Vaccine approaches using live, replicating viruses have shown potential problems with mixed formulations, presumably stemming from serotype competition and/or dominance (7, 12, 24, 29). In an effort to circumvent this problem, we are developing nonreplicating DNA and recombinant viral vector vaccines against dengue viruses. We previously demonstrated that a dengue virus 1 DNA vaccine expressing the premembrane (prM) and full-length envelope (E) genes induced neutralizing antibodies in mice and in nonhuman primates (21, 22) and provided partial protection from the corresponding live virus challenge (22). It has generally been recognized that inadequate uptake of DNA vaccines by host cells and poor expression of antigen(s) are the leading causes of limited success with naked DNA vaccines. One approach to circumvent these apparent problems is to use replication-deficient recombinant viral vectors as vehicles for delivering DNA vaccines. Venezuelan equine encephalitis (VEE) virus-based viral vectors are especially attractive because of their efficient infection of cells of dendritic morphology that migrate to lymph nodes (14) and the lack of widespread preexisting immunity to VEE virus in human populations. VEE virus replicon particles (VRP) have been used successfully to produce immune responses to a number of viral, bacterial (13, 19, 25, 31, 34), and cancer (8, 16, 30) antigens in preclinical animal studies. In this study, we compared a dengue virus 1 naked DNA vaccine with a vaccine based on VRP. We also evaluated a heterologous prime-boost vaccine regimen in cynomolgus macaques. To our knowledge, this is the first report where a VRP-based vaccine candidate has been used in a heterologous prime-boost regimen.
MATERIALS AND METHODS
Vaccines. (i) D1ME-DNA.
The dengue virus 1 DNA vaccine (D1ME-DNA) was described previously (22). It contains the prM and full-length E genes of dengue virus 1 (strain Western Pacific 74) in the pVR1012 plasmid vector.
(ii) D1ME-VRP.
The vaccine D1ME-VRP is a recombinant VRP expressing the dengue virus 1 (strain Western Pacific 74) prM and E genes. To clone the dengue virus genes into the VEE replicon plasmid pERK, the dengue virus 1 sequence from D1ME-DNA was amplified by reverse transcription-PCR. The forward primer retained only the final G residue of the start codon (5′-GGCTGTGACCATGCTCCTCATGCTGC-3′). The reverse primer contained a unique AscI restriction enzyme site (5′-AGGCGCGCCATTCTTCATGGTCCGAAACACCG-3′). The amplicon was inserted into pERK between the EcoRV and AscI cleavage sites, thereby reconstituting the ATG start codon. Preparation of VRP was done essentially as previously described by Pushko et al. (18). The VRP was purified by heparin affinity chromatography, using a 5-ml Hi-Trap heparin column (Amersham Biosciences, Piscataway, NJ), and suspended in 4% sucrose and 1% normal human serum.
Animals.
Fourteen cynomolgus monkeys of either sex, aged 5 to 18 years, were housed at the Naval Medical Research Center/Walter Reed Army Institute of Research animal facility in Silver Spring, MD. The animals were prescreened for the presence of dengue virus-specific antibody by enzyme-linked immunosorbent assay (ELISA). Only those that did not show evidence of preexisting anti-dengue virus antibodies were included in the study. Monkeys were divided into groups DDD, VVV, DDV, and control and immunized with D1ME-DNA or D1ME-VRP as shown in the study design (Table 1). D1ME-DNA (1 mg in 1 ml phosphate-buffered saline [PBS]) was administered intramuscularly, with 0.5 ml given in each of the two upper arms, using a needle-free Biojector system (20). D1ME-VRP (108 IU diluted in 1 ml PBS just prior to use) was delivered intramuscularly by using a 27-gauge needle. Animals were bled periodically from the saphenous vein, and sera and peripheral blood mononuclear cells (PBMC) were used to assess humoral and cellular immune responses. Twenty weeks after the final immunization, animals were challenged by subcutaneous injection of 105 PFU of live dengue virus 1 (Western Pacific 74 strain) in a 0.5-ml volume. Animals were bled daily for 10 days, and sera were used to determine the presence of virus in the circulation (viremia). The experiments reported herein were conducted in compliance with the Animal Welfare Act and in accordance with the principles set forth in the Guide for the Care and Use of Laboratory Animals (15a).
TABLE 1.
Study design for vaccine administration to and virus challenge of cynomolgus macaquesa
| Group | n | Dose 1 (day 0) | Dose 2 (day 28) | Dose 3 (day 117) |
|---|---|---|---|---|
| Control | 3 | PBS | PBS | PBS |
| DDD | 3 | D1ME-DNA | D1ME-DNA | D1ME-DNA |
| VVV | 4 | D1ME-VRP | D1ME-VRP | D1ME-VRP |
| DDV | 4 | D1ME-DNA | D1ME-DNA | D1ME-VRP |
Groups of cynomolgus monkeys (n = 3 or 4) were vaccinated with three doses of D1ME-DNA (DDD), three doses of D1ME-VRP (VVV), or two doses of D1ME-DNA and one dose of D1ME-VRP (DDV) on the days indicated. All animals were challenged with dengue virus (type 1) 5 months after the final vaccination (day 252).
Antibody analysis by ELISA and plaque reduction neutralization test (PRNT).
Induction of dengue virus 1-specific immunoglobulin G (IgG) and IgM antibody responses in the individual monkeys was measured by ELISA as previously described (1), except that polyethylene glycol-precipitated dengue virus 1 virions were used as the coating antigen and a peroxidase-labeled anti-human IgG or IgM was used as the conjugate. Quantitative measurements of dengue virus-specific total IgG and IgG subclasses in monkey sera were made by ELISA as previously described by Williamson et al. (33). Briefly, monkey serum samples, diluted 1:2,000 in blocking buffer, were bound in duplicate to 96-well microtiter plates (Immulon 2HB) precoated with polyethylene glycol-precipitated dengue virus 1 virions in 1× PBS. Captured anti-dengue virus antibody was detected using horseradish peroxidase-labeled anti-human IgG (Bethyl Corp., TX) or anti-human IgG subclass antibody (The Binding Site, CA). Standard curves for total IgG were generated using a human IgG ELISA quantitation kit (Bethel Corp., TX). Standard curves for subclasses were generated by capturing known quantities of kappa fragments of IgG1, IgG2, or IgG4 (Sigma) on plates coated with anti-human Fab IgG (Sigma) and detecting them with horseradish peroxidase-conjugated anti-human IgG1, IgG2, or IgG4 (The Binding Site, CA).
Virus-neutralizing antibody titers were determined by PRNT as previously described (22), using Vero cells and serial twofold dilutions of serum samples. Individual monkey serum samples were used to determine neutralization titers. A prevaccination monkey serum pool was used as the negative control. Fifty percent PRNT titers (PRNT50s) were determined by probit analysis using Minitab software.
T-cell analysis by ELISPOT assay.
Gamma interferon (IFN-γ)-secreting T cells were enumerated using a monkey IFN-γ enzyme-linked immunospot (ELISPOT) assay kit (specific for rhesus and cynomolgus monkeys) from Mabtech (Mariemont, OH), using the manufacturer's recommended procedures. Briefly, plates were coated aseptically with the anti-IFN-γ monoclonal antibody GZ-4, incubated overnight at 4°C, washed with PBS, and blocked with RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS). PBMC were isolated from monkey blood by gradient centrifugation on Ficoll-Paque. PBMC (2 × 105 per well) were plated out in triplicate and stimulated with a purified preparation of dengue virus 1. Plates were incubated at 37°C in a 5% CO2 atmosphere for 2 days. The cells were removed, and the plates were washed five times with PBS. Biotinylated anti-IFN-γ monoclonal antibody 7-B6-1 was added to the plates at a concentration of 1 μg/ml and incubated for 2 hours at room temperature. The plates were then washed and further incubated with alkaline phosphatase-labeled streptavidin. Following 1 hour of incubation at room temperature and subsequent washing, a substrate solution (BCIP/NBT-Plus) was added for development of spots. The reaction was stopped by rinsing the plates extensively with tap water. After the plates had air dried, spots were enumerated using an AID ELISPOT reader (Cell Technology, Columbia, MD).
Viremia.
Sera collected from daily bleeds following challenge were used to detect viremia. Three hundred microliters of serum was diluted to 1 ml in Eagle's minimal essential medium (EMEM) supplemented with 2% FBS, penicillin, and streptomycin. Two T-25 flasks of subconfluent monolayers of Vero cells were inoculated with 0.5 ml of diluted serum and incubated at room temperature for 1 hour with gentle rocking. Four milliliters of EMEM containing 5% FBS was added to each flask. Cells were incubated at 37°C in a 5% CO2 atmosphere for 14 days, with a medium change on day 7. The cells were then scraped off the flask, washed with PBS, and placed as spots on immunofluorescence slides. Cells were fixed using cold acetone and processed for indirect immunofluorescence using the mouse monoclonal antibody 7E11 (specific for nonstructural protein NS-1) and fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin. Cells were examined under an Olympus fluorescence microscope for the presence or absence of dengue virus-specific antigen. With this method, a single PFU of virus could be detected by using serial dilutions of a virus stock of known titer, which resulted in a sensitivity of detection of about 7 PFU/ml. Although it is not quantitative, we have found this method to be reliable, sensitive, and reproducible compared to direct plaque assay with viremic sera or detection of viral RNA by real-time reverse transcription-PCR.
Statistical analyses.
Data from antibody analyses, ELISPOT assays, and viremia measurements were analyzed by one-way analysis of variance and Tukey's honestly significant difference and/or Fisher's least-significant-difference method for multiple mean comparisons. The ELISPOT data were square root transformed before analysis. All statistical analyses were performed using Minitab software.
RESULTS
Antibody response.
Dengue virus 1-specific antibody responses for each vaccination regimen were determined by ELISA and PRNT. All vaccinated animals had seroconverted after the first dose, as evidenced by ELISA positivity, but none contained detectable virus-neutralizing antibody (not shown). After the second dose, only two animals vaccinated with D1ME-VRP (VV) showed moderate neutralizing antibodies against dengue virus 1. After the third dose of vaccine, all animals in the DDD group, VVV group, and DDV group had developed neutralizing antibodies.
There were no significant differences in total dengue virus-specific IgG between the DDD group (mean = 53.6 μg/ml) and the VVV group (mean = 53.5 μg/ml) 4 weeks after the final dose (Table 2). Dengue virus-specific IgG was about twofold higher in the DDV group. IgG subclass analysis revealed that both IgG1 and IgG2 levels were significantly higher in the DDV group than in the DDD group (P = 0.05). The DDV group also had higher IgG1 and IgG2 levels than the VVV group, but the difference was not statistically significant (P > 0.05). The dengue virus 1-specific IgG response to all vaccination regimens was predominantly of the IgG1 type, with smaller amounts of IgG2 and no measurable amounts of IgG4 (not shown). IgG3 was not measured because it has been reported that the macaque genome does not have an equivalent of human IgG3 (3, 26). It should be noted that the human-specific reagents used in this analysis have been reported to react with primate antibodies at about 10% efficiency (E. D. Williamson, personal communication).
TABLE 2.
Dengue virus 1-specific total IgG and IgG subclass levels in vaccinated animalsa
| Vaccine group | Animal | Antibody level (μg/ml)
|
|||||
|---|---|---|---|---|---|---|---|
| IgG | Group mean (SE) | IgG1 | Group mean (SE) | IgG2 | Group mean (SE) | ||
| DDD | 16676 | 78 | 53.6 (12.4) | 53 | 30.6 (11.3) | 11 | 4.0 (3.5) |
| 16678 | 37 | 16 | 0 | ||||
| AV9K | 46 | 23 | 1 | ||||
| VVV | 16904 | 106 | 53.5 (19.6) | 83 | 35.2 (16.9) | 20 | 6.0 (4.6) |
| 16796 | 48 | 28 | 2 | ||||
| 16642 | 49 | 27 | 2 | ||||
| AA4B | 11 | 3 | 0 | ||||
| DDV | 16620 | 70 | 101.5 (12.9) | 46 | 94.5 (18.6) | 12 | 27.5 (5.8) |
| 16871 | 111 | 110 | 25 | ||||
| 16881 | 94 | 88 | 37 | ||||
| AB1H | 131 | 134 | 36 | ||||
Serum samples obtained on day 140 (about 4 weeks after the final immunization) were tested for dengue virus 1-specific total IgG and IgG subclass antibodies as described in Materials and Methods.
The ability of vaccine-induced antibodies to neutralize dengue virus 1 was measured by PRNT. PRNT50s were determined by probit analysis. PRNT50s for individual animals before and after virus challenge, as well as at the time of virus challenge, are shown in Table 3. About 4 weeks after the final vaccination (day 140), all vaccinated animals, with the exception of one animal in the VVV group, demonstrated appreciable levels of dengue virus-neutralizing antibody. The neutralization titers declined somewhat by the time the animals were challenged. Although there was a general trend of higher neutralization titers in the prime-boost vaccinated group, the differences in mean titers among the groups were not statistically significant (P = 0.184). It should be noted that animal AA4B, which did not develop appreciable virus-neutralizing antibody, had low dengue virus 1-specific IgG compared to that of the other animals (Table 2). No neutralizing antibodies were detected in the control group inoculated with PBS (not shown).
TABLE 3.
Virus-neutralizing antibodies in vaccinated monkeysa
| Vaccine group | Animal | PRNT50
|
||
|---|---|---|---|---|
| Day 140 | Day 252 | Day 262 | ||
| DDD | 16676 | 1,569 | 983 | 6,181 |
| 16678 | 1,014 | <80 | 6,483 | |
| AV9K | 1,510 | 668 | 2,455 | |
| VVV | 16904 | 2,369 | 581 | 5,819 |
| 16796 | 1,321 | 582 | 3,605 | |
| 16642 | 1,933 | 1,475 | 4,163 | |
| AA4B | <80 | <40 | 4,584 | |
| DDV | 16620 | 2,178 | 1,028 | 5,151 |
| 16871 | 2,509 | 2,638 | 3,675 | |
| 16881 | 2,820 | 1,696 | 4,082 | |
| AA3W | 1,707 | 582 | 3,786 | |
Sera obtained on day 140, day 252 (day of virus challenge), and day 262 (10 days postchallenge) were used to determine virus neutralization titers by PRNT. PRNT50s were determined by probit analysis. “<x” denotes <50% neutralization at the lowest dilution (x) used in the assay. Data are representative of two independent determinations.
To assess dengue virus serotype cross-neutralizing antibodies, sera from animals 16676 (DDD group), 16904 (VVV group), and 16681 (DDV group) were tested for neutralization of dengue virus types 2, 3, and 4. On day 140, low-level neutralizing antibodies were observed for dengue virus 2 (PRNT50s of 0 to 170), dengue virus 3 (PRNT50s of 0 to 160), and dengue virus 4 (PRNT50s of 30 to 130). However, cross-neutralizing antibodies had waned to undetectable levels by day 252 (day of virus challenge).
There was a significant increase in neutralizing antibody titers after live virus challenge (at day 10 postchallenge) for all vaccination regimens, indicating an anamnestic antibody response, including for animal AA4B, for which neutralizing antibodies could not be demonstrated prior to virus challenge. An anamnestic antibody response was also evident by measurement of dengue virus-specific IgM and IgG after virus challenge. All vaccinated groups showed an increase in IgG levels without accumulation of IgM antibody (Fig. 1). Control animals showed a slow accumulation of IgM antibody without detectable IgG (a primary antibody response).
FIG. 1.
Primary and secondary antibody responses in control and vaccinated cynomolgus monkeys following virus challenge. Sera obtained from daily bleeds after virus challenge were diluted 1:100 (IgM) (A) or 1:1,000 (IgG) (B), and relative levels of IgG and IgM were determined by standard ELISA as described in the text. The mean optical density at 405 nm for each group ± standard error is shown.
Induction of cell-mediated immune response.
To monitor the cell-mediated immune response to various vaccine regimens and virus challenge, the frequency of IFN-γ-producing cells when PBMC were stimulated by dengue virus 1 antigen in vitro was measured by ELISPOT assay. The results (Table 4) are expressed as numbers of spots per 2 × 105 cells. Eight weeks after the final vaccine dose (day 168), the D1ME-VRP vaccine (VVV group) failed to elicit T-cell responses. The VVV group was similar to the control group of animals. The other two vaccine regimens (DDD and DDV) elicited moderate but significantly higher T-cell responses (P = 0.008) than those for the control group. The difference between the DDD and DDV groups was not statistically significant. These responses increased after dengue virus challenge (day 285 or 33 days postchallenge).
TABLE 4.
IFN-γ ELISPOT assay of cells from control and vaccinated cynomolgus monkeysa
| Vaccine group | Animal | No. of spots/2 × 105 PBMC
|
|
|---|---|---|---|
| Day 168 | Day 285 | ||
| Control | 16675 | 0 | 15 |
| 16864 | 8 | 35 | |
| AA3W | 2 | 18 | |
| DDD | 16676 | 60 | 171 |
| 16678 | 25 | 98 | |
| AV9K | 77 | 155 | |
| VVV | 16904 | 8 | 5 |
| 16796 | 4 | 24 | |
| 16642 | 9 | 29 | |
| AA4B | 10 | 12 | |
| DDV | 16620 | 111 | 179 |
| 16871 | 14 | 77 | |
| 16881 | 41 | 32 | |
| AB1H | 30 | 109 | |
PBMC (2 × 105) obtained on day 168 (about 7 weeks after the final vaccination) and day 285 (about 5 weeks after virus challenge) were stimulated in triplicate in vitro, using purified dengue virus 1 antigen, and IFN-γ-secreting cells were enumerated by ELISPOT assay as described in Materials and Methods. Numbers of spots were corrected for background spots visualized in the absence of dengue virus 1 antigen.
Protection from live virus challenge.
To determine the protective efficacy of vaccine-induced immune responses, all monkeys were challenged with wild-type dengue virus (type 1) 5 months after the last dose of vaccine. The animals were bled daily for 10 days, and viremia was assayed by cell culture inoculation of sera followed by immunofluorescence assay (Table 5). All mock-immunized monkeys developed viremia for 6 to 7 days following challenge (mean time of viremia = 6.3 days). All four DDV group animals were completely protected from viremia (mean time of viremia = 0 days). Two of three DDD group animals and three of four VVV group animals developed a single day of viremia, for group means of 0.66 and 0.75 day, respectively. All vaccination regimens showed significant protection from viremia (P = 0) compared to that of the unvaccinated control group. However, the differences among different vaccination regimens were not statistically significant.
TABLE 5.
Viremia in control and vaccinated animals after live virus challengea
| Vaccine group | Animal | Presence of virus in serum on day:
|
Total time (days) of viremia | Mean time (days) of viremia | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | ||||
| PBS | 16675 | − | + | − | + | + | + | + | + | + | − | − | 7 | 6.33 |
| 16864 | − | − | − | + | + | + | + | + | + | − | − | 6 | ||
| AA3W | − | − | + | − | − | − | + | + | + | + | + | 6 | ||
| DDD | 16676 | − | − | − | − | − | − | − | + | − | − | − | 1 | 0.66 |
| 16678 | − | − | − | − | − | − | − | − | − | − | − | 0 | ||
| AV9K | − | − | + | − | − | − | − | − | − | − | − | 1 | ||
| VVV | 16904 | − | − | − | − | + | − | − | − | − | − | 1 | 0.75 | |
| 16796 | − | − | − | − | − | − | − | − | − | − | − | 0 | ||
| 16642 | − | − | − | − | − | − | − | − | + | − | − | 1 | ||
| AA4B | − | − | − | − | − | − | + | − | − | − | − | 1 | ||
| DDV | 16620 | − | − | − | − | − | − | − | − | − | − | − | 0 | 0.00 |
| 16871 | − | − | − | − | − | − | − | − | − | − | − | 0 | ||
| 16881 | − | − | − | − | − | − | − | − | − | − | − | 0 | ||
| AB1H | − | − | − | − | − | − | − | − | − | − | − | 0 | ||
Animals were vaccinated and challenged with live dengue virus 1 as described in Materials and Methods. Viremia for each animal is shown. + and −, presence and absence of virus, respectively.
DISCUSSION
Heterologous prime-boost strategies involving priming by DNA vaccination and boosting with recombinant vectors encoding common antigens is a prominent approach to enhancing DNA vaccines (5, 6). Many viral vectors, including fowlpox virus (5, 11), modified vaccinia virus Ankara (15, 32), and adenovirus (27), have been used to enhance the immunogenicity of human immunodeficiency virus (HIV) DNA vaccines. Recombinant VRP are attractive because of their ability to infect antigen-presenting cells of dendritic morphology and because of the absence of widespread preexisting immunity to VEE virus in human populations. A VRP expressing protective antigen of Bacillus anthracis was shown to protect mice against anthrax spore challenge (19). Hevey et al. (31) demonstrated that in two animal models (guinea pigs and cynomolgus monkeys), a VRP expressing Marburg virus antigen produced antibody and protected animals from Marburg virus challenge. VRP expressing dual antigens of Lassa virus and Ebola virus produced antibodies to both antigens and protected animals from either challenge (17). This is especially relevant to dengue vaccines, where immunity to all four dengue virus serotypes is required. The ability to produce multivalent VRP will reduce the complexity of a tetravalent dengue vaccine.
Although VRP-based vaccines have been used in a number of animal models with a variety of antigens, to our knowledge a VRP has not been used as a boosting agent in a heterologous prime-boost regimen. In this study, we conducted a head-to-head comparison of three vaccination regimens using two vaccine constructs, including a prime-boost regimen using DNA and VRP vaccines. The prM and E genes of dengue virus 1 were expressed from a plasmid DNA vaccine or from a VRP. Our earlier studies with a BALB/c mouse model (unpublished) had shown that the DNA vaccine elicited neutralizing antibodies that persisted over long periods of time but induced only poor to modest T-cell responses, whereas the VRP vaccine elicited neutralizing antibodies that declined over time but induced strong T-cell responses. Mice immunized with a DNA prime-VRP boost regimen elicited both long-lasting neutralizing antibodies and strong T-cell responses. However, in the nonhuman primate model described in this study, the VRP vaccine failed to induce measurable T-cell responses. In contrast, the DNA vaccine induced moderate T-cell responses. The T-cell responses observed in animals vaccinated with the DNA prime-VRP boost regimen were probably due to the DNA component. This discrepancy between the two animal models is reminiscent of studies by Caley et al. (2) with an HIV vaccine and Wilson and Hart (34) with an Ebola virus vaccine. In both of these studies, whereas the VRP-based vaccines produced robust antibody and T-cell responses in murine models, only modest T-cell responses in a subset of vaccinated nonhuman primates were demonstrated. Because viral vectors are expected to efficiently present immunogens to the cellular arm of the immune system, the lack of robust T-cell responses to immunogens in the primate model needs further investigation. The DNA-vaccinated (DDD) and prime-boost-vaccinated (DDV) animals exhibited modest T-cell responses, as measured by IFN-γ ELISPOT assay. Modest T-cell responses have previously been reported for rhesus macaques vaccinated with the D1ME-DNA vaccine (22). These data indicate that the DNA vaccine is responsible for the T-cell response observed in those animals. It is interesting that the DNA vaccine induced only poor T-cell responses in mice.
We have demonstrated that all three vaccination regimens used in this study elicited neutralizing antibody responses in cynomolgus macaques. The predominant IgG in all vaccinated macaques was IgG1. This is in contrast to our previous study with mice injected with a dengue virus 2 DNA vaccine, live dengue virus 2, or a recombinant protein antigen (28). Although all antigen formats produced neutralizing antibodies in mice, the DNA vaccine and the live virus produced predominantly IgG2a responses, whereas the recombinant protein produced an almost exclusively IgG1 response. There have been no systematic studies on the relationship between virus neutralization and IgG subclass in nonhuman primates or humans. However, it is clear from this study that, in general, higher IgG1 and IgG2 responses are associated with higher neutralizing antibodies (not shown).
The prime-boost-vaccinated animals exhibited the highest neutralization titers and sustained high titers over a period of 20 weeks, at which time the animals were challenged. This vaccination regimen also preserved the T-cell response (compared to the VRP vaccine). Prime-boost-vaccinated animals were also completely protected from viremia upon challenge. Other vaccine regimens (DDD and VVV) also provided significant protection compared to that of control animals. Although a direct correlation between virus neutralization titers at the time of challenge and viremia has been difficult to establish, higher neutralization titers are usually associated with better protection from viremia. Although statistical significance could not be demonstrated for differences in neutralization titers (perhaps due to small group sizes), the generally higher (about twofold) neutralizing antibody titers in prime-boost-vaccinated animals may have contributed to the complete protection of these animals. The precise role of T-cell responses in dengue virus pathogenesis and immunity is not fully understood, making it difficult to assess the contribution of T-cell responses to protection from viremia. Taken together, these data suggest the utility of VEE virus replicons as well as the prime-boost vaccination regimen in the development of dengue vaccines.
Acknowledgments
We thank Craig Morrissette of Walter Reed Army Institute of Research for help with statistical analysis of data.
This work was funded by the Office of Naval Research Agile Vaccines Program.
The corresponding author is an employee of the U.S. Government, and this work was prepared as part of his official duties. Title 17 U.S.C. §105 provides that “copyright protection under this title is not available for any work of the United States Government.” Title 17 U.S.C. §101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person's official duties. The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, or the U.S. Government.
Footnotes
Published ahead of print on 22 August 2007.
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