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Clin Vaccine Immunol. Nov 2009; 16(11): 1624–1632.
Published online Sep 23, 2009. doi:  10.1128/CVI.00182-09
PMCID: PMC2772385

A Complex Adenovirus-Vectored Vaccine against Rift Valley Fever Virus Protects Mice against Lethal Infection in the Presence of Preexisting Vector Immunity[down-pointing small open triangle]

Abstract

Rift Valley fever virus (RVFV) has been cited as a potential biological-weapon threat due to the serious and fatal disease it causes in humans and animals and the fact that this mosquito-borne virus can be lethal in an aerosolized form. Current human and veterinary vaccines against RVFV, however, are outdated, inefficient, and unsafe. We have incorporated the RVFV glycoprotein genes into a nonreplicating complex adenovirus (CAdVax) vector platform to develop a novel RVFV vaccine. Mice vaccinated with the CAdVax-based vaccine produced potent humoral immune responses and were protected against lethal RVFV infection. Additionally, protection was elicited in mice despite preexisting immunity to the adenovirus vector.

Rift Valley fever virus (RVFV) is an arthropod-borne bunyavirus that can cause severe disease in humans. Disease symptoms range from benign flulike symptoms to more severe disease involving retinal lesions, hemorrhagic fever, or encephalitis, and disease may be fatal (1, 4, 37). Outbreaks among livestock, often signaled by mass spontaneous abortions, can have a significant economic impact. RVFV outbreaks historically have been limited to sub-Saharan Africa but are highly transmissible and have since spread into other regions, including Saudi Arabia and Yemen (2, 34, 36, 47, 48). Additionally, the severity of human RVFV outbreaks may be increasing; among identified cases in an outbreak in Sudan in late 2007, the mortality rate reached 35% (49). Human infection with RVFV occurs most commonly through contact with infected animals but can also result from mosquito bites during periods when the virus is circulating at high densities in these hosts. Accidental infection of scientists in a laboratory setting, however, indicates that the virus is fully capable of aerosol transmission (15, 39), and this has also been demonstrated with animal models (5, 7).

Control of the arthropod host and immunization of livestock might be effective approaches to limit natural outbreaks of the disease, but no effective and safe vaccine is yet available. More importantly, these approaches do not address the threat that RVFV poses as an agent for bioterrorism. The virus is widespread in diverse parts of Africa and can be propagated easily and efficiently in vitro (14). Because of its disease potential, aerosolized RVFV could be used as a weapon to threaten human life and to devastate livestock and the economy (11, 39). Release of aerosolized RVFV in confined spaces, such as public buildings and subways, may enhance spread of the virus among civilians. With such potential in mind, RVFV has been placed on the CDC's list of select agents and is an NIAID category A priority pathogen. Therefore, developing a vaccine protective against RVFV infection for human use is a critical strategy to address this threat.

It is widely agreed that an effective RVFV vaccine should elicit potent neutralizing antibodies and provide complete sterilizing immunity. Prior studies have shown that passive transfer of immune sera completely protects naïve mice from lethal challenge with RVFV (35). Yet several RVFV vaccines fail to elicit a potent neutralizing antibody response or are deemed inappropriate for human vaccination due to safety concerns. While live-attenuated virus vaccines have been used against RVFV for livestock, these vaccines are unacceptable for human use, since they are known to cause abortions in cattle (6) and are teratogenic in sheep (23). A formalin-inactivated RVFV vaccine was developed in the 1980s for use with military personnel (25), but it is very weakly immunogenic, requiring a series of booster immunizations (16, 31). Other attenuated virus vaccines have been developed and tested with various animal species (9, 28, 29, 43), but these vaccines are considered unsafe for human use given their potential to revert to the pathogenic virus.

In an attempt to develop safer and more potent RVFV vaccines, several subunit and recombinant vaccine approaches have been explored. RVFV is an enveloped phlebovirus of the Bunyaviridae family that carries two glycoproteins on its surface, and these viral components are the likely targets for a protective immune response. These N-terminal and C-terminal glycoproteins, named Gn and Gc, respectively, are encoded on the M segment of the genome and are synthesized as part of polyprotein precursors, which can also include an additional 14-kDa N-terminal component depending on selection of alternative translation initiation codons (17, 24). Antibodies against Gn and Gc effectively neutralize virus by blocking virus-receptor interaction and cell entry events and may also play a role in complement-mediated clearance of virus. It was shown previously that lysates of insect cells infected with a baculovirus expressing the M segment elicited a protective immune response to RVFV in mice (35). Importantly, protection against challenge could be provided to naïve mice via passive transfer of immune sera, thus demonstrating the key role of neutralizing antibodies in providing protection against RVFV infection and the importance of Gn and Gc as antigens in eliciting a potent humoral immune response.

Recently, Wallace et al. compared three different vaccination approaches for RVFV: a DNA vaccine, a recombinant-protein vaccine, and a recombinant lumpy skin disease virus (rLSDV) vector vaccine expressing Gn and Gc (44). Both a recombinant form of the Gc protein and the rLSDV-RVFV vector protected mice from challenge with RVFV after a single immunization. While the use of rLSDV-RVFV is feasible only as a potential veterinary vaccine against RVFV and sheeppox virus, the result supports the use of RVFV glycoproteins for a vaccine. Expression of the Gc protein alone using a Venezuelan equine encephalitis virus (VEEV) replicon provided protection against RVFV challenge in mice following a single injection (19). Mice were also protected from challenge following immunization with a VEEV expressing a 318-amino-acid fragment of Gc fused to the E2 glycoprotein of the vector. However, the authors reported difficulties in propagation of the Gn-expressing VEEV replicons and suggested that Gn vectors would be problematic for large-scale manufacturing. Another promising vaccine candidate is RVFV virus-like particles, assembled from envelope and nucleocapsid proteins of the virus but lacking an intact viral genome (20). These replication-deficient virus-like particles have recently been shown to protect against RVFV challenge in mice that received a series of three immunizations, but their production on a large scale remains a challenge (30).

To address the lack of a safe, effective, and practical RVFV vaccine, we have utilized a proven vaccine platform based on our complex adenovirus (CAdVax) vector system. Adenovirus (Ad) vectors are widely studied viral vectors for vaccines and gene therapy applications and have been tested in hundreds of clinical trials worldwide, with a favorable safety profile (18). Ad vectors are capable of expressing high levels of transgenes, are efficiently produced in large quantities, and are highly immunogenic as vaccines. An additional capability of the CAdVax platform is the expression of multiple antigen inserts independently. RVFV glycoproteins have shown promise as targets of neutralizing immunity and form the primary surface components of RVFV. Since neutralizing antibodies against the RVFV envelope glycoproteins are the key component of protective immunity against RVFV infection, we developed and tested a CAdVax vaccine to direct simultaneous, de novo expression of the Gn and Gc glycoproteins. We found this vaccine to be highly immunogenic in mice, eliciting strong immune responses and providing complete protection against lethal challenge with RVFV. The CAdVax-RVF vaccine also showed efficacy in animals that were previously immunized with a heterologous CAdVax vector, suggesting that preexisting immunity to the Ad type 5 (Ad5) vector has little effect on limiting induction of a potent immune response to CAdVax vector antigens in vaccinated mice. Here, we present the results of these experiments and discuss their implications for the development and large-scale production of a safe and effective RVFV vaccine.

MATERIALS AND METHODS

Cell lines and viruses.

BS-C-1, HEK293, and A549 cells were purchased from ATCC and were cultured as recommended. The virulent ZH501 strain of RVFV was obtained from the University of Texas Medical Branch (UTMB, Galveston, TX) and was propagated at UTMB in Vero E6 cells under biosafety level 4 (BSL4) containment.

Construction of CAdVax-RVF.

The coding sequences for the mature RVFV Gn and Gc proteins (41) (GenBank accession no. NC_002044, strain ZH548M12) were synthesized separately, each with the human CD4 signal sequence fused upstream and with codons optimized for human cells (Geneart, Regensburg, Germany). The Gn and Gc genes were inserted into CAdVax vectors as previously described, under the control of the cytomegalovirus (CMV) promoter (32). Gene sequences of the completed bivalent CAdVax-RVF vector were analyzed and confirmed by restriction enzyme digestion, PCR analysis using primers specific to the Gn and Gc sequences, and sequencing analysis. The final preparation of CAdVax-RVF was titrated on HEK293 cells by use of a serial dilution plaque assay, and results were scored as PFU per ml. These procedures have been described in detail previously (32, 33). As controls for individual expression of Gn and Gc, retrovirus vectors expressing each RVFV glycoprotein were constructed from the QCXIN retroviral expression system by use of BamHI cloning sites, according to the manufacturer's protocol (Clontech).

Immunoblot analysis.

HEK293 cells were infected with CAdVax-RVF or a control CAdVax vector expressing dengue virus glycoproteins, CAdVax-D (22), at a multiplicity of infection of 20 for 48 h. As a control for expression, retrovirus vectors independently expressing either Gn or Gc were used to infect BS-C-1 cells in a similar manner. After infection, cell lysates were prepared as previously described (21). Cell lysates (35 μg) from CAdVax-RVF- and control-treated cells were separated on 4 to 20% Tris-HCl gradient gels (Bio-Rad) and transferred to polyvinylidene difluoride membranes (Millipore). Membranes were probed with rabbit anti-RVFV(CT) (ProSci catalog no. 4521) or rabbit anti-RVFV(IN) (ProSci catalog no. 4519), polyclonal antibodies against the Gc or Gn glycoprotein, respectively. The proteins were detected using a horseradish peroxidase-conjugated secondary antibody (Kirkegaard & Perry Laboratories, Inc.) and a 3,3′,5,5′-tetramethylbenzidine liquid substrate system for membranes (Sigma-Aldrich).

Mouse vaccination and collection of immune sera.

Male, 6-week-old CD-1 mice (Charles River Laboratory, Raleigh, NC) were injected intraperitoneally (i.p.) with 1 × 108 PFU (1 × 1010 total viral particles) of CAdVax-RVF or control CAdVax-D on week 0. CAdVax-D is a vector with a backbone identical to that of CAdVax-RVF that expresses dengue virus glycoproteins (22). Some mice also received booster vaccinations of the same dose on week 10. For the preexisting vector immunity study, CD-1 mice (n = 8) were immunized by i.p. injection with 1 × 109 PFU of CAdVax-D on week −10. A matched group of eight mice was maintained as a vector-naïve control group. At week 0, both groups of mice were subdivided into two additional groups and received i.p. injections of a low dose (2 × 106 PFU) or a high dose (2 × 108 PFU) of CAdVax-RVF. The animals received a booster vaccination of CAdVax-RVF on week 15. Blood from vaccinated animals was collected biweekly from the retroorbital sinus, and immune sera were prepared as previously described (21). All animals were maintained according to NIH and IACUC regulations.

ELISA.

To eliminate the use of live RVFV, we used CAdVax-RVF to generate target antigens that require only BSL2 containment. Monolayers of BS-C-1 cells were transduced with CAdVax-RVF at a multiplicity of infection of 20 for 48 h in serum-free medium. Cells were then harvested, and lysates were prepared as previously described (21). The cell lysate was diluted in phosphate-buffered saline to a concentration of 5 μg/ml, and 50 μl per well was added to Maxisorp microtiter plates (Nunc, Rochester, NY). After incubation overnight at 37°C, the enzyme-linked immunosorbent assays (ELISAs) were performed as described previously (46). Antibody titers for each mouse were determined by calculating the dilution of serum that corresponded to an absorbance of twice the background. The background ELISA level was determined from the mean A450 value of CAdVax-D-immunized mouse serum at a 1:200 dilution against the lysates of CAdVax-RVF-infected BS-C-1 cells.

Statistical analysis.

To measure the statistical significance of antibody titers at different time points, a sample-dependent, two-tailed Student t test was conducted.

Ad neutralization assay.

Serial dilutions of heat-inactivated mouse sera were incubated with 100 PFU of a CAdVax vector expressing green fluorescent protein for 1 h at 37°C. Virus-serum mixtures were then added to each well of 96-well plates containing A549 cells and cultured for 72 h at 37°C with 5% CO2. Ad infection was scored by counting green fluorescent protein-expressing cells under a UV fluorescent microscope. Neutralizing antibody titers were determined by the inverse of the dilution of serum that neutralized 50% of the virus infectivity.

Mouse challenge infection with RVFV.

Mouse challenge studies with RVFV were performed in the Robert E. Shope BSL4 animal laboratory at UTMB. Vaccinated and control animals were inoculated with 100 PFU of the ZH501 strain of RVFV by i.p. injection. Mice were monitored for a period of 14 days after inoculation. All animals used in the challenge experiments were maintained under IACUC-approved protocols and NIH guidelines.

RESULTS

Design and characterization of CAdVax-RVF.

The Gn and Gc antigens of RVFV are the major viral surface glycoproteins and act as primary targets for neutralizing antibodies during infection. Our goal was to mimic an RVFV infection without disease by expressing Gn and Gc in transduced cells at high levels. To achieve expression of both Gn and Gc, we took advantage of the multiple-insert capability of the CAdVax vector and inserted both Gn and Gc genes into a single vector (Fig. (Fig.1),1), with both genes under the control of identical CMV immediate-early promoters and bovine growth hormone polyadenylation (BGHpA) signals. Placing the Gn and Gc genes at opposite ends of the CAdVax genome allows balanced expression of the two genes in a nearly 1:1 ratio.

FIG. 1.
Genome structure of the recombinant CAdVax-RVF vaccine. The dual-gene CAdVax-RVF vaccine is designed to express the RVFV Gn and Gc genes simultaneously. Each gene is controlled by its own CMV immediate-early promoter and BGHpA sequence. The CAdVax vector ...

CAdVax-RVF was produced and isolated using HEK293 cells, and the presence of both the Gn and the Gc gene in the bivalent-vaccine construct was confirmed by PCR using primers specific to the Gn or the Gc gene (Fig. (Fig.2A)2A) and by restriction enzyme analysis (not shown). The PCRs produced DNA fragments consistent with the sizes of the RVFV Gn and Gc genes (1,592 bp, and 1,525 bp, respectively) and the anticipated DNA fragment between the flanking CMV promoter and BGHpA sequences (Fig. (Fig.2A).2A). Although it is difficult to see the separation of the bands given the similarity in size of the Gn and Gc genes using the flanking primers, the specificity of the Gn and Gc genes was confirmed using specific primer sequences and further corroborated using CAdVax-D as a negative control. Restriction enzyme analysis and DNA sequencing were used to confirm the presence of both genes, consistent with the design shown in Fig. Fig.11.

FIG. 2.
In vitro analysis of purified CAdVax-RVF. (A) PCR analysis using primers specific for RVFV Gn and Gc genes, located in the right and left ends of the CAdVax genome, respectively. Primers specific for the flanking CMV and BGHpA sequences were used (Flank); ...

The expression of both Gn and Gc gene products in transduced cells was then analyzed by Western blotting (Fig. 2B and C). Antibodies used for immunoblotting were specific to either the amino terminus [anti-RVFV(IN)] (Fig. (Fig.2B)2B) or the center [anti-RVFV(CT)] (Fig. (Fig.2C)2C) of the polyprotein precursor of the RVFV M segment and recognized either the Gn or the Gc glycoprotein, respectively. The anti-RVFV(IN) antibody against Gn detected a protein band in cells transduced with CAdVax-RVF, while no specific band was detected in mock- or control vector-transduced cells. Similarly, the anti-RVFV(CT) antibody detected a protein band in cells transduced with CAdVax-RVF at the anticipated size of Gc but not in the mock- or control CAdVax vector-transduced cells. In addition, cells transduced with retrovirus vector controls independently expressing either Gn or Gc served as an internal positive control for the recognition of each RVFV glycoprotein. The sizes of the bands are consistent with correct processing of the Gn (57-kDa) and Gc (55-kDa) proteins. There is some residual background binding observed with the polyclonal anti-RVFV(CT) antibody, especially in the retrovirus vector-transduced cells, but binding to a protein band at the anticipated size of Gc is clearly evident in the CAdVax-RVF- and the Gc retrovirus vector-transduced cells. Collectively, these data indicated that the bivalent CAdVax-RVF vaccine contained both Gn and Gc genes and that both gene products were expressed in transduced cells.

CAdVax-RVF induces protective immune responses in vaccinated mice.

To measure the immunogenicity of CAdVax-RVF, CD-1 mice were vaccinated with 1 × 108 PFU CAdVax-RVF or CAdVax-D as a control. Mouse sera were collected at 2-week intervals, and seroconversion was analyzed using ELISA against cell lysates containing recombinant Gn and Gc proteins (see Materials and Methods). Sera from mice vaccinated with CAdVax-RVF showed strong reactivity against the RVFV glycoprotein antigen, in contrast to sera from animals vaccinated with control CAdVax-D (Fig. (Fig.3A).3A). Due to the nonreplicating nature of the CAdVax platform and the inability of CAdVax to express Ad proteins, background binding against Ad antigens was negligible. The anti-RVFV antibody titers were detectable 2 weeks after vaccination and reached peak levels around 8 weeks. By week 10, the antibody titers appeared to begin to decline (two-tailed, sample-dependent Student t test P value of <0.0001) but remained at a level significantly higher than that at prevaccination. This suggested that the primary immune response may be in transition to the memory immune response phase and that booster immunization might be beneficial at the 10-week time point.

FIG. 3.
CAdVax-RVF induces antibody responses against Gn and Gc in vaccinated mice. (A) CD-1 mice were vaccinated with 1 × 108 PFU of CAdVax-RVF or CAdVax-D on week 0. (B) A second group of CD-1 mice was vaccinated as described above on week 0, except ...

To assess the protective capacity of the immune response generated by CAdVax-RVF, the same animals described above for Fig. Fig.3A3A were challenged with 100 PFU of the virulent ZH501 strain of RVFV 11 weeks after vaccination. The lethal ZH501 strain used for challenge has over 99% amino acid homology to the ZH548M12 strain used to generate antigens for CAdVax-RVF. Although antibody levels in these vaccinated animals had begun to decline by week 11, all vaccinated mice (n = 8) survived the lethal infection, while all control mice died between 4 and 10 days after infection (Table (Table1).1). Therefore, we can conclude that the CAdVax-RVF vaccine is immunogenic in mice and is capable of inducing protective immunity against lethal RVFV infection after only a single-dose vaccination.

TABLE 1.
Vaccinated mice survive lethal RVFV challengea

CAdVax-RVF induces long-term immunity against RVFV.

To assess the long-term protective immunity developed against RVFV, we investigated the feasibility of booster vaccination with the same CAdVax-RVF vaccine. We vaccinated a group of CD-1 mice at week 0 with CAdVax-RVF or CAdVax-D. As before, we found that the anti-Gn/Gc antibody titers reached peak levels at about 8 weeks and began to decline at 10 weeks postvaccination (Fig. (Fig.3B).3B). In this experiment, however, the animals received booster immunizations on week 10 with the same CAdVax-RVF vaccine. This induced a robust increase in the anti-Gn/Gc antibody titers of animals that received CAdVax-RVF, which was maintained through week 26 (up to 4 months postboost) (Fig. (Fig.3B).3B). This result confirmed the feasibility of boosting the immune response with the same CAdVax-RVF vaccine to elicit a durable antibody response.

We tested long-term protection with a challenge experiment at 27 weeks after vaccination. Similarly to the previous challenge experiment, all animals vaccinated with CAdVax-RVF survived challenge, with no evidence of acute or delayed disease for up to 14 days after challenge infection (Table (Table1).1). On the other hand, animals vaccinated with CAdVax-D died from lethal infection within 3 to 10 days. Based on these results, we conclude that a prime-boost vaccination with the CAdVax-RVF is effective and offers long-term protection of mice against lethal RVFV infection.

CAdVax-RVF vaccination protects mice with preexisting vector immunity.

A major concern regarding Ad5-based vaccines (such as CAdVax) is the possibility that preexisting immunity against Ad might interfere with vaccine efficacy. To investigate this concern, we performed CAdVax-RVF immunizations of mice with preexisting antivector antibodies (Fig. (Fig.4).4). Since Ad5 does not replicate in mice, we inoculated CD-1 mice with 1.1 × 109 PFU of CAdVax-D on week −10 to induce an antivector immune response. Ten weeks after inoculation (week 0), both ELISA and Ad neutralization tests showed that all vector-immune mice had very high levels of antivector antibodies compared to those of vector-naïve animals (Fig. (Fig.4B4B).

FIG. 4.
Vaccination in the presence of antivector immunity. (A) Schematic of experimental design. Mice were primed with 1 × 109 PFU of an unrelated CAdVax vector on week −10 to induce antivector immune responses. Naïve animals were maintained ...

On week 0, the same animals were separated into two groups and vaccinated with either a low dose (2 × 106 PFU) or a high dose (2 × 108 PFU) of the CAdVax-RVF vaccine (Fig. (Fig.4A).4A). As a control, naïve mice were also vaccinated on week 0 with matching doses of the CAdVax-RVF vaccine. ELISA for antibodies against Gn/Gc showed that the vector-naïve mice appeared to mount similar antibody responses, regardless of whether the animals received a low- or a high-dose immunization (Fig. (Fig.4C).4C). This suggests that both low-dose and high-dose vaccinations with CAdVax induce strong immunity in animals with no prior antivector immunity. The vector-immune animals, however, showed dose-dependent differences in antibody response profiles. Anti-RVFV titers in vector-immune mice receiving the low dose of CAdVax-RVF were elevated 2 weeks after vaccination but quickly declined over the next several weeks (Fig. (Fig.4C).4C). Because the same low dose of vaccine was highly immunogenic in the vector-naïve mice, it appears that the preexisting vector immunity limited the efficacy of a low dose of CAdVax-RVF. In contrast, vector-immune mice that received the high dose of CAdVax-RVF showed high levels of antibody responses, which were maintained over the next 14 weeks (Fig. (Fig.4C).4C). These data demonstrate that the limitation of high levels of preexisting vector immunity can be overcome by an increase in vaccine dose.

On week 15, all animals received a booster immunization with the same corresponding dose of CAdVax-RVF. The vector-immune animals that received the low-dose CAdVax-RVF immunizations mounted very weak responses on week 16 after the boost, consistent with the hypothesis that the lower dose is insufficient to overcome the limitations of preexisting vector immunity. Vector-naïve mice that received either low-dose or high-dose booster immunizations mounted strong secondary immune responses detected on week 16. Vector-immune mice that had received high-dose immunizations appeared to mount less vigorous responses after the boost, which represented a third exposure to Ad (Fig. (Fig.4C4C).

To gauge the protective efficacy of CAdVax-RVF in the face of preexisting vector immunity, the animals described above for Fig. Fig.44 were challenged on week 17 with 100 PFU of RVFV. Vector-naïve animals that had received a prime-boost vaccination with either a low dose or a high dose of CAdVax-RVF all survived lethal RVFV infection, whereas all mice vaccinated with the control CAdVax-D died from RVFV infection (Table (Table2).2). However, only one of four vector-immune mice that received the low-dose prime-boost of vaccine survived the virus challenge. In contrast, three of four vector-immune animals vaccinated with the high dose of CAdVax-RVF survived the RVFV challenge, suggesting that an increased dose of vaccine could overcome the limiting effects of preexisting vector immunity. The anti-Gn/Gc antibody titer of the vector-immune, high-dose prime-boost mouse that died was below 2.0 log10, whereas the three animals in this group that survived RVFV challenge had titers of 3.5 to 4.0 log10.

TABLE 2.
Survival of mice vaccinated against RVFV in the presence of preexisting vector immunitya

DISCUSSION

The CAdVax vaccine platform is a practical and safe vector that has shown promise when tested for vaccine activity against a number of emerging viral pathogens. We have studied the feasibility of developing CAdVax vaccines against Ebola virus (46), Marburg virus (45), multistrain filoviruses (40), dengue viruses (22, 31a), West Nile virus (34), and H5N1 avian influenza viruses (21). These CAdVax-based vaccines each induced cellular and humoral immune responses against the corresponding viruses and were protective against lethal virus infections in animal challenge models. Here we show that the CAdVax system can be applied successfully to RVFV. Expression of the RVFV glycoproteins de novo from the CAdVax-RVF vector mimics an actual RVFV infection, which induces potent immune responses against the viral proteins. These immune responses elicited a strong antibody response and were 100% protective against lethal RVFV infection in vaccinated mice. We also show protection against lethal RVFV infection even in the presence of high levels of antivector immunity. Due to the inherent safety profile, high immunogenicity, and complete protective efficacy of the CAdVax vector platform, the CAdVax-RVF vaccine may play an important role in providing protective immunity against RVFV infection.

Unlike other recombinant RVFV vaccines, such as live-attenuated RVFV or alphavirus replicons (4, 19, 44), the CAdVax-RVF vaccine does not replicate inside the body and therefore has an increased level of safety inherent in its design. Increased vaccine safety is a major issue given the dangerous side effects of the current veterinary RVFV vaccine, which causes spontaneous abortions in cattle (6) and birth defects in sheep (23). Furthermore, the need for other virus-based vaccines to replicate inside the body after immunization in order to achieve an efficient immune response runs a risk of causing disease symptoms, especially in patients with compromised immune systems. In contrast, the nonreplicating CAdVax-RVF vaccine carries little to no risk of causing disease symptoms and is considered safe. With Good Laboratory Practices (41a) toxicology and biodistribution studies, we have shown previously that repeated administration of as much as 2 × 1013 CAdVax virus particles per dose in New Zealand White rabbits was well tolerated by this highly sensitive animal model (data not shown). Furthermore, since CAdVax vaccines do not need to replicate in order to induce immune responses, the immunity is achieved rapidly after immunization. Here we show rapid development of antibodies against Gn and Gc glycoproteins only 2 weeks after a single CAdVax-RVF vaccine immunization and a sustained and durable secondary response following a booster immunization at week 10.

The primary criticism of Ad5-based vaccines is that preexisting immunity to Ad5 in a given population may interfere with the efficacy of the vaccine. To bypass anti-Ad5 immunity, some researchers have promoted the use of alternative human Ad serotypes (3, 10) or Ad vectors of nonhuman serotypes (26, 38, 50). Due to antigenic differences between Ad serotypes, these alternative serotype vectors can be effective in the presence of acute immune responses against Ad5 in animal models, although their potency often does not match that of Ad5 (3, 10, 27). However, a growing body of evidence from recent clinical trials suggests that the issue of Ad5 preexisting immunity may not be as critical as was initially thought and that using an alternative serotype to bypass preexisting immunity may not be necessary. For example, an influenza vaccine clinical trial reported that there was no correlation between preexisting Ad5 neutralizing antibody titers and the potency of an intranasally delivered Ad5-based influenza vaccine (42). Earlier data from Merck's Ad5-based human immunodeficiency virus (HIV) vaccine clinical trials showed that by simply increasing the dose of the vaccine, strong immune responses against HIV could be generated even in Ad5-seropositive individuals (12). Additional support also stems from recently generated data from our group whereby macaques administered our CAdVax-PanFilovirus vaccine and subsequently challenged with 1,000 times the lethal dose of Ebola or Marburg virus showed complete protection in the presence of preexisting immunity at both a baseline (low) and an acute (high) level of Ad5-specific antibodies (unpublished data). These data are complemented by the CAdVax-RVF results presented here, showing that the CAdVax-RVF vaccine was both immunogenic and protective against lethal infection in mice with high levels of preexisting antivector immunity. Even the failed phase IIb clinical trials for Merck's Ad5-based HIV vaccine demonstrated that preexisting immunity did not interfere with the Ad5 vector's ability to induce immune responses against HIV antigens in humans (8, 13). In 3,000 subjects with low, medium, and high levels of preexisting immunity against Ad5, the vaccine induced comparable levels (with less than 15% difference) of immune response against HIV antigens in all subjects. However, the clinical trial failed because the nonneutralizing immunity induced by the vaccine did not protect vaccinated individuals from acquiring HIV. Additionally, trial participants with higher levels of Ad5 neutralizing antibodies prior to vaccination appeared to have a higher incidence of HIV infection than those who were seronegative for Ad5 neutralizing antibodies (8, 13). Although it remains highly debatable whether anti-Ad5 immunity could play a role in increasing susceptibility to HIV infection in vaccine recipients, these data strongly contradict the original belief that preexisting immunity against Ad5 would prevent the induction of immune responses against the recombinant antigen.

The CAdVax-RVF vaccine expresses the Gn and Gc glycoproteins of RVFV as separate recombinant proteins, each fused to an exogenous N-terminal CD4 signal sequence. Gn and Gc on the surface of RVFV particles are likely targets for neutralization, having been shown to be essential and sufficient for immune protection, as suggested in earlier published investigations using baculovirus and sheeppox expression of these same two RVFV genes (35, 38). In evaluating immune responses to CAdVax-RVF, we therefore focused on generating antibodies against Gn and Gc capable of neutralizing live virus. Previous studies with other CAdVax vaccines have demonstrated the capability of these vectors to induce cytotoxic-T-lymphocyte responses against the targeted pathogen (21, 22, 45, 46). We do not rule out the possible contribution of cytotoxic-T-lymphocyte responses in the clearance of virally infected cells; however, it may not be necessary for protection against RVFV, as it has been shown previously that passive transfer of immune sera is sufficient to protect mice from challenge with RVFV (35).

The protective response elicited by CAdVax-RVF is interesting in light of RVFV glycoprotein processing. In the course of infection, Gn and Gc originate from a common polyprotein precursor through processing that includes signal peptidase, and this processing can lead to the generation of alternative products. Because CAdVax-RVF synthesizes the two proteins as separate units through incorporation of a signal peptide from CD4 and lacks native processing signals, the Gn and Gc proteins are expressed without the potential for producing these alternative products. During natural infection, these alternative products may act to divert immune responses away from neutralizing epitopes on full-length Gn and Gc that are structurally involved in receptor binding. By presenting conformationally relevant Gn and Gc with CAdVax-RVF, we may provide an improved environment in which to elicit neutralizing antibodies against conformationally sensitive epitopes. Since CAdVax-RVF was effective in protecting mice against challenge, it will be of interest in future studies to further characterize the expressed proteins and their heterodimeric interactions with each other, the types of immune responses induced against each glycoprotein, and their roles in protecting animals from lethal infection with RVFV.

Acknowledgments

We thank Jessica McCoy and Qiaoliang Fang for their help in the GenPhar laboratory. We also gratefully acknowledge Missy Worthy for her help with the virus challenge experiments in the Robert E. Shope BSL4 facility at UTMB.

Footnotes

[down-pointing small open triangle]Published ahead of print on 23 September 2009.

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