Evaluation of infectivity and transmission of different Asian foot-and-mouth disease viruses in swine.

Most isolates of foot-and-mouth disease virus (FMDV) display a broad host range. Since the late 1990s, the genetic lineage of PanAsia topotype FMDV serotype O has caused epidemics in the Far East, Africa, the United Kingdom, France, the Netherlands, and numerous other countries throughout Europe and Asia. In contrast, there are several FMDV isolates that exhibit a more restricted host range. A Cathay topotype isolate of FMDV serotype O from the 1997 epizootic in Taiwan (O/TAW/97) demonstrated restricted host specificity, only infecting swine. Methods used to evaluate infectivity and pathogenicity of FMDV isolates in cattle are well-documented, but there has been less progress studying transmission and pathogenicity of FMDV isolates in pigs. In previous studies designed to examine pathogenicity, various chimeric viruses derived from O/TAW/97 were intradermally inoculated in the heel bulb of pigs. Subsequent quantitative scoring of disease and evaluation of virus released into nasal secretions and blood was assessed. Here we prove the usefulness of this method in direct and contact inoculated pigs to evaluate infectivity, pathogenicity and transmission of different Asian FMDV isolates. Virus strains within the Cathay topotype were highly virulent in swine producing a synchronous disease in inoculated animals and were efficiently spread to in-contact naïve pigs, while virus strains from the PanAsia topotype displayed more heterogeneous properties.

Non-specific antiviral response detected in RNA-treated cultured cells of the sandfly, Lutzomyia longipalpis.

Lutzomyia longipalpis is the principal vector of visceral leishmaniasis in the Americas, and can also transmit some viruses. To help develop a gene-silencing system for this sandfly, we transfected cultured embryonic cells with various double-stranded RNAs using West Nile virus (WNV) virus-like particles (VLPs) expressing luciferase as the target RNA to demonstrate effective gene knock-down. When luciferase dsRNA was introduced into these cells, they produced the expected reduction in VLP-encoded luciferase, suggesting specific silencing of the luciferase gene. Surprisingly, we found that unrelated dsRNAs, which included those specific for several L. longipalpis gene sequences and Escherichia coli beta-galactosidase, diminished replication of the VLP-encoded genome. These results are the first indication for a nucleic acid-induced, non-specific antiviral response in this important insect vector.

Studies of genetically defined chimeras of a European type A virus and a South African Territories type 2 virus reveal growth determinants for foot-and-mouth disease virus.

The three South African Territories (SAT) types of foot-and-mouth disease virus (FMDV) display great genetic and antigenic diversity, resulting from the independent evolution of these viruses in different geographical localities. For effective control of the disease in such areas, the use of custom-made vaccines is required. To circumvent the tedious process of vaccine strain selection, an alternative in the control process is being investigated. Specifically, it is proposed to replace the antigenic determinants of an infectious genome-length cDNA copy of a good SAT vaccine strain with those of appropriate field strains, producing custom-made FMDV chimeras for use in vaccine production. Here the construction of an infectious genome-length cDNA copy of the SAT2 vaccine strain, ZIM/7/83, is described, created utilizing an exchange-cassette strategy with an existing A(12) genome-length cDNA clone. The virus derived from this cDNA (designated vSAT2) displayed excellent growth properties in cell culture, indicating its potential usefulness in the production of custom-made vaccine strains. Evaluation of the growth of various SAT2/A12 chimeras created during the derivation of SAT2 infectious cDNA suggested incompatibilities between the non-structural proteins of ZIM/7/83 and the 5' UTR of A(12).

Engineering better vaccines for foot-and-mouth disease.

Although efficacious and safe, current vaccines for FMD suffer from drawbacks. Among these are that the immune response to the vaccine interferes with the ability to detect vaccinated animals that have subsequently become infected and could carry and shed the virus, creating an obstacle to re-instating disease-free status to countries/regions that vaccinate to control outbreaks. Multiple diagnostic tests are available to identify animals that have been infected with FMDV by detection of antibodies to viral non-structural proteins (NSP) that are present in low concentration in traditional vaccines and are poorly immunogenic in vaccine preparations. However, these tests are not 100% reliable. To circumvent this problem, we have developed a new generation of vaccines that express the "empty capsid" subunit of the virus, in the absence of one of the most immunogenic NSPs, 3Dpol. Here we describe delivery of the empty capsid subunits by recombinant replication-defective human adenovirus type 5 (Ad5). These Ad5-vectored empty capsid vaccines can protect pigs from FMDV challenge as early as 7 days post-vaccination. A second problem with current FMD vaccines is that they do not induce protective immunity quickly, a drawback that is likely to be shared by our Ad5-vectored empty capsid vaccine. To overcome this problem, we have developed a prophylactic antiviral treatment consisting of an Ad5 encoding porcine interferon alpha (pIFNalpha). Administration of Ad5-pIFNalpha protects swine from FMD as early as one day post-administration. The combination of this antiviral treatment and the empty capsid subunit vaccine should induce rapid and complete protection from FMD, and could overcome current diagnostic problems.

Procedures for preventing the transmission of foot-and-mouth disease virus to pigs and sheep by personnel in contact with infected pigs.

The most effective method of containing an outbreak of foot-and-mouth disease (FMD) is by the culling of livestock. However, qualified people must diagnose the disease before the culling can begin, and they must avoid susceptible animals after having been in contact with infected premises, to prevent them from transmitting the virus. To test the effectiveness of biosecurity procedures in preventing the transmission of FMD virus (O/UK/35/2001) investigators contacted and sampled pigs inoculated with FMD virus for approximately 45 minutes and then contacted and sampled sentinel pigs and sheep after either using no biosecurity procedures, or washing their hands and donning clean outerwear, or showering and donning clean outerwear. The virus was detected in the nasal secretions of one investigator immediately after the postmortem investigation of the inoculated pigs but was not detected in samples collected between approximately 12 and 84 hours later. After the contaminated personnel had showered and changed into clean outerwear they did not transmit the strain of FMD virus to susceptible pigs and sheep.

Comparisons of the complete genomes of Asian, African and European isolates of a recent foot-and-mouth disease virus type O pandemic strain (PanAsia).

During the last 12 years, a strain of foot-and-mouth disease (FMD) virus serotype O, named PanAsia, has spread from India throughout Southern Asia and the Middle East. During 2000, this strain caused outbreaks in the Republic of Korea, Japan, Russia (Primorsky Territory), Mongolia and South Africa (KwaZulu-Natal Province), areas which last experienced FMD outbreaks in 1934, 1908, 1964, 1974 and 1957, respectively. In February 2001, the PanAsia strain spread to the United Kingdom where, in just over 7 months, it caused outbreaks on 2030 farms. From the UK, it quickly spread to the Republic of Ireland, France and the Netherlands. Previous studies that utilized RT-PCR to sequence the VP1-coding region of the RNA genomes of approximately 30 PanAsia isolates demonstrated that the UK virus was most closely related to the virus from South Africa (99.7 % nucleotide identity). To determine if there was an obvious genetic reason for the apparently high level of fitness of this new strain, and to further analyse the relationships between the PanAsia viruses and other FMDVs, complete genomes were amplified using long-range PCR techniques and the PCR products were sequenced, revealing the sequences for the entire genomes of five PanAsia isolates as well as an animal-passaged derivative of one of them. These genomes were compared to two other PanAsia genomes. These analyses revealed that all portions of the genomes of these isolates are highly conserved and provided confirmation of the close relationship between the viruses responsible for the South Africa and UK outbreaks, but failed to identify any genetic characteristic that could account for the unprecedented spread of this strain.

Early protection against homologous challenge after a single dose of replication-defective human adenovirus type 5 expressing capsid proteins of foot-and-mouth disease virus (FMDV) strain A24.

Previously we demonstrated that two doses of a replication-defective human adenovirus serotype 5 (Ad5) carrying the capsid (P1) and 3C protease coding regions of a laboratory strain of FMDV (A12) completely protected five of six swine challenged with homologous virus. The objective of the current study was to evaluate the efficacy of one dose of an Ad5-vectored vaccine expressing the P1 coding region of an FMDV field strain. A replication-defective Ad5 containing the P1 coding region of FMDV A24 and the 3C coding region of A12 (Ad5A24) was constructed and evaluated for its ability to induce neutralizing antibodies and protect swine against homologous challenge after a single vaccination. Animals were challenged 7, 14 or 42 days after vaccination. Control groups included animals inoculated with commercial vaccine or phosphate-buffered saline. All vaccinated swine were completely protected against homologous challenge at 7, 14 or 42 days after vaccination. Based on these results, we conclude that a single inoculation of Ad5-vectored vaccines could be used as a tool to control FMD in outbreak situations.

Prospects, including time-frames, for improved foot and mouth disease vaccines.

Inactivated foot and mouth disease (FMD) vaccines have been used successfully as part of eradication programmes. However, there are a number of concerns with the use of such vaccines and the recent outbreaks of FMD in disease-free countries have increased the need for improved FMD control strategies. To address this requirement, new generation FMD vaccines are being developed. Currently, one of the most promising of these vaccine candidates utilises an empty viral capsid subunit delivered to animals by a live virus vector. This candidate, a replication-defective recombinant human adenovirus containing the capsid and 3C proteinase coding regions of FMD virus (FMDV), induces an FMDV-specific neutralising antibody response in inoculated animals. Upon challenge with a virulent animal-passaged homologous virus, swine and cattle vaccinated with this recombinant adenovirus are protected from clinical signs of FMD as well as from FMDV replication. One inoculation of a high dose of this vaccine candidate protected swine from challenge as early as seven days after vaccination.

Subcellular distribution of the foot-and-mouth disease virus 3A protein in cells infected with viruses encoding wild-type and bovine-attenuated forms of 3A.

Picornavirus infection induces the proliferation and rearrangement of intracellular membranes in response to the synthesis of nonstructural proteins, including 3A. We have previously shown that changes in 3A are associated with the inability of a Taiwanese strain of foot-and-mouth disease virus (FMDV) (OTai) to grow in bovine cells and cause disease in cattle, although the virus grows to high titers in porcine cells and is highly virulent in pigs (C. W. Beard and P. W. Mason, 2000, J. Virol. 74, 987-991). To study if differences in the distribution of 3A could account for the species specificity of OTai, we compared the localization of the OTai 3A with a bovine-virulent 3A (serotype A12) in keratinocytes prepared from the tongues of cattle and pigs. Following either infection of keratinocytes or transfection with 3A we were unable to discern differences in 3A distribution in either species of keratinocyte, independent of the strain of virus (or 3A) utilized. In both cell types, 3A distributed in a pattern that overlapped with an endoplasmic reticulum (ER) marker protein, calreticulin (CRT). Furthermore, although FMDV infection or transfection with 3A did not result in a gross redistribution of CRT, both virus infection and 3A transfection disrupted the Golgi. Other picornaviruses that disrupt Golgi function are sensitive to brefeldin A (BFA), a fungal metabolite that interferes with retrograde transport between the Golgi and the ER. Interestingly, BFA has little effect on FMDV replication, suggesting that FMDV may acquire cellular membranes into its replication complexes in a manner different from that of other picornaviruses.

Immune responses and protection against foot-and-mouth disease virus (FMDV) challenge in swine vaccinated with adenovirus-FMDV constructs.

A replication-defective adenovirus 5 encoding foot-and-mouth disease virus (FMDV) capsid and 3C proteinase coding regions (Ad5-FMDV3CWT) was used to vaccinate swine. A single inoculation utilizing 1 x 10(8) plaque forming units (pfu) or an inoculation of 1 x 10(8) followed by a boost of 5 x 10(8) pfu Ad5-FMDV3CWT were tested, along with an inoculation and boost using an adenovirus encoding the FMDV capsid coding region and an inactive form of the 3C proteinase (Ad5-FMDV3CMUT). Sera collected from these animals were examined for the presence of FMDV-specific antibodies using immunoprecipitation, neutralization, and ELISA assays specific for IgM, IgG1 and IgG2. Efficacy studies were performed by placing the vaccinated swine in contact with an FMDV-infected swine and monitoring for signs of disease and changes in serum antibody levels. Ad5-FMDV3CMUT, which is unable to produce FMDV capsid structures, did not elicit FMDV-neutralizing antibodies or protect against FMD. Single inoculation with Ad5-FMDV3CWT generated FMDV-specific neutralizing antibodies, and reduced clinical signs in challenged swine, but failed to completely protect the majority of swine from FMD. Swine which received a primary vaccination with Ad5-FMDV3CWT followed by the boost at 4 weeks generated high levels of FMDV-neutralizing antibodies resulting in complete protection of five of the six swine and limited disease in the remaining animal. Increased efficacy of the two-dose regimen was associated with heightened levels of FMDV-specific IgG1 and IgG2 antibodies.

Generation and characterization of a mammalian cell line continuously expressing Japanese encephalitis virus subviral particles.

We have generated a cell line (F cells) producing a secreted form of Japanese encephalitis virus (JEV) subviral particle (extracellular particles [EPs]) that contains the JEV envelope glycoprotein (E) and a precursor (prM) of the virion membrane protein (M). The F cells were engineered to synthesize these JEV products from a cDNA encoding a mutated (furin proteinase resistant) form of prM, since stable cell lines expressing E and the authentic form of prM could not be obtained, due (in part) to the cell-fusing ability of EPs containing E and M. Our biochemical alteration of the prM protein was critical for the successful production of EP-producing cell lines. EPs produced by F cells share the biochemical properties of empty viral particles produced by JEV-infected cells, except that the F-cell EPs lack hemagglutinating activity and M. F-cell EPs were recognized by a panel of monoclonal antibodies to E, and EPs were shown to be useful as vaccine candidates in mice and as diagnostic reagents in evaluating human immune responses to JE vaccination. The amounts of E antigen released into the culture fluid of F cells were similar to those found in virion fractions of JEV-infected cell culture fluids or JEV-infected weanling mouse brains (the current source of antigen used to produce human vaccines for JE). Thus, the F-cell line would appear to be a useful source of antigen for JE vaccines and diagnostics.

Emergence in Asia of foot-and-mouth disease viruses with altered host range: characterization of alterations in the 3A protein.

In 1997, an epizootic in Taiwan, Province of China, was caused by a type O foot-and-mouth disease virus which infected pigs but not cattle. The virus had an altered 3A protein, which harbored a 10-amino-acid deletion and a series of substitutions. Here we show that this deletion is present in the earliest type O virus examined from the region (from 1970), whereas substitutions surrounding the deletion accumulated over the last 29 years. Analyses of the growth of these viruses in bovine cells suggest that changes in the genome in addition to the deletion, per se, are responsible for the porcinophilic properties of current Asian viruses in this lineage.

Safety and immunogenicity of NYVAC-JEV and ALVAC-JEV attenuated recombinant Japanese encephalitis virus--poxvirus vaccines in vaccinia-nonimmune and vaccinia-immune humans.

A controlled, randomized, double-blind clinical trial evaluated whether two attenuated recombinant poxviruses with identical Japanese encephalitis virus (JEV) gene insertions, NYVAC-JEV and ALVAC-JEV, were safe and immunogenic in volunteers. Groups of 10 volunteers distinguished by vaccinia immune status received two doses of each vaccine. The vaccines appeared to be equally safe and well tolerated in volunteers, but more reactogenic than licensed formalin-inactivated JE and placebo vaccines given as controls. NYVAC-JEV and ALVAC-JEV vaccine recipients had frequent occurrence of local warmth, erythema, tenderness, and/or arm pain after vaccination. There was no apparent effect of vaccinia immune status on frequency or magnitude of local and systemic reactions. NYVAC-JEV elicited antibody responses to JEV antigens in recipients but ALVAC-JEV vaccine poorly induced antibody responses. However, NYVAC-JEV vaccine induced neutralizing antibody responses only in vaccinia-nonimmune recipients while vaccinia-immune volunteers failed to develop protective antibodies (5/5 vs. 0/5 seroconversion, p<0.01). These data suggest that preexisting immunity to poxvirus vector may suppress antibody responses to recombinant gene products.

High-efficiency utilization of the bovine integrin alpha(v)beta(3) as a receptor for foot-and-mouth disease virus is dependent on the bovine beta(3) subunit.

We have previously reported that Foot-and-mouth disease virus (FMDV), which is virulent for cattle and swine, can utilize the integrin alpha(v)beta(3) as a receptor on cultured cells. Since those studies were performed with the human integrin, we have molecularly cloned the bovine homolog of the integrin alpha(v)beta(3) and have compared the two receptors for utilization by FMDV. Both the alpha(v) and beta(3) subunits of the bovine integrin have high degrees of amino acid sequence similarity to their corresponding human subunits in the ectodomains (96%) and essentially identical transmembrane and cytoplasmic domains. Within the putative ligand-binding domains, the bovine and human alpha(v) subunits have a 98.8% amino acid sequence similarity while there is only a 93% similarity between the beta(3) subunits of these two species. COS cell cultures, which are not susceptible to FMDV infection, become susceptible if cotransfected with alpha(v) and beta(3) subunit cDNAs from a bovine or human source. Cultures cotransfected with the bovine alpha(v)beta(3) subunit cDNAs and infected with FMDV synthesize greater amounts of viral proteins than do infected cultures cotransfected with the human integrin subunits. Cells cotransfected with a bovine alpha(v) subunit and a human beta(3) subunit synthesize viral proteins at levels equivalent to those in cells expressing both human subunits. However, cells cotransfected with the human alpha(v) and the bovine beta(3) subunits synthesize amounts of viral proteins equivalent to those in cells expressing both bovine subunits, indicating that the bovine beta(3) subunit is responsible for the increased effectiveness of this receptor. By engineering chimeric bovine-human beta(3) subunits, we have shown that this increase in receptor efficiency is due to sequences encoding the C-terminal one-third of the subunit ectodomain, which contains a highly structured cysteine-rich repeat region. We postulate that amino acid sequence differences within this region may be responsible for structural differences between the human and bovine beta(3) subunit, leading to more efficient utilization of the bovine receptor by this bovine pathogen.

Japanese encephalitis DNA vaccine candidates expressing premembrane and envelope genes induce virus-specific memory B cells and long-lasting antibodies in swine.

Swine are an important amplifier of Japanese encephalitis (JE) virus in the paradomestic environment. In this study, two JE DNA vaccine candidates were evaluated for immunogenicity in swine. Both vaccine plasmids encode a cassette consisting of the signal of premembrane (prM), prM, and envelope (E) coding regions of JE virus. One plasmid, designated pcJEME, is based on a commercial vector (pcDNA3), whereas the other plasmid, designated pNJEME, is based on a vector (pNGVL4a) designed to address some of the safety concerns of DNA vaccine use. No differences were detected in the immunogenicity of these two plasmids in mice or swine. Swine immunized with the DNA vaccines at a dose of 100 to 450 microgram at an interval of 3 weeks developed neutralizing and hemagglutination-inhibitory (HAI) antibody titers of 1:40 to 1:160 at 1 week after the second immunization. However, swine administered two doses of a commercial JE vaccine (formalin-inactivated virus preparation; JEVAX-A) developed low (1:10) or undetectable antibody responses after their boost. Interestingly, serum antibody titers elicited by DNA vaccines in swine were higher than those detected in mice. Eight days after boosting with viral antigen (JEVAX-A) to detect an anamnestic response, swine immunized two times with the DNA vaccine showed a >100-fold elevation in HAI titer, indicating a strong recall of antibody response. Swine maintained detectable levels of HAI antibody for at least 245 days after two immunizations with a DNA vaccine. These results indicate that these DNA vaccines are able to induce virus-specific memory B cells and long-lasting antibodies in swine, which were of higher levels than those obtained with a commercial formalin-inactivated JE vaccine.

Genetic determinants of altered virulence of Taiwanese foot-and-mouth disease virus.

In 1997, a devastating outbreak of foot-and-mouth disease (FMD) in Taiwan was caused by a serotype O virus (referred to here as OTai) with atypical virulence. It produced high morbidity and mortality in swine but did not affect cattle. We have defined the genetic basis of the species specificity of OTai by evaluating the properties of genetically engineered chimeric viruses created from OTai and a bovine-virulent FMD virus. These studies have shown that an altered nonstructural protein, 3A, is a primary determinant of restricted growth on bovine cells in vitro and significantly contributes to bovine attenuation of OTai in vivo.

A DNA vaccine expressing dengue type 2 virus premembrane and envelope genes induces neutralizing antibody and memory B cells in mice.

A dengue DNA vaccine candidate was developed and evaluated for immunogenicity in mice. The vaccine, designated pcD2ME, is a pcDNA3-based plasmid encoding the signal sequence of premembrane (prM), prM and envelope (E) genes of the New Guinea C strain of dengue type 2 virus. CHO-K1 cells transfected with pcD2ME expressed prM and E as determined by immunochemical staining with monoclonal antibodies. BALB/c mice inoculated intramuscularly with 100 microg of pcD2ME two or three times at an interval of 2 weeks developed a low level of neutralizing antibody (1:10 at a 90% plaque reduction). Immunization twice with 10 microg or 1 microg of pcD2ME or three times with 100 microg of pcDNA3 did not induce detectable levels of neutralizing antibody. Mice immunized two or three times with 100 microg of pcD2ME raised neutralizing antibody titers to 1:40 or greater on days 4 and 8 after challenge with 3x10(5) plaque forming units (PFU) of the New Guinea C strain of dengue type 2 virus, showing strong anamnestic responses to the challenge. In contrast, mice immunized two or three times with 100 microg of pcDNA3 developed no detectable neutralizing antibody on days 4 and 8 after challenge. These results indicate that immunization with pcD2ME induces neutralizing antibody and dengue type 2 virus-responsive memory B cells in mice.

Development of DNA vaccines for foot-and-mouth disease, evaluation of vaccines encoding replicating and non-replicating nucleic acids in swine.

We have developed naked DNA vaccine candidates for foot-and-mouth disease (FMD), an important disease of domestic animals. The virus that causes this disease, FMDV, is a member of the picornavirus family, which includes many important human pathogens, such as poliovirus, hepatitis A virus, and rhinovirus. Picornaviruses are characterized by a small (7-9000 nucleotide) RNA genome that encodes capsid proteins, processing proteinases, and enzymes required for RNA replication. We have developed two different types of DNA vaccines for FMD. The first DNA vaccine, pP12X3C, encodes the viral capsid gene (P1) and the processing proteinase (3C). Cells transfected with this DNA produce processed viral antigen, and animals inoculated with this DNA using a gene gun produced detectable antiviral immune responses. Mouse inoculations with this plasmid, and with a derivative containing a mutation in the 3C proteinase, indicated that capsid assembly was essential for induction of neutralizing antibody responses. The second DNA vaccine candidate, pWRMHX, encodes the entire FMDV genome, including the RNA-dependent RNA polymerase, permitting the plasmid-encoded viral genomes to undergo amplification in susceptible cells. pWRMHX encodes a mutation at the cell binding site, preventing the replicated genomes from causing disease. Swine inoculated with this vaccine candidate produce viral particles lacking the cell binding site, and neutralizing antibodies that recognize the virus. Comparison of the immune responses elicited by pP12X3C and pWRMHX in swine indicate that the plasmid encoding the replicating genome stimulated a stronger immune response, and swine inoculated with pWRMHX by the intramuscular, intradermal, or gene gun routes were partially protected from a highly virulent FMD challenge.

The anamnestic neutralizing antibody response is critical for protection of mice from challenge following vaccination with a plasmid encoding the Japanese encephalitis virus premembrane and envelope genes.

For Japanese encephalitis (JE), we previously reported that recombinant vaccine-induced protection from disease does not prevent challenge virus replication in mice. Moreover, DNA vaccines for JE can provide protection from high challenge doses in the absence of detectable prechallenge neutralizing antibodies. In the present study, we evaluated the role of postchallenge immune responses in determining the outcome of JE virus infection, using mice immunized with a plasmid, pcDNA3JEME, encoding the JE virus premembrane (prM) and envelope (E) coding regions. In the first experiment, 10 mice were vaccinated once (five animals) or twice (remainder) with 100 micrograms of pcDNA3JEME. All of these mice showed low (6 of 10) or undetectable (4 of 10) levels of neutralizing antibodies. Interestingly, eight of these animals showed a rapid rise in neutralizing antibody following challenge with 10,000 50% lethal doses of JE virus and survived for 21 days, whereas only one of the two remaining animals survived. No unimmunized animals exhibited a rise of neutralizing antibody or survived challenge. Levels of JE virus-specific immunoglobulin M class antibodies were elevated following challenge in half of the unimmunized mice and in the single pcDNA3JEME-immunized mouse that died. In the second experiment, JE virus-specific primary cytotoxic T-lymphocyte (CTL) activity was detected in BALB/c mice immunized once with 100 micrograms of pcDNA3JEME 4 days after challenge, indicating a strong postchallenge recall of CTLs. In the third experiment, evaluation of induction of CTLs and antibody activity by plasmids containing portions of the prM/E cassette demonstrated that induction of CTL responses alone were not sufficient to prevent death. Finally, we showed that antibody obtained from pcDNA3JEME-immunized mice 4 days following challenge could partially protect recipient mice from lethal challenge. Taken together, these results indicate that neutralizing antibody produced following challenge provides the critical protective component in pcDNA3JEME-vaccinated mice.