Ebola virus is an aggressive pathogen that causes a highly lethal hemorrhagic fever syndrome in humans and nonhuman primates. Typically, Ebola virus infection runs its course within 14 to 21 days. Infection initially presents with nonspecific flu-like symptoms such as fever, myalgia, and malaise. As the infection progresses, patients exhibit severe bleeding and coagulation abnormalities, including gastrointestinal bleeding, rash, and a range of hematological irregularities, such as lymphopenia and neutrophilia. Cytokines are released when reticuloendothelial cells encounter virus, which can contribute to exaggerated inflammatory responses that are not protective. Damage to the liver, combined with massive viremia, leads to disseminated intravascular coagulopathy. The virus eventually infects microvascular endothelial cells and compromises vascular integrity. The terminal stages of Ebola virus infection usually include diffuse bleeding, and hypotensive shock accounts for many Ebola virus fatalities (Sullivan et al., 2003).
The Ebola virus genome is 19 kb long, with seven open reading frames encoding structural proteins, including the virion envelope glycoprotein (GP), nucleoprotein (NP), and matrix proteins VP24 and VP40; nonstructural proteins, including VP30 and VP35; and the viral polymerase. The GP open reading frame of Ebola virus gives rise to two gene products, a soluble 60- to 70-kDa protein (sGP) and a full-length 150- to 170-kDa protein (GP) that inserts into the viral membrane through transcriptional editing (Sullivan et al., 2003).
4. Microbial Pathogenesis
Mononuclear phagocytes are the first targets of infection relevant to disease pathogenesis, followed by connective tissues and parenchymal cells. Cytokines are released when reticuloendothelial cells encounter virus, which can contribute to exaggerated nonprotective inflammatory responses. Ebola virus GP plays a vital role in infection. Virual GP appears to form a trimeric complex and binds preferentially to endothelial cells Infection of endothelial cells also induces a cytopathic effect and damage to the endothelial barrier that, together with cytokine effects, leads to the loss of vascular integrity. Cytokine dysregulation and virus infection may synergize at the endothelial surface, promoting hemorrhage and vasomotor collapse. Severity of infection is influenced by age, immune status, and viral virulence. Infection with species-adapted viruses may be lethal. The amount and location of fibin deposits varies with animal species. Strains show differing levels of virulence both across species and by route of administration. Microvascular damage and activation of the clotting cascade occurs. Death is secondary to massive cell death, fluid shifts, hemorrhages, and vascular abnormalities (Sullivan et al., 2003a).
Both adaptive and innate inflammatory systems respond to infection (Sullivan et al., 2003a). Although antibody titres correlate with the protective response, many studies in non-human primates have suggested that the passive transfer of antibody is insufficient to provide long-lasting protection against Ebola virus (Sullivan et al., 2003b). In rodent studies with adapted Ebola virus, passive transfer of antibodies or adoptive transfer of cytotoxic T cells showed protection when given before infection. A more sensitive but less quantitative CD4 lympho-proliferative response correlated with protection in a DNA/ADV prime–boost study. In addition to the antibody response induced by an effective vaccine, both CD4 and CD8 responses were observed after the challenge. The fact that CD4 responses were not observed before challenge whereas CD8 responses were more consistently seen beforehand suggests that the CD8 response is likely to have an important role in protection in non-human primates (Sullivan et al., 2003b).
Molecule Role Annotation :
Cynomolgus macaques were immunized with DNA/rAd5 vaccines expressing ZEBOV (GP) and SEBOV glycoprotein (GP) prior to lethal challenge with BEBOV. Vaccinated subjects developed robust, antigen-specific humoral and cellular immune responses against the GP from ZEBOV as well as cellular immunity against BEBOV GP, and immunized macaques were uniformly protected against lethal challenge with BEBOV (Hensley et al., 2010).
Molecule Role Annotation :
Cynomolgus macaques were immunized with DNA/rAd5 vaccines expressing ZEBOV (GP) and SEBOV glycoprotein (GP) prior to lethal challenge with BEBOV. Vaccinated subjects developed robust, antigen-specific humoral and cellular immune responses against the GP from ZEBOV as well as cellular immunity against BEBOV GP, and immunized macaques were uniformly protected against lethal challenge with BEBOV (Hensley et al., 2010).
Molecule Role Annotation :
Guinea pigs infected with a single intranasal inoculation of HPIV3/EboGP-NP showed no apparent signs of disease yet developed a strong humoral response specific to the EV proteins. When these animals were challenged with an intraperitoneal injection of 10(3) PFU of EV, there were no outward signs of disease, no viremia or detectable EV antigen in the blood, and no evidence of infection in the spleen, liver, and lungs. In contrast, all of the control animals died or developed severe EV disease following challenge (Bukreyev et al., 2006).
>NP_066250.1 membrane-associated protein [Zaire ebolavirus]
MAKATGRYNLISPKKDLEKGVVLSDLCNFLVSQTIQGWKVYWAGIEFDVTHKGMALLHRLKTNDFAPAWS
MTRNLFPHLFQNPNSTIESPLWALRVILAAGIQDQLIDQSLIEPLAGALGLISDWLLTTNTNHFNMRTQR
VKEQLSLKMLSLIRSNILKFINKLDALHVVNYNGLLSSIEIGTQNHTIIITRTNMGFLVELQEPDKSAMN
RMKPGPAKFSLLHESTLKAFTQGSSTRMQSLILEFNSSLAI
Molecule Role :
Protective antigen
Molecule Role Annotation :
VP24 expressed from alphavirus replicons induced a protective immune response in BALB/c mice when challenged with Ebola virus Zaire (Wilson et al., 2001).
>NP_066249.1 minor nucleoprotein [Zaire ebolavirus]
MEASYERGRPRAARQHSRDGHDHHVRARSSSRENYRGEYRQSRSASQVRVPTVFHKKRVEPLTVPPAPKD
ICPTLKKGFLCDSSFCKKDHQLESLTDRELLLLIARKTCGSVEQQLNITAPKDSRLANPTADDFQQEEGP
KITLLTLIKTAEHWARQDIRTIEDSKLRALLTLCAVMTRKFSKSQLSLLCETHLRREGLGQDQAEPVLEV
YQRLHSDKGGSFEAALWQQWDRQSLIMFITAFLNIALQLPCESSAVVVSGLRTLVPQSDNEEASTNPGTC
SWSDEGTP
Molecule Role :
Protective antigen
Molecule Role Annotation :
VP30 expressed from alphavirus replicons induced a protective immune response in BALB/c mice when challenged with Ebola virus Zaire (Wilson et al., 2001).
Molecule Role Annotation :
VP35 expressed from alphavirus replicons induced a protective immune response in C57BL/6 mice when challenged with Ebola virus Zaire (Wilson et al., 2001).
>NP_066245.1 matrix protein [Zaire ebolavirus]
MRRVILPTAPPEYMEAIYPVRSNSTIARGGNSNTGFLTPESVNGDTPSNPLRPIADDTIDHASHTPGSVS
SAFILEAMVNVISGPKVLMKQIPIWLPLGVADQKTYSFDSTTAAIMLASYTITHFGKATNPLVRVNRLGP
GIPDHPLRLLRIGNQAFLQEFVLPPVQLPQYFTFDLTALKLITQPLPAATWTDDTPTGSNGALRPGISFH
PKLRPILLPNKSGKKGNSADLTSPEKIQAIMTSLQDFKIVPIDPTKNIMGIEVPETLVHKLTGKKVTSKN
GQPIIPVLLPKYIGLDPVAPGDLTMVITQDCDTCHSPASLPAVIEK
Molecule Role :
Protective antigen
Molecule Role Annotation :
Vaccination of BALB/c or C57BL/6 mice with eVLPs, composed of the EBOV glycoprotein and matrix viral protein (VP)40 with a lipid membrane, in conjunction with QS-21 adjuvant resulted in mixed IgG subclass responses, a Th1-like memory cytokine response, and protection from lethal EBOV challenge (Warfield et al., 2005).
VP40 expressed from alphavirus replicons induced a protective immune response in BALB/c mice when challenged with Ebola virus Zaire (Wilson et al., 2001).
Description:
The recombinant chimpanzee adenovirus Type 3-vectored Ebola vaccine, VRC-EBOADC086-00-VP (cAd3-EBO S), is composed of a cAd3 vector that encodes Ebola Sudan wild type glycoprotein (WT GP).(Ledgerwood et al., 2017)
The bivalent cAdVaxE(GPs/z) vaccine includes the SEBOV glycoprotein (GP) and ZEBOV GP genes together in a single complex adenovirus-based vaccine (cAdVax) vector (Wang et al., 2006).
h. Immunization Route
Intramuscular injection (i.m.)
i.
Mouse Response
Vaccine Immune Response Type:
VO_0003057
Immune Response:
Vaccination of mice with the bivalent cAdVaxE(GPs/z) vaccine led to efficient induction of EBOV-specific antibody and cell-mediated immune responses to both species of EBOV (Wang et al., 2006).
Challenge Protocol:
Mice were challenged with a lethal dose of ZEBOV (30,000 times the 50% lethal dose) (Wang et al., 2006).
Efficacy:
the cAdVax technology demonstrated induction of a 100% protective immune response in mice, as all vaccinated C57BL/6 and BALB/c mice survived challenge with a lethal dose of ZEBOV (Wang et al., 2006).
A panfilovirus vaccine based on a complex adenovirus (CAdVax) technology that expresses multiple antigens from five different filoviruses de novo (Swenson et al., 2008).
f. Immunization Route
Intramuscular injection (i.m.)
g.
Macaque Response
Vaccination Protocol:
The macaques in the vaccine groups (five per group) were anesthetized by intramuscular injection of ketamine HCl (10 mg/kg of body weight), followed by intramuscular vaccination with an equal mixture of 1 × 10^10 PFU of each vaccine component: EBO2, EBO7, M8, and M11 (resulting in 4 × 10^10 total PFU per animal). Control animals received 4 × 10^10 PFU of the HC4 vaccine vector, also via the intramuscular route (Swenson et al., 2008).
Vaccine Immune Response Type:
VO_0000287
Challenge Protocol:
Group 1 was inoculated subcutaneously with MARV Musoke, while group 2 was inoculated intramuscularly with ZEBOV, using approximately 1,000 PFU of each filovirus. EBOV and MARV each have different established routes of administration (intramuscular and subcutaneous, respectively) (Swenson et al., 2008).
Efficacy:
All vaccinated animals showed no detectable viremia or hematology abnormalities. We can conclude that the multivalent filovirus vaccine was 100% protective against lethal MARV Musoke and ZEBOV challenges (Swenson et al., 2008).
A multivalent vaccine candidate (EBO7) that expresses the glycoproteins of Zaire ebolavirus (ZEBOV) and Sudan ebolavirus (SEBOV) in a single complex adenovirus-based vector (CAdVax) (Pratt et al., 2010).
f. Immunization Route
Intramuscular injection (i.m.)
g.
Macaque Response
Vaccination Protocol:
For the parenteral challenge studies, cynomolgus macaques were vaccinated intramuscularly (i.m.) on day zero with a 1:1 mixture of 1 × 10^10 PFU each of EBO7 and M8 (total 2 × 10^10 PFU). Control animals received an i.m. injection of 2 × 10^10 PFU of HC4. For the initial aerosol infection experiments, cynomolgus macaques were vaccinated by i.m. injection of 1 × 10^10 PFU of EBO7 or 1 × 10^10 PFU of HC4 (Pratt et al., 2010).
Vaccine Immune Response Type:
VO_0003057
Challenge Protocol:
For the aerosol infection group, twenty-eight days after vaccination, animals were anesthetized and exposed to a target dose of 1,000 PFU of either aerosolized ZEBOV or aerosolized SEBOV. For the parenteral challenge studies, six weeks after the boosting vaccinations, the macaques were anesthetized by i.m. injection of Telazol (2 to 6 mg/kg of body weight) and then inoculated i.m. with SEBOV or ZEBOV challenge stock (Pratt et al., 2010).
Efficacy:
EBO7 vaccine provided protection against both Ebola viruses by either route of infection. Significantly, protection against SEBOV given as an aerosol challenge, which has not previously been shown, could be achieved with a boosting vaccination (Pratt et al., 2010).
Plasmids containing the sGP cDNAs are used to subclone the relevant inserts into CMV expression vectors, which utilize the bovine growth hormone polyadenylation sequence. The plasmid pCRII-sGP is digested with EcoRI and treated with Klenow enzyme, and the resulting fragment is inserted into the BamHI/Bg/II CMV plasmid, which has been incubated with Klenow fragment and calf intestinal phosphate (CIP), and phenol chloroform extracted (Xu et al., 1998).
f. Description
sGP is a secreted or transmembrane form of glycoprotein (Xu et al., 1998).
g.
Guinea pig Response
Vaccination Protocol:
Two groups of guinea pigs were immunized by injection of 0.5 mg/ml in each hind leg (two injections at each time point) with the plasmed expression vectors. Animals were challenged by inoculation with a stock of Ebola virus that had been passaged once in Vero E6 cells and serially passaged by intraperitoneal injection of slpeen homogenates in Hartley guinea pigs seven times. Immunized guinea pigs were injected intraperitoneally with 0.5 ml of a 1:1000 dillution of spleen cell homogenate in Hanks' balanced salt solution 122 days after the initial plasmid DNA injection. Survival was determined 10 days later at which times animals were killed for serologic and pathologic analysis (Xu et al., 1998).
Persistence:
None noted
Immune Response:
A broad immune response was conferred by sGP which induced both cellular and humoral immunity to the membrane-associated GP. The ability of vectors expressing GP to confer immunity may be explained by the generation of the lower molecular weight degradation products, which could provide sufficient protein for antigen presentation to induce detectable, cellular and humoral immune responses in guinea pigs (Xu et al., 1998).
Side Effects:
None noted
Efficacy:
For the first group of 6 giunea pigs, animals were challenged within 2 months after the initial immunization. Five of six of the immunized subjects survived in contrast to 0/6 control subjects. In the second group, guinea pigs were challenged 4 months after the initial immunization. Three of the five guinea pigs immunized with sGP showed no ill effects following the viral challenge (Xu et al., 1998).
6. Ebola virus DNA vaccine DNA/rAd5 encoding ZEBOV and SEBOV antigens
Vaccination Protocol:
Four cynomolgus macaques were injected at 4–6 week intervals with GP(Z) and GP(S/G) DNA, followed by a rest period, and boosted after one year with rAd5 vectors containing the EBOV matched insert (Hensley et al., 2010).
Vaccine Immune Response Type:
VO_0000286
Immune Response:
DNA/rAd prime-boost EBOV immunization generated antigen-specific CD4+ T cell immunity against proteins expressed by the vaccine insert. The magnitude of antigen-specific CD4+ T cells was uniform across the four immunized macaques (Hensley et al., 2010).
Efficacy:
DNA/rAd5 immunization of cynomolgus macaques protects against infection when animals are challenged with a virus species homologous to the vaccine inserts (Hensley et al., 2010).
Immune Response:
The mice had low neutralizing antibody responses, yet were protected from challenge. This, as indicated by earlier studies, may be due to gene gun vaccination, which generally elicits a TH2-type response in BALB/c mice (Riemenschneider et al., 2003).
Efficacy:
Two vaccinations with the EBOV GP DNA elicited consistently high antibody responses and conferred complete protection from EBOV challenge (Riemenschneider et al., 2003).
8. Ebola virus DNA vaccine encoding ZEBOV GP and SEBOV GP
Vaccination Protocol:
DNA immunizations were administered by Biojector IM injection, bilateral deltoid, with a mixture of 2 mg each of two plasmid vectors encoding GP(Z) and GP(S/G). DNA immunizations were administered at 0, 4, 8, and 14 weeks. Each subject received a boost with 10^11 particle units (PU) of rAd5 GP(Z) at 12 months following the final DNA priming immunization (Hensley et al., 2010).
Challenge Protocol:
All animals were challenged by the intramuscular route with 1,000 TCID50 of BEBOV, 7 weeks post rAd5 GP boost. The challenge virus used in this study was isolated from blood specimen #200706291 from a fatal case infected during the 2007 EBOV outbreak in Bundibugyo district, Uganda. The virus was isolated on Vero E6 cells and passaged twice prior to initiating these experiments (Hensley et al., 2010).
Efficacy:
Vaccinated subjects developed robust, antigen-specific humoral and cellular immune responses against the GP from ZEBOV as well as cellular immunity against BEBOV GP, and immunized macaques were uniformly protected against lethal challenge with BEBOV (Hensley et al., 2010).
Efficacy:
Mice were completely protected from challenge with mouse adapted EBOV with a priming dose of 0.5 microgram of GP DNA followed by three or four subsequent vaccinations with 1.5 micrograms of DNA. Partial protection could be observed for at least 9 months after three immunizations with 0.5 microgram of the GP DNA vaccine (Vanderzanden et al., 1998).
Immune Response:
The mice had low neutralizing antibody responses, yet were protected from challenge. This, as indicated by earlier studies, may be due to gene gun vaccination, which generally elicits a TH2-type response in BALB/c mice (Riemenschneider et al., 2003).
Efficacy:
Two vaccinations with the EBOV NP DNA elicited consistently high antibody responses and conferred complete protection from EBOV challenge (Riemenschneider et al., 2003).
11. Ebola virus recombinant adenovirus vaccine AdC7-ZGP encoding GP
Immune Response:
AdC7 vaccine stimulated robust T and B cell responses to ZEBOV in naïve mice (Kobinger et al., 2006).
Efficacy:
Mice were immunized with a single dose of 5 × 10^10 particles per animal as performed previously and vaccinated animals were challenged with 200 LD50 of the mouse-adapted strain of ZEBOV 21 days later. All control mice (vehicle and AdHu5-LacZ) died between days 5 and 9 post-challenge. In contrast, all mice vaccinated with AdC7-ZGP survived the challenge with mouse-adapted ZEBOV. Weight loss was observed only from control groups (vehicle and AdHu5-LacZ). Complete protection following vaccination with 5 × 10^10 particles of AdC7-ZGP was demonstrated with challenge doses of ZEBOV up to 200,000 LD50, which was the highest dose tested (Kobinger et al., 2006).
To make ADV-GP, the BamHI/EcoRI fragment of GP(Z) was digested from PGEM-3Zf(-)-GP, treated with Klenow, and inserted into HindIII/XbaI/Kle/CIP-treated pRc/CMV plasmid. The resulting plasmid was digested by NruI/DraIII and treated with Klenow. The NruI/DraIII/Kle fragment containing the CMV enhancer, GP(Z) DNA and bovine growth hormone polyadenylation signal was inserted into the BgIII site of the adenoviral shuttle plasmid pAdBgIII26. The adenovirus, a first generation dl 309-based Ad5 vector, contained a deletion in E1 to render the vector replication defective and a partial deletion/substitution in E3, which disrupts the coding sequences for the E3 proteins with a relative molecular mass of 14,700, 14,500 and 10,400, respectively (Sullivan et al., 2000).
g. Virulence
h. Description
ADV−GP consists of an adenoviral (ADV) vector encoding the Ebola glycoprotein (GP) (Sullivan et al., 2003).
i.
Monkey Response
Host Strain:
Cynomolgus macaque
Vaccination Protocol:
Cynomolgus macaques were immunized with ADV−GP and ADV−NP, followed by boosting 9 weeks later (Sullivan et al., 2003).
Persistence:
None noted
Side Effects:
None noted
Challenge Protocol:
One week after the boost, animals were challenged with either a low or high dose of a 1995 isolate of Ebola virus Zaire.
Efficacy:
In the saline-injected control animals these doses were uniformly fatal 6−12 days afterwards. In contrast, the ADV−GP/NP immunized monkeys were completely protected. Analysis of the cell-mediated and humoral immune responses revealed significant increases in the CD8+ T-cell response to Ebola antigens by intracellular cytokine staining for interferon (IFN)-, seen before exposure to virus, in contrast to control animals where no response was seen. Similarly, antibody titres to the virus were stimulated in vaccinated animals, which minimally increased after the viral challenge (Sullivan et al., 2003).
Efficacy:
Expression of specific GPs alone vectored by rAd are sufficient to confer protection against lethal challenge in a relevant nonhuman primate model (Sullivan et al., 2006).
Immune Response:
Ad-CAGoptZGP at a dose of 1×10^5 IFU/mouse elicited a frequency of 1.3±0.3% positive IFN-γ producing CD8+ T cells. Overall, the average frequency of positive IFN-γ producing CD8+ T cells was slightly higher with a lower dose of Ad-CAGoptZGP than with a higher dose of Ad-CMVZGP (Richardson et al., 2009).
Efficacy:
All mice vaccinated with doses of 1×10^4, 1×10^5 and 1×10^6 IFU/mouse of Ad-CAGoptZGP were fully protected from the viral challenge with no weight loss or other clinical signs of disease (Richardson et al., 2009).
15. Ebola virus recombinant vector vaccine Ad-CMVZGP encoding the glycoprotein
Immune Response:
Vaccination following viral challenge produced enhanced T and B cell immune responses with a low dose of Ad-CAGoptZGP. The frequency of IFN-γ+CD8 T cells with Ad-CMVZGP at 1×10^7 IFU/mouse was at 1.6±0.4%, on average (Richardson et al., 2009).
Efficacy:
Protection following Zaire ebola virus challenge was complete in mice vaccinated with Ad-CMVZGP at 1×10^6 and 1×10^7 IFU/mouse but was only partially protective at 1×10^5 IFU/mouse (Richardson et al., 2009).
16. Ebola virus recombinant vector vaccine EBO7 encoding GP from SEBOV and ZEBOV
Efficacy:
Significantly, protection against SEBOV given as an aerosol challenge, which has not previously been shown, could be achieved with a boosting vaccination (Pratt et al., 2010).
17. Ebola virus recombinant vector vaccine pVSVXN2∆G/ZEBOVsGP encoding GP
Efficacy:
The rVSV expressing the Zaire Ebola virus transmembrane glycoprotein mediated protection in mice against a lethal Zaire Ebola virus challenge (Garbutt et al., 2004).
a replication competent vesicular stomatitis virus vector (VSVΔG) (Jones et al., 2005)
g. Immunization Route
Intramuscular injection (i.m.)
h.
Macaque Response
Vaccine Immune Response Type:
VO_0000286
Efficacy:
A single intramuscular injection of the EBOV or MARV vaccine elicited completely protective immune responses in nonhuman primates against lethal EBOV or MARV challenges (Jones et al., 2005).
An adenovirus vectored vaccine encoding Ebola virus ZGP providing protection in Macaque model. (Wong et al., 2015)
i.
Macaque Response
Vaccination Protocol:
NHPs were treated at either 30 mins or 24 hrs after exposure. Control NHPs were treated with phosphate-buffered saline. Specific treatment procedures described in articles. (Wong et al., 2015)
Immune Response:
All surviving cynomolgus macaques treated 30 minutes or 24 hours after EBOV challenge had elevated anti-ZGP IgG levels by 14 days after infection, and levels remained elevated until the termination of the experiment 28 days after infection. ZGP-specific IgG was not detected in nonsurviving NHPs A4 and A8 but was detected in animal A3 by 7 days after infection. For nonsurviving and control animals in the 24-hour treatment groups, ZGP-specific IgG was not detected, with the exception of animal E2. Surviving animals in the 24-hour treatment groups developed elevated anti-ZGP IgG levels by 14 days after infection, which remained elevated 28 days after infection. (Wong et al., 2015)
Challenge Protocol:
NHPs were challenged with a uniformly lethal dose of 1000 plaque-forming units of EBOV (Ebola virus H.sapiens-tc/COD/1995/Kikwit-9510621; GenBank accession number AY354458). (Wong et al., 2015)
Efficacy:
Six of 9 NHPs (67%) survived the challenge when treated 30 minutes after exposure, whereas 1 of 4 (25%) survived challenge when treated 24 hours after infection. The 3 nonsurviving NHPs treated 30 minutes after challenge had a delayed time to death of 13, 11, and 13 days after infection, surviving nearly twice as long as the control animals, which died 7 days after infection. In contrast, the 3 nonsurviving cynomolgus macaques treated 24 hours after challenge died 7, 7, and 9 days after infection, which is similar to times of death for the control animals. Nonsurviving, treated rhesus macaques died 11 and 16 days after infection, which is longer than the control animals. (Wong et al., 2015)
The Ebola NP and GP genes from the Mayinga strain of Ebola virus were derived from pSP64- and pGEM3Zf(-)-based plasmids. The BamHI±EcoRI (2.3 kb) and BamHI±KpnI (2.4 kb) fragments containing the NP and GP genes, respectively, were subcloned into a shuttle vector digested with BamHI and EcoRI within a polylinker sequence flanked by ClaI sites. For cloning of the GP gene, overhanging ends produced by KpnI (in the GP fragment) and EcoRI (in the shuttle vector) were made blunt by incubation with T4 DNA polymerase. From the shuttle vector, NP or GP genes were transferred as ClaI-fragments into the ClaI site of the replicon clone, resulting in plasmids encoding the NP or GP gene in place of the VEE structural protein genes (Pushko et al., 2000).
d. Virulence
e. Description
This immunogen is composed of RNA replicon particles derived from an attenuated strain of Venezuelan equine encephalitis virus (VEEV) expressing EBOV glycoprotein and nucleoprotein (Geisbert et al., 2002).
f.
Monkey Response
Host Strain:
Cynomolgus macaques
Vaccination Protocol:
Groups of three cynomolgus macaques were vaccinated with VRP that expressed EBOV GP, EBOV NP, a mixture of EBOV GP and EBOV NP, or a control antigen (influenza hemagglutinin) that has no effect on EBOV immunity. Animals were vaccinated by subcutaneous injection of 10^7 focus-forming units of VRP in a total of 0.5 mL at one site. Vaccinations were repeated 28 days after the first injection and 28 days after the second (Geisbert et al., 2002).
Persistence:
None noted
Side Effects:
None noted
Efficacy:
These results indicate that rodent models of EBOV hemorrhagic fever do not consistently predict efficacy of candidate vaccines in nonhuman primates, perhaps because the disease course in rodents differs from that reported in human and nonhuman primates (Geisbert et al., 2002).
Description:
All animals, including the four untreated macaques, were challenged with 1,000 PFU of EBOV 49 days after the third vaccine dose. At postchallenge day 3, all animals became ill; two animals from each vaccination group (i.e., GP, NP, GP + NP, influenza HA) died on day 6, and the remaining animals died on day 7 (Geisbert et al., 2002).
The Ebola GP genes from the Mayinga strain of Ebola virus were derived from pGEM3Zf(-)-based plasmid. The BamHI±KpnI (2.4 kb) fragment containing the GP gene was subcloned into a shuttle vector. From the shuttle vector, GP gene was transferred as ClaI-fragment into the ClaI site of the replicon clone, resulting in plasmids encoding the GP gene in place of the VEE structural protein genes (Pushko et al., 2000).
f. Virulence
g.
Mouse Response
Host Strain:
BALB/c
Vaccination Protocol:
VRP were diluted in PBS and administered to 6±8 week old BALB/c mice. Groups of 10 BALB/c mice were inoculated on days 0 and 28 with two doses of NP-VRP, GP-VRP, or a mixture of both. Challenge was carried out 4 weeks after final immunization with VRP. Mice were challenged i.p. with mouse-adapted Ebola virus. To determine subsequent viral titers in the serum, liver, and spleen, two mice were taken from VRP-vaccinated or control groups on each of days 1±5 after challenge, anesthetized and exsanguinated. Portions of the liver and spleen were removed aseptically, weighed, and ground in a sterile mortar. Viral titers in the sera and tissues were determined by plaque assay (Pushko et al., 2000).
Persistence:
None noted
Side Effects:
None
Efficacy:
GP-VRP was effective in protecting BALB/c mice against a lethal challenge with mouse-adapted Ebola virus (Pushko et al., 2000). Nine out of ten animals vaccinated with GP-VRP were protected (Pushko et al., 2000).
h.
Guinea pig Response
Host Strain:
strain 2 and strain 13
Vaccination Protocol:
VRP were diluted in PBS and administered to inbred, strain 2 or strain 13 guinea pigs. Groups of five guinea pigs were inoculated subcutaneously (s.c.) at day 0 with a total of 0.5 ml containing 10^7 IU VRP at one (strain 2) or two (strain 13) dorsal sites. Challenge was carried out 4 weeks after final immunization with VRP. Guinea pigs were challenged s.c. with 1000 LD50 of guinea pig- adapted Ebola virus. Animals were observed daily for 60 days, and morbidity (determined as changes in behavior, appearance, and weight) and survival were recorded. Blood samples were taken
on the days indicated after challenge and viremia levels were determined by plaque assay (Pushko et al., 2000).
Persistence:
None noted
Side Effects:
None noted
Efficacy:
At day 7 after challenge, both VRP-vaccinated groups had lower viremia titers than control animals. All mockvaccinated animals or NP-VRP-vaccinated animals became ill, and died at days 8±11 after challenge. However, three out of five guinea pigs vaccinated with GP-VRP showed no signs of illness and survived challenge, and the remaining two showed increased survival times. No clear relationship with survival and antibody titers was observed, as the pre-challenge ELISA and PRNT50 titers of the two GP-VRP-inoculated animals that died were equivalent to those of the three survivors (Pushko et al., 2000).
The Ebola NP gene from the Mayinga strain of Ebola virus were derived from pSP64-based plasmid. The BamHI±EcoRI (2.3 kb) fragment containing the NPgene, was subcloned into a shuttle vector digested with BamHI and EcoRI within a polylinker sequence flanked by ClaI sites. From the shuttle vector, NP gene was transferred as ClaI-fragments into the ClaI site of the replicon clone, resulting in plasmids encoding the NP gene in place of the VEE structural protein genes (Pushko et al., 2000).
g. Immunization Route
Intramuscular injection (i.m.)
h.
Guinea pig Response
Host Strain:
strain 2 and strain 13
Vaccination Protocol:
VRP were diluted in PBS and administered to inbred, strain 2 or strain 13 guinea pigs. Groups of five guinea pigs were inoculated subcutaneously (s.c.) at day 0 with a total of 0.5 ml containing 10^7 IU VRP at one (strain 2) or two (strain 13) dorsal sites. Challenge was carried out 4 weeks after final immunization with VRP. Guinea pigs were challenged s.c. with 1000 LD50 of guinea pig- adapted Ebola virus. Animals were observed daily for 60 days, and morbidity (determined as changes in behavior, appearance, and weight) and survival were recorded. Blood samples were taken on the days indicated after challenge and viremia levels were determined by plaque assay (Pushko et al., 2000).
Efficacy:
At day 7 after challenge, NP-VRP-vaccinated group had lower viremia titers than control animals. All mock vaccinated animals or NP-VRP-vaccinated animals became ill, and died at days 8±11 after challenge (Pushko et al., 2000).
i.
Mouse Response
Host Strain:
BALB/c
Vaccination Protocol:
VRP were diluted in PBS and administered to 6±8 week old BALB/c mice. Groups of 10 BALB/c mice were inoculated on days 0 and 28 with two doses of NP-VRP, GP-VRP, or a mixture of both. Challenge was carried out 4 weeks after final immunization with VRP. Mice were challenged i.p. with mouse-adapted Ebola virus. To determine subsequent viral titers in the serum, liver, and spleen, two mice were taken from VRP-vaccinated or control groups on each of days 1±5 after challenge, anesthetized and exsanguinated. Portions of the liver and spleen were removed aseptically, weighed, and ground in a sterile mortar. Viral titers in the sera and tissues were determined by plaque assay (Pushko et al., 2000).
Efficacy:
NP-VRP was effective in protecting BALB/c mice against a lethal challenge with mouse-adapted Ebola virus (Pushko et al., 2000). All mice vaccinated with NP-VRP survived the challenge with no signs of illness (Pushko et al., 2000).
Description:
A mouse CMV (MCMV) vector expressing a CD8+ T cell epitope from the nucleoprotein (NP) of Zaire ebolavirus (ZEBOV) (MCMV/ZEBOV-NP(CTL)) (Tsuda et al., 2011).
A mouse CMV (MCMV) vector expressing a CD8+ T cell epitope from the nucleoprotein (NP) of Zaire ebolavirus (ZEBOV) (MCMV/ZEBOV-NP(CTL)) (Tsuda et al., 2011).
g. Immunization Route
Intramuscular injection (i.m.)
h.
Mouse Response
Vaccination Protocol:
Mice were vaccinated with rCMV- EBOV (Tsuda et al., 2011).
Vaccine Immune Response Type:
VO_0003057
Challenge Protocol:
Mice were challenged with a lethal dose of ZEBOV (Tsuda et al., 2011).
Efficacy:
The vaccine induced high levels of long-lasting (>8 months) CD8+ T cells against ZEBOV NP in mice. Importantly, all vaccinated animals were protected against lethal ZEBOV challenge (Tsuda et al., 2011).
Description:
New vectors were generated that contain EBOV viral protein 40 (VP40) or EBOV nucleoprotein (NP) as a second antigen expressed by the same rVSV vector that encodes the heterologous GP (Marzi et al., 2011).
Description:
New vectors were generated that contain EBOV viral protein 40 (VP40) or EBOV nucleoprotein (NP) as a second antigen expressed by the same rVSV vector that encodes the heterologous GP (Marzi et al., 2011).
Vaccination Protocol:
The guinea pigs were vaccinated intraperitoneally with a single dose of 2 ×10^5 PFU to guinea pigs of rVSV (Marzi et al., 2011).
Vaccine Immune Response Type:
VO_0003057
Challenge Protocol:
The guinea pigs were subsequently challenged with 1000 LD50 of GPA-ZEBOV [20] or boosted with the same dose of rVSV and challenged 3 weeks later (Marzi et al., 2011).
Efficacy:
After applying a 2-dose immunization approach, we observed an improved cross-protection rate, with 5 of 6 guinea pigs surviving the lethal ZEBOV challenge if vaccinated with rVSV-expressing SEBOV-GP and -VP40 (Marzi et al., 2011).
A recombinant vesicular stomatitis virus (rVSV) vector-based monovalent vaccine platform expressing a filovirus glycoprotein (Falzarano et al., 2011).
h. Immunization Route
Intramuscular injection (i.m.)
i.
Macaque Response
Vaccination Protocol:
Cynomolgus macaques were vaccinated with an rVSV vaccine expressing either the glycoprotein of Zaire ebolavirus (ZEBOV) or Côte d'Ivoire ebolavirus (CIEBOV) (Falzarano et al., 2011).
Vaccine Immune Response Type:
VO_0003057
Challenge Protocol:
The macaques were challenged with Bundibugyo ebolavirus (BEBOV)(Falzarano et al., 2011).
Efficacy:
A single vaccination with the ZEBOV-specific vaccine provided cross-protection (75% survival) against subsequent BEBOV challenge, whereas vaccination with the CIEBOV-specific vaccine resulted in an outcome similar to mock-immunized animals (33% and 25% survival, respectively) (Falzarano et al., 2011).
28. V920
a. Manufacturer:
BioProtection Systems Corporation and Department of Health and Human Services
Replicon RNAs were packaged into particles. Briefly, capped replicon RNAs were produced in vitro by T7 runoff transcription of NotI-digested plasmid templates using the RiboMAX T7 RNA polymerase kit. BHK cells were cotransfected with the replicon RNAs and two RNAs expressing the VEE virus structural proteins. The cell culture supernatants were harvested approximately 30 h after transfection and the replicon particles were concentrated and partially purified by centrifugation through a 20% sucrose cushion. Packaged VRPs were suspended in phosphatebuffered saline and titers were determined as immunofluorescent foci after infection of Vero cells as described using either EBOV-immune rabbit serum or mouse monoclonal antibodies to VP24 (Z-AC01-BG11-01), VP35 (M-HC01-AF11), or VP40 (M-HD06-AD10) (Wilson et al., 2001).
f. Virulence
g. Description
VP24 is an Ebola virus protein. It is membrane associated and is most likely located on the inside of the membrane. The function of VP24 is not known but it may serve as a minor matrix protein, facilitating the interaction of VP40 and/or GP with the RNP complex, or function in the uncoating of the virion during infection (Wilson et al., 2001).
h.
Mouse Response
Host Strain:
BALB/c and C57BL/6
Vaccination Protocol:
Groups of 10 BALB/c or C57BL/6 mice per experiment were subcutaneously injected at the base of the neck with 2(10^6) focus-forming units of VRPs encoding the EBOV-Z genes, or with a control replicon encoding the Lassa N gene. Booster immunizations were administered at 1-month intervals (Wilson et al., 2001).
Persistence:
None noted
Side Effects:
None noted
Efficacy:
Vaccination with VRPs encoding the EBOV-Z VP24 protein protected the majority (90±95%) of the BALB/c mice from lethal EBOV challenge. In a similar experiment, two inoculations of VRPs encoding the EBOV-Z VP24 protein also protected 5/5 BALB/c mice from a 3(10^4) LD50 challenge dose and 5/5 BALB/c mice from a 3(10^6) LD50 challenge dose. None of the C57BL/6 mice were protected. Most of the mice had detectable EBOV-Z-specific serum antibodies after vaccination with VRPs encoding the EBOV-Z VP protein (Wilson et al., 2001). These results indicate that the VP24 protein may be an important component of a vaccine designed to protect humans from Ebola hemorrhagic fever.
Replicon RNAs were packaged into particles. Briefly, capped replicon RNAs were produced in vitro by T7 runoff transcription of NotI-digested plasmid templates using the RiboMAX T7 RNA polymerase kit. BHK cells were cotransfected with the replicon RNAs and two RNAs expressing the VEE virus structural proteins. The cell culture supernatants were harvested approximately 30 h after transfection and the replicon particles were concentrated and partially purified by centrifugation through a 20% sucrose cushion. Packaged VRPs were suspended in phosphatebuffered saline and titers were determined as immunofluorescent foci after infection of Vero cells as described using either EBOV-immune rabbit serum or mouse monoclonal antibodies to VP24 (Z-AC01-BG11-01), VP35 (M-HC01-AF11), or VP40 (M-HD06-AD10) (Wilson et al., 2001).
f. Virulence
g. Description
VP30 is an Ebola virus protein. It associates with the genomic RNA in a ribonucleoprotein complex. The VP30 protein is not essential for replication, but it is necessary for efficient transcription in this system. It has also recently been shown to be essential for the recovery of infectious EBOV-Z from cloned cDNAs (Wilson et al., 2001).
h.
Mouse Response
Host Strain:
BALB/c and C57BL/6
Vaccination Protocol:
Groups of 10 BALB/c or C57BL/6 mice per experiment were subcutaneously injected at the base of the neck with 2(10^6) focus-forming units of VRPs encoding the EBOV-Z genes, or with a control replicon encoding the Lassa N gene. Booster immunizations were administered at 1-month intervals (Wilson et al., 2001).
Persistence:
None noted
Side Effects:
None noted
Efficacy:
Three injections of VRPs encoding EBOV-Z VP30 induced protection from lethal disease in 85% of the BALB/c mice examined. However, when the vaccination schedule was decreased to two injections, only 55% of the mice immunized with VP30 survived challenge. None of the C57BL/6 mice were protected. Most of the mice had detectable EBOV-Z-specific serum antibodies after vaccination with VRPs encoding the EBOV-Z VP protein (Wilson et al., 2001). These results indicate that the VP30 protein may be an important component of a vaccine designed to protect humans from Ebola hemorrhagic fever (Wilson et al., 2001).
Replicon RNAs were packaged into particles. Briefly, capped replicon RNAs were produced in vitro by T7 runoff transcription of NotI-digested plasmid templates using the RiboMAX T7 RNA polymerase kit. BHK cells were cotransfected with the replicon RNAs and two RNAs expressing the VEE virus structural proteins. The cell culture supernatants were harvested approximately 30 h after transfection and the replicon particles were concentrated and partially purified by centrifugation through a 20% sucrose cushion. Packaged VRPs were suspended in phosphatebuffered saline and titers were determined as immunofluorescent foci after infection of Vero cells as described using either EBOV-immune rabbit serum or mouse monoclonal antibodies to VP24 (Z-AC01-BG11-01), VP35 (M-HC01-AF11), or VP40 (M-HD06-AD10) (Wilson et al., 2001).
f. Virulence
g. Description
VP35 is an Ebola virus protein. It associates with the genomic RNA in a ribonucleoprotein complex. It is essential for replication and encapsidation of the EBOV genome. The VP35 protein has also recently been shown to be essential for the recovery of infectious EBOV-Z from cloned cDNAs. In addition to being an essential component of the replication complex, VP35 was also recently implicated as an interferon antagonist. VP35 may therefore facilitate viral replication in infected cells by blocking the induction of antiviral immune responses normally induced by the production of interferon (Wilson et al., 2001).
h.
Mouse Response
Host Strain:
BALB/c and C57BL/6
Vaccination Protocol:
Groups of 10 BALB/c or C57BL/6 mice per experiment were subcutaneously injected at the base of the neck with 2(10^6) focus-forming units of VRPs encoding the EBOV-Z genes, or with a control replicon encoding the Lassa N gene. Booster immunizations were administered at 1-month intervals (Wilson et al., 2001).
Persistence:
None noted
Side Effects:
None noted
Efficacy:
The VP35 protein was not efficacious in the BALB/c mouse model, as only 20 and 26% of the mice were protected from lethal challenge after either two or three doses, respectively. The mean day of death of the VP-vaccinated mice that succumbed to the EBOV challenge was within 1 day of the control Lassa N-vaccinated mice. C57BL/6 mice were protected from lethal EBOV challenge after vaccination with the EBOV-Z VP35 protein, with 70% of the mice protected after three inoculations. When the viral titers were measured 5 days after challenge, vaccination with VRPs encoding the EBOV-Z VP35 protein reduced the viral load by at least 4 log10 compared to control mice. Most of the mice had detectable EBOV-Z-specific serum antibodies after vaccination with VRPs encoding the EBOV-Z VP protein (Wilson et al., 2001). These results indicate that the VP35 protein may be an important component of a vaccine designed to protect humans from Ebola hemorrhagic fever (Wilson et al., 2001).
Replicon RNAs were packaged into particles. Briefly, capped replicon RNAs were produced in vitro by T7 runoff transcription of NotI-digested plasmid templates using the RiboMAX T7 RNA polymerase kit. BHK cells were cotransfected with the replicon RNAs and two RNAs expressing the VEE virus structural proteins. The cell culture supernatants were harvested approximately 30 h after transfection and the replicon particles were concentrated and partially purified by centrifugation through a 20% sucrose cushion. Packaged VRPs were suspended in phosphatebuffered saline and titers were determined as immunofluorescent foci after infection of Vero cells as described using either EBOV-immune rabbit serum or mouse monoclonal antibodies to VP40 (M-HD06-AD10) (Wilson et al., 2001).
f. Virulence
g. Description
VP40 is an Ebola virus protein. It is membrane-associated and is most likely located on the inside of the membrane. VP40 has been shown to associate with cell membranes, where it is believed to be involved in maturation of the virus by inducing viral assembly at the plasma membrane of infected cells (Wilson et al., 2001).
h.
Mouse Response
Host Strain:
BALB/c and C57BL/6
Vaccination Protocol:
Groups of 10 BALB/c or C57BL/6 mice per experiment were subcutaneously injected at the base of the neck with 2 x10^6 focus-forming units of VRPs encoding the EBOV-Z genes, or with a control replicon encoding the Lassa N gene. Booster immunizations were administered at 1-month intervals (Wilson et al., 2001).
Persistence:
None noted
Side Effects:
None noted
Efficacy:
Vaccination with VRPs encoding the VP40 protein protected 85 and 70% of the BALB/c mice after either two or three injections, respectively. None of the C57BL/6 mice were protected, however most of the mice had detectable EBOV-Z-specific serum antibodies after vaccination with VRPs encoding the EBOV-Z VP protein (Wilson et al., 2001). These results indicate that the VP30 protein may be an important component of a vaccine designed to protect humans from Ebola hemorrhagic fever.
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