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Vaccine Comparison

Chimeric SIN/VEE Virus SIN-83 Defective adenovirus expressing VEEV E2 glycoprotein Live attenuated vaccine TC-83 Live attenuated VEE vaccines live TC-83 VEE Vaccine with DHEA Recombinant RNA replicons from attenuated VEE virus VEE virus complex-specific monoclonal antibody VEE virus DNA vaccine encoding 26S VEE virus DNA vaccine pSTU-TRDF encoding VEEV E3–E2–6K VEE virus DNA vaccine VEEV IA/B parent encoding structural genes VEE Virus PE2/E1 mutant vaccine VEE virus recombinant vector vaccine RAd/VEEV VEE virus recombinant vector vaccine RAd/VEEV#2 VEE virus recombinant vector vaccine RAd/VEEV#3 encoding TC-83
Vaccine Information Vaccine Information Vaccine Information Vaccine Information Vaccine Information Vaccine Information Vaccine Information Vaccine Information Vaccine Information Vaccine Information Vaccine Information Vaccine Information Vaccine Information Vaccine Information
  • Vaccine Ontology ID: VO_0004111
  • Type: Recombinant vector vaccine
  • Antigen: All structural proteins derived from VEEV TC-83 (Paessler et al., 2003).
  • Preparation: The parental pToto1101 plasmid, encoding the SINV genome, and the pTC-83 plasmid, encoding the genome of VEEV TC-83, were obtained from Charles M. Rice (Rockefeller University, New York, N.Y.) and Richard Kinney (Centers for Disease Control, Fort Collins, Colo.), respectively. Fragments containing the SINV subgenomic promoter and the 5′ untranslated region (UTR) of the VEEV subgenomic RNA were generated by PCR amplification, cloned into the pRS2 plasmid for sequencing, and then used for generating the cDNA clone of the chimeric SIN-83S virus genome. The plasmid construct pSIN83 (Paessler et al., 2003) contained the promoter for SP6 RNA polymerase, followed by nucleotides (nt) 1 to 7601 of the SINV genome, nt 7536 to 11382 of VEEV TC-83 (with an additional C→T mutation of nt 7555), an AGGCCTTGGG sequence, and a 355-nt sequence containing the SINV 3′UTR (starting from nt 11394), poly(A) followed by an XhoI restriction site. Plasmids pZPC and pSH, containing infectious cDNAs of VEEV strains ZPC738 (subtype ID) and SH3 (subtype IC), respectively. The plasmids were purified and linearized. RNAs were synthesized and transfected into BHK-21 cells (Bredenbeek et al., 1993). Viruses were harvested after development of cytopathic effects, usually at 24 h following electroporation.
  • Virulence: None of the chimeric SIN/VEE viruses caused any detectable disease in adult mice after either intracerebral (i.c.) or subcutaneous (s.c.) inoculation, and all chimeras were more attenuated than the vaccine strain, VEEV TC83, in 6-day-old mice after i.c. infection (Paessler et al., 2003).
  • Description: The chimeric SIN/VEE viruses contain the replicative machinery from another alphavirus, Sindbis virus (SINV), and the structural genes from VEEV. The prototype chimeric virus SIN83 is capable of replicating in tissue culture and exhibits a safe and highly attenuated phenotype in mice and hamsters but induces a protective immune response against VEEV (Paessler et al., 2003). It is safe and efficacious in adult mice and hamsters and is potentially useful as VEEV vaccin. In addition, immunized animals provide a useful model for studying the mechanisms of the anti-VEEV neuroinflammatory response, leading to the reduction of viral titers in the CNS and survival of animals.
  • Vaccine Ontology ID: VO_0004116
  • Type: Recombinant vector vaccine
  • Antigen: VEEV E2 glycoprotein
  • POLS_EEVVT Structural polyprotein (p130) gene engineering:
    • Type: Recombinant protein preparation
    • Description: Recombinant defective type 5 adenoviruses expressing the E3E26K structural genes of VEEV were prepared and examined (Phillpotts et al., 2005).
    • Detailed Gene Information: Click Here.
  • Preparation: To make recombinant viruses, the core gene, the first three nucleotides at the 5′-end of the VEE E3 gene, the 3′-end of the 6K gene and the E1 gene were removed by restriction digestions. An initiation codon, the three nucleotides deleted from E3, the nucleotides removed from the 3′-end of the 6K gene and a termination codon were all added by ligation of oligonucleotide linkers. The E3–E2–6K fragment was then cloned into plasmid pMV101, derived from pMV100 by the addition of a unique Eco R1 restriction site at an Xba I site located downstream of the promoter sequence, to generate plasmid pVEEV. Plasmid pMV100, which contains the CMV major immediate early promoter and a polyadenylation signal. Three sites within the VEEV E2 glycoprotein were mutated by site-directed mutagenesis from the sequence found in the attenuated TC-83 strain to the sequence found in the virulent TrD strain. Each of the E3–E2–6K sequences from plasmids pVEEV, pVEEV#2 and pVEEV#3 were inserted into AHuman adenovirus type 5 (Ad5) dl309 to make recombinant adenoviruses. Ad5 dl309 has a deleted E1a gene (Jones et al., 1979) and is replication defective, requiring E1a complementation for replication. The E1a gene product may be supplied in trans by stably transfected 293 cells. A second deletion is found in the E3 region of this virus (Bett et al., 1995). The Ad5 vector is designed to enter mammalian cells and express proteins but it is defective for production of infectious progeny virus. All of the recombinant adenoviruses were purified by three rounds of terminal dilution in 293 cells cultured in 96-well plates. The VEEV inserts in the recombinant adenoviruses were characterised by sequencing (Phillpotts et al., 2005).

    To prepare virulent virus stocks, suckling mice were infected intracerebrally of each supplied virus. Infected brains were harvested, prepared as tissue suspensions and clarified by centrifugation. The titre of each VEEV strain was determined by plaque formation under a carboxymethyl cellulose overlay in Vero cells (Phillpotts et al., 2005).
  • Description: Recombinant defective type 5 adenoviruses, expressing the E3E26K structural genes of VEEV were examined for their ability to protect mice against airborne challenge with virulent virus. After intranasal administration, good protection was achieved against the homologous serogroup 1A/B challenge virus (strain Trinidad donkey). There was less protection against enzootic serogroup II and III viruses, indicating that inclusion of more than one E3E26K sequence in a putative vaccine may be necessary. These studies confirm the potential of recombinant adenoviruses as vaccine vectors for VEEV and will inform the development of a live replicating adenovirus-based VEEV vaccine, deliverable by a mucosal route and suitable for use in humans (Phillpotts et al., 2005).
  • Vaccine Ontology ID: VO_0004105
  • Type: Live, attenuated vaccine
  • Antigen: Stocks of TC-83 were prepared by propagation from a vial of vaccine for human use (National Drug Company, Philadelphia PA, Lot 4, run 2). Virulent VEEV strains were prepared as suckling mouse brain suspensions by standard methods.
  • Preparation: Stocks of TC-83 were prepared by propagation from a vial of vaccine for human use (National Drug Company, Philadelphia PA, Lot 4, run 2). Virulent VEEV strains were prepared as suckling mouse brain suspensions by standard methods. In vitro virus propagation and plaque assays were performed in BHK21 cells, grown in Glasgow minimal essential medium. A hyperimmune rabbit antiserum raised against TC-83 virus was kindly provided by T. Webber, DET, at CBD, Porton Down.
  • Virulence: none
  • Description: TC-83 has proven safe and effective for immunisation of horses (Eddy et al., 1972; Walton et al., 1972)and has US Food and Drug Administration Investigational New Drug status for use in Humans (IND No. 142). Vaccination with TC-83 virus produced solid protection against subcutaneous challenge with Venezuelan equine encephalitis (VEEV) viruses from homologous and heterologous serogroups, but breakthrough infection and disease occurred after airborne challenge.
  • Vaccine Ontology ID: VO_0004112
  • Type: Live, attenuated vaccine
  • Preparation: The generation of isogenic molecular clones and viral stocks for this study was previously described. Briefly, a full-length cDNA clone of the wild-type Trinidad donkey strain of VEE (TrD), pV3000 (Davis et al., 1989), served as the template. VEEV clones with either single or multiple mutations were constructed for site-directed mutagenesis of a M13 subclone of the glycoprotein genes in pV3000. Infectious VEEV RNA, transcribed in vitro from these clones (e.g. pV3519), was used to produce virus (e.g. termed V3519) by transfection of baby hamster kidney (BHK) cells (Davis et al., 1991). Virus stocks tested in these studies were obtained directly from transfected BHK culture supernatant fluids and were used after appropriate dilution without further passage. Parent V3000 virus was passaged twice in BHK cells after collection from transfection supernatant fluids.
  • Description: Molecular clones of vaccine candidates were constructed by inserting either three independently attenuating mutations or a PE2 cleavage-signal mutation with a second-site resuscitating mutation into full-length cDNA clones. Vaccine candidate viruses were recovered through DNA transcription and RNA transfection of cultured cells, and assessed in rodent and non-human primate models. Based on results from this assessment, one of the PE2 cleavage-signal mutants, V3526, was determined to be the best vaccine candidate for further evaluation for human use.
  • Vaccine Ontology ID: VO_0004261
  • Type: Live, attenuated vaccine
  • Status: Research
  • Adjuvant: DHEA vaccine adjuvant
    • VO ID: VO_0001340
    • Description: DHEA (Sigma Chemical Co., St. Louis, MO, U.S.A.) was dissolved in 50% ethanol. Mice were injected with DHEA (10 mg/Kg), in 0.3 mL, subcutaneously (s.c.), 4 hours before vaccination (Negrette et al., 2001).
  • Immunization Route: Intraperitoneal injection (i.p.)
  • Vaccine Ontology ID: VO_0004109
  • Type: Recombinant vector vaccine
  • Preparation: A plasmid encoding the VEE replicon vector was described previously (Davis et al., 1996; Pushko et al., 1997). The Ebola NP and GP genes from the Mayinga strain of Ebola virus were derived from pSP64- and pGEM3Zf(ÿ)-based plasmids (Sanchez et al., 1993; Sanchez et al., 1989). The fragments containing the NP and GP genes, respectively, were subcloned into a shuttle vector(Davis et al., 1996; Grieder et al., 1995). From the shuttle vector, NP or GP genes were transferred the replicon clone, resulting in plasmids encoding the NP or GP gene in place of the VEE structural protein genes.

    Transcripts of the replicon were pooled and cotransfected as described previously (Pushko et al., 1997). Transfected cells were incubated and harvested. Culture supernatants were clarified by centrifugation and VRP particles were concentrated and partially purified by centrifugation. VRP titers were determined as immunofluorescent foci after infection of BHK cells in eight-well chamber slides as previously described (Pushko et al., 1997; Pifat et al., 1988). Positive cells were counted, and the titers of the VRP preparations were calculated. Production and titration of Lassa N±VRP were described previously (Pushko et al., 1997). For expression assays, BHK cells were infected with NP- or GP±VRP, or cotransfected with replicon and helper RNAs. Cells were incubated before harvesting. Polypeptides were separated and Western blotting was carried out. VEE proteins in western blots were detected with sera from guinea pigs immunized with the live-attenuated TC-83 vaccine.
  • Description: RNA replicons derived from an attenuated strain of Venezuelan equine encephalitis virus (VEE), an alphavirus, were configured as candidate vaccines for Ebola hemorrhagic fever. The Ebola nucleoprotein (NP) or glycoprotein (GP) genes were introduced into the VEE RNA downstream from the VEE 26S promoter in place of the VEE structural protein genes. The resulting recombinant replicons, expressing the NP or GP genes, were packaged into VEE replicon particles (NP–VRP and GP–VRP, respectively). The immunogenicity of NP–VRP and GP–VRP and their ability to protect against lethal Ebola infection were evaluated in BALB/c mice and in two strains of guinea pigs. The GP–VRP alone, or in combination with NP–VRP, protected both strains of guinea pigs and BALB/c mice, while immunization with NP–VRP alone protected BALB/c mice, but neither strain of guinea pig. Passive transfer of sera from VRP-immunized animals did not confer protection against lethal challenge. However, the complete protection achieved with active immunization with VRP, as well as the unique characteristics of the VEE replicon vector, warrant further testing of the safety and efficacy of NP–VRP and GP–VRP in primates as candidate vaccines against Ebola hemorrhagic fever.
  • Vaccine Ontology ID: VO_0004104
  • Type: Monoclonal antibody
  • Antigen: No VEE virus antigens were used for this vaccination approach. The monoclonal antibodies are specifically designed against VEEV surface glycoproteins E1 and E2.
  • Preparation: The monoclonal antibodies (MAB) are: (1). the VEEV surface glycoprotein E2-specific monoclonal antibodies 1A4A-1; (2). the VEEV surface glycoprotein E2-specific monoclonal antibodies 1A3B-7; (3) the glycoprotein E1-specific MAB 3B2A-9; (4) 4. the E1-specific MAB MH2. All MAB were produced and purified by protein G affinity chromatography as previously described (Phillpotts et al., 2002).

    Strains of VEEV from serogroups IA/B (Trinidad donkey; TrD), IC (P676), ID (3880), IE (Menall), IF (78v-3531), II (Fe37c), IIIA (Mucambo BeAn8), IV (Pixuna BeAn356445), V (Cabassou CaAr508) and IV (AG80-663) are used.
    For the preparation of virus stocks, suckling mice were infected intracerebrally with a 1/1000 dilution of each virus. Infected brains were harvested at 24 h, prepared as 10% tissue suspensions in L15MM and clarified by centrifugation at 10,000 × g for 15 min. Alternatively viruses were propagated in Vero cell monolayers inoculated with an m.o.i. of 0.01 and harvested when 50–75% of the cell sheet showed evidence of virus cytopathic effect (CPE). The titre of each VEEV strain was determined by plaque formation under a carboxymethyl cellulose overlay in BHK21 cells.
  • Virulence: Not virulent
  • Description: Using a murine model to study monoclonal antibody (MAB) a VEEV complex-specific, glycoprotein E2-binding MAB was identified, able to protect against disease induced by exposure to aerosolised VEEV from serogroups I, II and IIIA (mouse-virulent strains). There was no synergy in protection between anti-E1 and anti-E2 MAB. Assays of MAB virus neutralising activity in a homologous (mouse fibroblast) cell line suggested that neutralisation played a significant role in protection in addition to the previously reported mechanism of Fc receptor-binding [Mathews et al., 1985. J. Virol. 55, 594–600]. Development of an analogous human MAB with identical VEEV epitope specificity may be informed and monitored by reference to these properties (Phillpotts, 2006).
  • Vaccine Ontology ID: VO_0004488
  • Type: DNA vaccine
  • Status: Research
  • Host Species as Laboratory Animal Model: Mouse
  • 26S mRNA gene engineering:
  • Vector: pWRG7077 (Riemenschneider et al., 2003)
  • Immunization Route: Intramuscular injection (i.m.)
  • Vaccine Ontology ID: VO_0004490
  • Type: DNA vaccine
  • Status: Research
  • Host Species as Laboratory Animal Model: Mouse
  • C-E3-E2-E1-6K gene engineering:
    • Type: DNA vaccine construction
    • Detailed Gene Information: Click Here.
  • Vector: pJW4304 prime, adenovirus-based vector boost (Perkins et al., 2006)
  • Immunization Route: Intramuscular injection (i.m.)
  • Vaccine Ontology ID: VO_0004421
  • Type: DNA vaccine
  • Status: Research
  • Host Species as Laboratory Animal Model: Mouse
  • Antigen: VEEV subtype IA/B capsid protein (C) and envelope glycoproteins (E1 and E2) (Dupuy et al., 2009)
  • E1 glycoprotein gene engineering:
    • Type: DNA vaccine construction
    • Detailed Gene Information: Click Here.
  • E2 envelope protein gene engineering:
    • Type: DNA vaccine construction
    • Detailed Gene Information: Click Here.
  • Vector: pES (Dupuy et al., 2009)
  • Immunization Route: Intradermal injection (i.d.)
  • Type: Live, attenuated vaccine
  • Status: Research
  • Host Species as Laboratory Animal Model: Mouse
  • E1 glycoprotein gene engineering:
    • Type: Gene mutation
    • Description: This PE2/E1 mutant is from Venezuelan equine encephalitis virus (Davis et al., 1995).
    • Detailed Gene Information: Click Here.
  • PE2 gene engineering:
    • Type: Gene mutation
    • Description: This PE2/E1 mutant is from Venezuelan equine encephalitis virus (Davis et al., 1995).
    • Detailed Gene Information: Click Here.
  • Immunization Route: intranasal immunization
  • Vaccine Ontology ID: VO_0004422
  • Type: Recombinant vector vaccine
  • Status: Research
  • Host Species as Laboratory Animal Model: Mouse
  • Antigen: E3–E2–6K from VEEV strain TC-83 (Phillpotts et al., 2005)
  • Vector: recombinant adenovirus (Phillpotts et al., 2005)
  • Immunization Route: Intramuscular injection (i.m.)
  • Vaccine Ontology ID: VO_0004423
  • Type: Recombinant vector vaccine
  • Status: Research
  • Host Species as Laboratory Animal Model: Mouse
  • Antigen: E3-E2-6K from VEEV TC-83 with two mutations in the major neutralisation site of E. (Phillpotts et al., 2005)
  • Vector: recombinant adenovirus (Phillpotts et al., 2005)
  • Immunization Route: Intramuscular injection (i.m.)
  • Vaccine Ontology ID: VO_0004424
  • Type: Recombinant vector vaccine
  • Status: Research
  • Host Species as Laboratory Animal Model: Mouse
  • Antigen: E3-E2-6K from VEEV TC-83 with two mutations in the major neutralisation site of E2 and an additional mutation at the N-terminus of E2 (Phillpotts et al., 2005)
  • Vector: recombinant adenovirus (Phillpotts et al., 2005)
  • Immunization Route: Intramuscular injection (i.m.)
Host Response Host Response Host Response Host Response Host Response Host Response Host Response Host Response Host Response Host Response Host Response Host Response Host Response Host Response

Mouse Response

  • Host Strain: NIH Swiss
  • Vaccination Protocol: Six-week-old, female NIH Swiss mice (12 per group) were inoculated on day 0 s.c. into the medial thigh with chimeric SIN/VEE viruses SIN-83. The live VEE TC-83 vaccine virus was used as control for comparison. One half of the animals (six per group) received an additional booster on day 28, which was performed in the same way as the initial immunization. All of the animals were bled on days 1, 2, and 3 and at 4 and 8 weeks after immunization. Serum samples from the first 3 days after immunization were tested for the presence of infectious virus by a plaque assay on BHK-21 cells (Paessler et al., 2003).
  • Persistence: To compare the virulence of the VEE TC-83 and SIN-83 viruses, 6-day-old mice were inoculated i.c. or s.c. with different doses of each virus ranging from 2 × 104 to 2 × 106 PFU. VEEV TC-83 was virulent for weanling mice regardless of the inoculation route. VEEV TC-83 was less pathogenic for weanling mice after s.c. inoculation (mortality rate, 10 to 20%). However, many of the surviving animals developed clinical disease and/or CNS sequelae. None of the SIN-83-inoculated animals had detectable clinical illness. Animals infected with VEEV TC-83 at the age of 6 days were highly inhibited in their growth compared to those infected with SIN-83 or compared to the noninfected control group of the same age (Paessler et al., 2003).
  • Immune Response: After 28 days, VEEV-specific neutralizing antibodies in the sera of SIN-83 and VEE TC-83 immunized groups. However, the titers in VEEV TC-83-immunized animals were higher. This can be explained by the higher replication levels of this virus in cell culture.
  • Side Effects: none
  • Challenge Protocol: Challenge studies to determine the protection against clinical encephalitis in the mouse model. Fifteen 6-week-old, female NIH Swiss mice were vaccinated with 5 x 105 PFU of each chimeric virus or PBS alone (control) in a total volume of 100 µl. After vaccination, each cohort of 15 animals was maintained for 8 weeks without any manipulation. Immunized animals were then challenged with VEEV subtype ID strain ZPC738 by using three different inoculation methods: (i) s.c. inoculation into the medial thigh with 106 PFU (roughly 106 50% lethal dose) per animal in 0.1 ml of PBS (five mice per group), (ii) i.c. inoculation into the left brain hemisphere with 2 x 105 PFU per animal in 20 µl of PBS (five mice per group), and (iii) intranasal (i.n.) inoculation with 2 x 105 PFU per animal in 20 µl of PBS (five mice per group). Mice were observed for clinical illness (for anorexia and/or paralysis) and/or death twice daily for a period of 2 months.

    Challenge studies to determine protection against viral replication in the brain following i.c. or i.n. inoculation with ZPC738. After a group of mice was vaccinated, the first challenge with ZPC738 was performed using two different inoculation methods: (i) i.c. inoculation into the left brain hemisphere with 2 x 105 PFU in 20 µl of PBS and (ii) i.n. inoculation with 2 x 105 PFU in 20 µl of PBS. Two animals per group were euthanized on days 3, 7, and 28 after infection, and lungs, livers, spleens, kidneys, and brains were collected for viral titration or histological examinations. In addition, 10 animals per group were housed for 28 days after i.n. challenge with ZPC738, without any manipulation. On day 28, all animals from this group received the second i.n. dose of 2 x 105 PFU of ZPC738. Two animals per group were euthanized on days 3, 7, and 28 postchallenge, and organs were collected as described above.
  • Efficacy: All vaccinated mice were protected against lethal encephalitis following i.c., s.c., or intranasal (i.n.) challenge with the virulent VEEV ZPC738 strain (ZPC738). In spite of the absence of clinical encephalitis in vaccinated mice challenged with ZPC738 via i.n. or i.c. route, high levels of infectious challenge virus in the central nervous system (CNS) were regularly detected. However, infectious virus was undetectable in the brains of all immunized animals at 28 days after challenge (Paessler et al., 2003).

Mouse Response

  • Host Strain: Balb/c
  • Vaccination Protocol: Balb/c mice, 6–8 weeks old (Charles River Laboratories, UK) were immunised intranasally under halothane anaesthesia on days 0, 7 and 21 with 107.0 pfu of each recombinant adenovirus in 50 μl PBS.
  • Immune Response: The ability of serum to neutralise VEEV was examined in standard plaque reduction assays. Briefly, dilutions of serum and VEEV suspended in L15MM were mixed and incubated at 4 °C overnight. Residual infectious virus was estimated by plaque assay in Vero cells. A reduction in plaque numbers of equal to or greater than 50% was considered indicative of virus neutralisation.

    Immunisation with RAd/VEEV#3 provides cross-protection against other epizootic and enzootic strains
  • Challenge Protocol: Seven days after the final immunisation, the animals were challenged via the airborne route by exposure for 20 min to a polydisperse aerosol generated by a Collison nebuliser (Phillpotts et al., 1997). Mice were contained loose within a closed box during airborne challenge. The virus dose was calculated by sampling the air in the box and assuming a respiratory minute volume for mice of 1.25 ml/g (Guyton, A.C., 1947). After challenge, mice were observed twice daily for clinical signs of infection (piloerection, hunching, inactivity, excitability and paralysis) by an observer who quantified these and was unaware of the treatment allocations. In accordance with UK Home Office requirements and as previously described, humane endpoints were used (Wright et al., 1998). These experiments therefore record the occurrence of severe disease rather than mortality. Even though it is rare for animals infected with virulent VEEV and showing signs of severe illness to survive, our use of humane endpoints should be considered when interpreting any virus dose expressed here as 50% lethal doses (LD50).
  • Efficacy: Optimal protection within the VEEV IA/B serogroup depends upon sequence homology
  • Description: Balb/c mice, 6-8 weeks old

Mouse Response

  • Host Strain: outbred Porton TO
  • Vaccination Protocol: Subcutaneous (s.c.) inoculations of TC-83 virus in L15 MM, L15 MM alone, or virulent VEEV in L15 MM were delivered into the scruff of the neck in a volume of 100 μl. The number of s.c. LD50/ml was determined by titration using five mice per dilution.
  • Challenge Protocol: Infection by the airborne route was achieved using a specially constructed small animal exposure box(Phillpotts et al., 1997). Briefly, mice were exposed loose in a box of 80 l capacity, vented through a HEPA filter, to a virus-containing aerosol produced using a Collison nebuliser (8 l/min). The virus suspending fluid was L15 MM with 2% trehalose and the exposure time was 20 min. The air in the box was sampled with a glass impinger running at 1 l/min and the quantity of virus present in the Collison spray reservoir at the end of each run was determined by back-titration. A titration experiment was performed for each virus (five mice per dilution) in order to calculate the number of LD50 delivered by a given dilution of virus in the spray. As there was a linear relationship between the quantity of virus in the Collison reservoir and the impinger, in subsequent experiments the dose of virus delivered was calculated from titration of the impinger contents. Data for each virus at each time point were from a single exposure.
  • Efficacy: Vaccination with TC-83 virus produced solid protection against subcutaneous challenge with Venezuelan equine encephalitis (VEEV) viruses from homologous and heterologous serogroups, but breakthrough infection and disease occurred after airborne challenge.
  • Description: Infection by the airborne route was achieved using a specially constructed small animal exposure box(Phillpotts et al., 1997).

Mouse Response

  • Host Strain: C57BL/6
  • Vaccination Protocol: Female C57BL/6 mice (8–10 weeks) were inoculated s.c. with 0.2 ml of cell culture medium containing either no virus, or plaque forming units (pfu) of the virulent V3000 virus or one of the mutant viral strains. Groups of animals inoculated with either a single human dose of TC-83 or three human doses of C-84 on days 0, 7 and 28 were used as comparisons. The degree of attenuation of the viral strains was assessed during the 14-day observation period after inoculation. On day 49 after inoculation, surviving mice were bled from the retro-orbital sinus under methoxyflurane (Pitman-Moore, Mundelein, IL) anesthesia.
  • Challenge Protocol: On day 55 after the primary inoculation, animals were challenged with a calculated dose of 105 pfu of V3000 or 104 pfu of TrD by aerosol exposure or by intraperitoneal inoculation. For aerosol challenge, animals were exposed for 10 min to an infectious aerosol generated by a Collison nebulizer within a Plexiglass chamber contained within a Class III biological safety cabinet located in a Biosafety Level 3 laboratory. Viral doses delivered by aerosol were calculated by standard procedures(Hart et al., 1997). Protection was assessed by monitoring animals for 28 days post-infection. Additional groups of animals were inoculated with selected viruses (V3526 or V3528) or TC-83 by aerosol and then challenge by aerosol with V3000 on day 55 post-inoculation to evaluate the ability of these viruses to induce mucosal immunity and protect against aerosol challenge.
  • Efficacy: All single mutants tested in mice were fully attenuated and induced protective immune responses (data not shown).
  • Description: female 8-10 weeks

Mouse Response

  • Host Strain: NMRI-IVIC
  • Vaccination Protocol: Mice were immunized intraperitoneally (i.p.) with 0.05 mL of live TC-83 VEE virus suspension, containing 1.7 x 106 PFU/mL, in 0.4% BABS. Virus stock from IVIC was replicated in VERO cell culture and contained 106.78 LD50. DHEA (Sigma Chemical Co., St. Louis, MO, U.S.A.) was dissolved in 50% ethanol. Mice were injected with DHEA (10 mg/Kg), in 0.3 mL, subcutaneously (s.c.), 4 hours before vaccination. Control mice were injected with the diluent (Negrette et al., 2001).
  • Immune Response: In mice treated with a single dose of DHEA, a significant increase (p<0.01) of antibody titers (Control = 1:1.680 vs. DHEA treated group = 1:4.320) was detected at week 2 after vaccination with TC-83 virus (Negrette et al., 2001).
  • Challenge Protocol: Live VEE virus (10 LD50) was injected in mice treated with DHEA, 21 days after immunization, and 2 to 5 days later they were sacrificed to determine viral titers in serum and brain (Negrette et al., 2001).
  • Efficacy: MIce immunized with the TC-83 virus and DHEA had reduced viremia in the blood and brain after challenge (Negrette et al., 2001).

Mouse Response

  • Host Strain: BALB/c
  • Vaccination Protocol: VRP were diluted and administered to BALB/c mice. Groups of mice were inoculated subcutaneously (s.c.).
  • Side Effects: none
  • Challenge Protocol: Challenge was carried out 4 weeks after final immunization with VRP. Mice were challeged i.p. with mouse-adapted Ebola virus. Animals wre observed daily for 60 days and morbidity and survival were recorded.
  • Efficacy: The GP-VRP alone, or in combination with NP-VRP, protected BALB/c mice. Immunization with NP-VRP alone protected BALB/c mice. Passive transfer of sera from VRP-immunized animals did not confer protection against lethal challenge.

Mouse Response

  • Host Strain: Balb/c mice
  • Vaccination Protocol: Balb/c mice, 3–4 (s.c. challenge) or 6–8 (airborne challenge) weeks old (Charles River Laboratories, UK) were passively immunized intraperitoneally (i.p.) 24 h before exposure to virulent virus with MAB diluted in PBS.
  • Side Effects: No side effect
  • Challenge Protocol: The challenge route was either s.c. (100 μl virus-containing suspension in PBS) or via the airborne route, by exposure for 20 min to a poly disperse aerosol generated by a Collison nebuliser (Phillpotts et al., 1997). Mice were contained loose within a closed box during airborne challenge. The virus dose was calculated by sampling the air in the box and assuming a respiratory minute volume for mice of 1.25 ml/g (Guyton, A.C., 1947). After challenge, mice were observed twice daily for clinical signs of infection (piloerection, hunching, inactivity, excitability and paralysis) by an observer who quantified these and was unaware of the treatment allocations. These clinical signs are typical of VEEV infection and occurred in all mice challenged with VEEV.
    In previous studies (Phillpotts et al., 2002) their association with VEEV infection as the cause of death has been confirmed by the isolation of virus from brain and other internal organs. In accordance with UK Home Office requirements and as previously described, humane endpoints were used (Wright et al., 1998). These experiments therefore record the occurrence of severe disease rather than mortality. Even though it is rare for animals infected with virulent VEEV and showing signs of severe illness to survive, our use of humane endpoints should be considered when interpreting any virus dose expressed here as 50% lethal doses (LD50).
  • Efficacy: MAB MH2, 3B2A-9 and 1A3B-7 may be broadly reactive and potentially able to protect against a range of VEEV strains. There was also the possibility that the combination of E1- and E2-specific MAB could lead to synergy in protection. MAB 1A4A-1 while not broadly reactive, was able to bind strongly to, neutralise and protect against challenge with some VEEV strains. It was therefore included in these experiments as a positive control.
  • Description: The challenge route was either s.c. (100 μl virus-containing suspension in PBS) or via the airborne route, by exposure for 2

Mouse Response

  • Vaccine Immune Response Type: VO_0000286
  • Efficacy: DNA vaccines to four dissimilar pathogens that are known biowarfare agents, Bacillus anthracis, Ebola (EBOV), Marburg (MARV), and Venezuelan equine encephalitis virus (VEEV), can elicit protective immunity in relevant animal models. In addition, a combination of all four vaccines is shown to be equally as effective as the individual vaccines for eliciting immune responses in a single animal species (Riemenschneider et al., 2003).

Mouse Response

  • Vaccine Immune Response Type: VO_0000286
  • Efficacy: Particle-mediated epidermal delivery of a DNA vaccine encoding the E2 glycoprotein of VEEV can be boosted with a mucosally-delivered Ad-based vaccine encoding the same E2 glycoprotein, resulting in a significant increase in protection against airborne VEEV (Perkins et al., 2006).

Mouse Response

  • Vaccine Immune Response Type: VO_0000286
  • Immune Response: The vaccine was able to induce increased total anti-VEEV IA/B antibody responses and increased neutralizing antibody responses (Dupuy et al., 2009).
  • Efficacy: The VEEV IA/B parent construct protected 80% of the mice from the lethal challenge, which is the same level of protection elicited by this DNA vaccine in a previous study. As in the previous study, mice vaccinated with the VEEV IA/B parent displayed signs of illness after challenge including ruffled fur, loss of appetite, and inactivity; however, the surviving mice completely recovered before the end of the 28 day post-challenge observation period (Dupuy et al., 2009).

Mouse Response

  • Persistence: A PE2/E1 mutant is highly attenuated in mice (Davis et al., 1995).
  • Efficacy: A PE2/E1 mutant induces complete protection in mice from challenge with wild type VEE virus (Davis et al., 1995).

Mouse Response

  • Vaccine Immune Response Type: VO_0000286
  • Immune Response: Intranasal immunisation of mice with this recombinant virus produced a vigorous antibody response, with local IgA also detectable in respiratory secretions accompanied by highly significant protection against airborne VEEV challenge (Phillpotts et al., 2005).
  • Efficacy: This vaccine provided protection against low dose challenge (73 LD50); 4 out of 6 challenged mice survived (Phillpotts et al., 2005).

Mouse Response

  • Vaccine Immune Response Type: VO_0000286
  • Immune Response: Intranasal immunisation of mice with this recombinant virus produced a vigorous antibody response, with local IgA also detectable in respiratory secretions accompanied by highly significant protection against airborne VEEV challenge (Phillpotts et al., 2005).
  • Efficacy: This vaccine provided protection against low dose challenge (73 LD50); 4 out of 6 challenged mice survived (Phillpotts et al., 2005).

Mouse Response

  • Vaccine Immune Response Type: VO_0000286
  • Immune Response: Intranasal immunisation of mice with this recombinant virus produced a vigorous antibody response, with local IgA also detectable in respiratory secretions accompanied by highly significant protection against airborne VEEV challenge (Phillpotts et al., 2005).
  • Efficacy: This vaccine provided protection against low and intermediate dose challenge (73 LD50 and 640 LD50, respectively); 5 out of 6 mice survived low dose challenge and 3 out of 5 mice survived intermediate dose challenge (Phillpotts et al., 2005).

Monkey Response

  • Host Strain: Macaca fascicularis
  • Vaccination Protocol: The non-human primate model monkey used to test the safety and efficacy of VEEV vaccines was previously described (Pratt et al., 2003). Briefly, 30 healthy cynomolgus macaques were s.c. implanted with radiotelemetry devices to monitor body temperatures. During the pre-vaccination and pre-challenge periods (day −10 to day 0) and the 21 days after vaccination and challenge, body temperatures were recorded every 15 min. An autoregressive integrated moving average model (BMDP Statistics Software 1992) for each monkey was developed using the averaged hourly body temperature data over a baseline-training period (day −10 to day −3) and was used to forecast normal body temperature values during the vaccination and challenge time periods. Significant temperature elevations were used to compute fever duration (number of hours or days of significant temperature elevation) and fever-hours (sum of the significant temperature elevations).

    Monkeys were randomly divided into six groups (N=5) and each monkey received a single s.c. 0.5 ml dose of a vaccine candidate (V3524, V3526 or V3528), TC-83, V3000 or virus-free cell culture medium. On day 35 or 36 after inoculation, bronchial lavage and blood samples were collected for N antibody titrations.
  • Side Effects: fever for V3524, V3526, V3528, TC-83. Four days of significant fever for wild type V3000
  • Challenge Protocol: On days 42 or 43, monkeys were anaesthetized and exposed for to an infectious aerosol of V3000. Monkeys were bled daily for 6 days after both immunization and challenge to monitor viremias and lymphocyte counts. On day 14 post-challenge, serum was collected for N antibody titrations. Virus dosages, viremias, and N antibody titers were determined in similar manner to those for the rodent studies. Statistical evaluation of the groups of monkeys was made using analysis of variance followed by multiple comparisons using the Tukey studentized range test (SAS ver. 6.10, Cary, NC).
  • Efficacy: Monkeys vaccinated with V3526 or V3528 were well protected against aerosol challenge with few to no signs of fever, lymphopenia, or viremia—similar to the group of monkeys previously inoculated with V3000. The group of monkeys vaccinated with TC-83 was also in this category, but one monkey that did not have pre-challenge N antibody titers did develop fever responses similar to the mock-inoculated monkeys. Unlike the groups of monkeys inoculated with V3526, V3528, TC-83, or V3000, the group of monkeys inoculated with V3524 was not as well protected against aerosol challenge with V3000 and was in a distinct fever grouping. It suggested a higher degree of infection and viral replication in these monkeys. Additionally, viremia was present in one V3524-inoculated monkey.
  • Description: 30 healthy cynomolgus macaques (Macaca fascicularis, 4.2–6.7 kg), screened negative by ELISA for previous exposure to alphaviruses

Guinea pig Response

  • Host Strain: strain 2 or strain 13
  • Vaccination Protocol: VRP were diluted and administered to BALB/c mice. Groups of guinea pigs were inoculated subcutaneously (s.c.).
  • Side Effects: none
  • Challenge Protocol: Challenge was carried out 4 weeks after final immunization with VRP. Guinea pigs were challeged s.c. with guinea pig-adapted Ebola virus. Animals wre observed daily for 60 days and morbidity and survival were recorded.
  • Efficacy: The GP-VRP alone, or in combination with NP-VRP, protected both strains of guinea pigs. Immunization with NP-VRP alone protected neither strain of guinea pigs. Passive transfer of sera from VRP-immunized animals did not confer protection against lethal challenge.

Hamster Response

  • Host Strain: golden hamster
  • Vaccination Protocol: Three 6-week-old female Syrian golden hamsters per viral strain were vaccinated s.c. in the right medial thigh with 5 x 105 PFU of SAAR/TRD, SIN/ZPC, SIN/TRD, or TC83 strain or PBS alone. Blood samples were obtained daily for the first 3 days after infection, and the animals were observed twice daily for 21 days. Serum viremia was determined by using a plaque assay on BHK-21 cells as previously described. The presence of neutralizing antibody in hamster serum samples was determined via a plaque reduction neutralization test, as described for the murine experiments
  • Side Effects: none
  • Challenge Protocol: Three weeks after vaccination, the hamsters were challenged s.c. in the medial thigh with ZPC738 at a dose of 106 PFU in a total volume of 100 µl of PBS (roughly 5 x 106 50% lethal dose). The animals were observed for 28 days, and deaths or cases of clinical illness were documented.
  • Efficacy: Hamsters vaccinated with chimeric SIN/VEE viruses were also protected against s.c. challenge with ZPC738.
  • Description: Six- to 8-week-old female Syrian golden hamsters (Mesocricetus auratus) were purchased from Harlan and acclimatized in the facility for a week prior to infection.

Hamster Response

  • Host Strain: Syrian
  • Vaccination Protocol: Female Syrian (7–9 weeks) were inoculated s.c. with 0.2 ml of cell culture medium containing either no virus, or plaque forming units (pfu) of the virulent V3000 virus or one of the mutant viral strains. Groups of animals inoculated with either a single human dose of TC-83 or three human doses of C-84 on days 0, 7 and 28 were used as comparisons. The degree of attenuation of the viral strains was assessed during the 14-day observation period after inoculation. On day 49 after inoculation, surviving hamsters were bled by cardiac puncture under tiletamine-zolazepam (50 mg/kg, Aveco Co., Inc., Fort Dodge, IA) anesthesia.
  • Challenge Protocol: On day 55 after the primary inoculation, animals were challenged with a calculated dose of 105 pfu of V3000 or 104 pfu of TrD by aerosol exposure or by intraperitoneal inoculation. For aerosol challenge, animals were exposed for 10 min to an infectious aerosol generated by a Collison nebulizer within a Plexiglass chamber contained within a Class III biological safety cabinet located in a Biosafety Level 3 laboratory. Viral doses delivered by aerosol were calculated by standard procedures(Hart et al., 1997). Protection was assessed by monitoring animals for 28 days post-infection. Additional groups of animals were inoculated with selected viruses (V3526 or V3528) or TC-83 by aerosol and then challenge by aerosol with V3000 on day 55 post-inoculation to evaluate the ability of these viruses to induce mucosal immunity and protect against aerosol challenge.
  • Efficacy: All single mutants, when tested in hamsters, ranged from fully virulent to partially attenuated. In general, hamsters that survived did generate protective immunity against aerosol challenge.
  • Description: female 7-9 weeks
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