VIOLIN Logo
VO Banner
Search: for Help
About
Introduction
Statistics
VIOLIN News
Your VIOLIN
Register or Login
Submission
Tutorial
Vaccine & Components
Vaxquery
Vaxgen
VBLAST
Protegen
VirmugenDB
DNAVaxDB
CanVaxKB
Vaxjo
Vaxvec
Vevax
Huvax
Vaccine Mechanisms
Vaximmutordb
Vaxism
Vaxar
Vaccine Literature
VO-SciMiner
Litesearch
Vaxmesh
Vaxlert
Vaccine Design
Vaxign
Community Efforts
Vaccine Ontology
ICoVax 2012
ICoVax 2013
Advisory Committee
Vaccine Society
Vaxperts
VaxPub
VaxCom
VaxLaw
VaxMedia
VaxMeet
VaxFund
VaxCareer
Data Exchange
V-Utilities
VIOLINML
Help & Documents
Publications
Documents
FAQs
Links
Acknowledgements
Disclaimer
Contact Us
UMMS Logo

Vaccine Detail

Live attenuated VEE vaccines
Vaccine Information
  • 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.
Host Response

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

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

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
References
BMDP Statistics Software 1992: . BMDP Statistics Software. 467. BMDP Statistics Software Release 7. 1992. , .
Davis et al., 1989: Davis NL, Willis LV, Smith JF, Johnston RE. In vitro synthesis of infectious venezuelan equine encephalitis virus RNA from a cDNA clone: analysis of a viable deletion mutant. Virology. 1989 Jul; 171(1); 189-204. [PubMed: 2525837].
Davis et al., 1991: Davis NL, Powell N, Greenwald GF, Willis LV, Johnson BJ, Smith JF, Johnston RE. Attenuating mutations in the E2 glycoprotein gene of Venezuelan equine encephalitis virus: construction of single and multiple mutants in a full-length cDNA clone. Virology. 1991 Jul; 183(1); 20-31. [PubMed: 2053280 ].
Hart et al., 1997: Hart MK, Pratt W, Panelo F, Tammariello R, Dertzbaugh M. Venezuelan equine encephalitis virus vaccines induce mucosal IgA responses and protection from airborne infection in BALB/c, but not C3H/HeN mice. Vaccine. 1997 Mar; 15(4); 363-9. [PubMed: 9141206 ].
Pratt et al., 2003: Pratt WD, Davis NL, Johnston RE, Smith JF. Genetically engineered, live attenuated vaccines for Venezuelan equine encephalitis: testing in animal models. Vaccine. 2003 Sep 8; 21(25-26); 3854-62. [PubMed: 12922119].