• 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_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.

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 × 10^4 to 2 × 10^6 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 10^5 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 10^6 PFU (roughly 10^6 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 10^5 PFU per animal in 20 µl of PBS (five mice per group), and (iii) intranasal (i.n.) inoculation with 2 x 10^5 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 10^5 PFU in 20 µl of PBS and (ii) i.n. inoculation with 2 x 10^5 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 10^5 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: 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: 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