Development and evaluation of a replicon particle vaccine expressing the E2 glycoprotein of bovine viral diarrhea virus (BVDV) in cattle
© Loy et al.; licensee BioMed Central Ltd. 2013
Received: 23 July 2012
Accepted: 22 January 2013
Published: 28 January 2013
Bovine viral diarrhea virus is one of the most significant and costly viral pathogens of cattle worldwide. Alphavirus-derived replicon particles have been shown to be safe and highly effective vaccine vectors against a variety of human and veterinary pathogens. Replicon particles are non-propagating, DIVA compatible, and can induce both humoral and cell mediated immune responses. This is the first experiment to demonstrate that Alphavirus-based replicon particles can be utilized in a standard prime/boost vaccination strategy in calves against a commercially significant bovine pathogen.
Replicon particles that express bovine viral diarrhea virus sub-genotype 1b E2 glycoprotein were generated and expression was confirmed in vitro using polyclonal and monoclonal antibodies specific to E2. Vaccine made from particles was generated in Vero cells and administered to BVDV free calves in a prime/boost regimen at two dosage levels. Vaccination resulted in neutralizing antibody titers that cross-neutralized both type 1 and type 2 BVD genotypes following booster vaccination. Additionally, high dose vaccine administration demonstrated some protection from clinical disease and significantly reduced the degree of leukopenia caused by viral infection.
Replicon particle vaccines administered in a prime/boost regimen expressing BVDV E2 glycoprotein can induce cross-neutralizing titers, reduce leukopenia post challenge, and mitigate clinical disease in calves. This strategy holds promise for a safe and effective vaccine to BVDV.
KeywordsBVD Bovine viral diarrhea virus Alphavirus replicon Replicon particle Vaccine
Bovine viral diarrhea virus (BVDV) is an enveloped, positive strand RNA virus in the genus Pestivirus (family Flaviviridae), and is the causative agent of bovine viral diarrhea (BVD). BVD is one of the most economically significant bovine diseases in the world. Production losses, estimated on a population level, are thought to be $10–40 million US per million calvings . Classically, BVDV has been associated with acute enteric disease; however, BVDV is now understood to be responsible for a broad range of clinical illnesses in cattle including respiratory disease, reproductive loss, and fetal infections . The BVDV E2 gene encodes a 53 kDa major structural glycoprotein that contains a neutralizing epitope that varies among strains . Monoclonal antibodies specific to E2 demonstrate virus neutralizing (VN) ability against both cytopathic and noncytopathic strains of BVDV . Current strategies to reduce losses caused by BVD in infected herds include vaccination with modified live (MLV) or inactivated vaccines and elimination of persistently infected animals. There are no commercially available recombinant or vectored vaccines, and thus producers are limited to either modified live MLV or inactivated vaccine approaches . Additionally, some success has been shown experimentally using BVD pseudovirions, which have a deletion in the structural genes, and are rescued using homologous helper RNA in trans.
A replicon-based expression system has been derived from the alphavirus (family Togaviridae) Venezuelan Equine Encephalitis (VEE) virus [7, 8]. Alphavirus-based replicon particles have numerous advantages for vaccine development including; accurate production of native proteins and a propagation-defective nature that makes the system only capable of a single infection cycle . This viral vector has previously been used to express genes from numerous pathogens including human strains of influenza virus, simian immunodeficiency virus, Norwalk virus, Ebola virus, smallpox virus, Lassa virus, and equine arteritis virus [10–16]. RP have a robust safety profile. It has been demonstrated that RP are not shed from vaccinated animals and as such cannot spread to unvaccinated animals. Furthermore, RP have been tested for the presence of replication competent virus that may arise by recombination during production of RP and have been shown to lack the ability to revert to virulence . Alphavirus RP have also been tested in cattle against foot and mouth disease virus (FMDV) (Kurt Kamrud, personal communication) but have not been tested against a commercially-significant cattle disease in the United States using a prime/boost vaccination strategy. Therefore, it is proposed that alphavirus RP that express the E2 glycoprotein from BVDV would provide a novel, safe, and effective approach to control BVD.
Seven (7) calves, 8 weeks of age, were sourced from a BVD free herd. Each animal was tested and found negative for BVDV antibodies by viral neutralization assay (VN), BVDV antigen in earnotch samples (BVD immunohistochemistry), and were negative for circulating virus in whole blood by PCR. Three (3) calves were randomly assigned to each of 2 blocks (experimental groups) and one (1) calf was assigned to a single block (placebo). The calves were acclimated for 7 days before onset of the trial. Calves were injected intramuscularly (IM) with 2 mL of 5 × 106 infectious units (IU)/mL RP (1 × 107 IU total), 5 × 105 IU/mL RP (1 × 106 IU total), or a placebo control of RP diluent (1% normal bovine serum, 5% sucrose in PBS). Personnel sampling and scoring the animals were blinded to the treatment groups.
Serum was collected from calves on days 0, 21, 28, 35, 42, 49, and on 56. Whole blood was collected in tubes containing EDTA 1 day prior to challenge through 14 days post challenge (a total of 16 days). On day 42 animals were challenged with 1×106 TCID50/mL of BVD1b strain NY1 intranasally (USDA distributed efficacy challenge material). Neutralizing antibody responses were assessed using representative viruses from Type 1 and Type II BVDV strains (Singer and 296c, respectively) using day 0, 21, 28, and 42 serum samples. Clinical disease was scored on a scale from 0–3 each day. White blood cell (WBC) counts were assessed on whole blood samples collected in tubes containing EDTA using an automated cell counter (Iowa State University, Veterinary Pathology). The degree of leukopenia following virus challenge was assessed by subtracting the maximum reduction in circulating WBCs from the baseline level (calculated from a mean of two samples prior to challenge).
In summary, these data demonstrate that RP expressing BVD1b NY1 E2 glycoprotein can induce dose dependent immune responses specific to BVDV. Importantly, the E2 RP induce a cross-reactive VN antibody response to Type 1a and Type 2 BVD strains. This response was significantly higher in animals that received the 1 × 107 IU E2 RP dosage level. Furthermore, the vaccine impacted clinical parameters post-challenge with a homologous BVDV strain and reduced the degree of leukopenia post-challenge when compared to the control. The current study does not address if the heterologous neutralization titers indicate efficacy against heterologous challenge, however, this will be addressed in future experiments. We believe that BVDV E2 RP represent an attractive alternative to MLV or inactivated vaccine approaches because of the efficacy demonstrated here, the safety profile of the vector, and because E2 RP may allow differentiation of infected and vaccinated animals capable of supporting BVDV eradication programs.
The use of biohazardous materials and the calf experimental protocol were reviewed and approved by the ISU Institutional Biosafety Committee (IBC) and the Institutional Animal Care and Use Committee (9-11-7225-B).
The authors would like to thank Kara Burrack, Kay Kimpston-Burkgren, Lyle Kesl, and Kara Jimenez for their contributions.
- Houe H: Economic impact of BVDV infection in dairies. Biologicals 2003,31(2):137-143. 10.1016/S1045-1056(03)00030-7PubMedView ArticleGoogle Scholar
- Evermann JF, Barrington JM: Clinical features. In Bovine Viral Diarrhea Virus: Diagnosis, Management,and Control. 1 edition. Edited by: Goyal SM, Ridpath JF. Ames: Wiley-Blackwell; 2005:105-120.View ArticleGoogle Scholar
- Hansen TR, Smirnova NP, Van Campen HF, Shoemaker ML, Ptitsyn AA, Bielefeldt-Ohmann H: Maternal and fetal response to fetal persistent infection with bovine viral diarrhea virus. Am J Reprod Immunol 2011,64(4):293-306.Google Scholar
- Donis RO, Corapi WF, Dubovi EJ: Neutralizing monoclonal antibodies to bovine viral diarrhoea virus bind to the 56K to 58K glycoprotein. J Gen Virol 1988, 69: 77-86. 10.1099/0022-1317-69-1-77PubMedView ArticleGoogle Scholar
- Rodning SP, Marley MS, Zhang YF, Eason AB, Nunley CL, Walz PH, Riddell KP, Galik PK, Brodersen BW, Givens MD: Comparison of three commercial vaccines for preventing persistent infection with bovine viral diarrhea virus. Theriogenology 2010,73(8):1154-1163. 10.1016/j.theriogenology.2010.01.017PubMedView ArticleGoogle Scholar
- Zemke J, König P, Mischkale K, Reimann I, Beer M: Novel BVDV-2 mutants as new candidates for modified-live vaccines. Vet Microbiol 2010, 142: 69-80. 10.1016/j.vetmic.2009.09.045PubMedView ArticleGoogle Scholar
- Pushko P, Parker M, Ludwig GV, Davis NL, Johnston RE, Smith JF: Replicon-helper systems from attenuated Venezuelan equine encephalitis virus: expression of heterologous genes in vitro and immunization against heterologous pathogens in vivo. Virology 1997, 239: 389-401. 10.1006/viro.1997.8878PubMedView ArticleGoogle Scholar
- Kamrud KI, Custer M, Dudek JM, Owens G, Alterson KD, Lee JS, Groebner JL, Smith JF: Alphavirus replicon approach to promoterless analysis of IRES elements. Virology 2007, 360: 376-387. 10.1016/j.virol.2006.10.049PubMedPubMed CentralView ArticleGoogle Scholar
- Vander Veen RL, Harris DL, Kamrud KI: Alphavirus replicon vaccines. Anim Health Res Rev 2012, 13: 1-9. 10.1017/S1466252312000011PubMedView ArticleGoogle Scholar
- Balasuriya UBR, Heidner HW, Davis NL, Wagner HM, Hullinger PJ, Hedges JF, Williams JC, Johnston RE, Wilson WD, Liu IK, et al.: Alphavirus replicon particles expressing the two major envelope proteins of equine arteritis virus induce high level protection against challenge with virulent virus in vaccinated horses. Vaccine 2002, 20: 1609-1617. 10.1016/S0264-410X(01)00485-6PubMedView ArticleGoogle Scholar
- Balasuriya UBR, Heidner HW, Hedges JF, Williams JQ, Davis NL, Johnston RE, Maclachlan NJ: Expression of the Two major envelope proteins of equine arteritis virus as a heterodimer is necessary for induction of neutralizing antibodies in mice immunized with recombinant Venezuelan equine encephalitis virus replicon particles. J Virol 2000, 74: 10623-10630. 10.1128/JVI.74.22.10623-10630.2000PubMedPubMed CentralView ArticleGoogle Scholar
- Davis NL, Caley IJ, Brown KW, Betts MR, Irlbeck DM, McGrath KM, Connell MJ, Montefiori DC, Frelinger JA, Swanstrom R, et al.: Vaccination of macaques against pathogenic Simian immunodeficiency virus with Venezuelan equine encephalitis virus replicon particles. J Virol 2000, 74: 371-378. 10.1128/JVI.74.1.371-378.2000PubMedPubMed CentralView ArticleGoogle Scholar
- Harrington PR, Yount B, Johnston RE, Davis N, Moe C, Baric RS: Systemic, mucosal, and heterotypic immune induction in mice inoculated with Venezuelan equine encephalitis replicons expressing Norwalk virus-like particles. J Virol 2002, 76: 730-742. 10.1128/JVI.76.2.730-742.2002PubMedPubMed CentralView ArticleGoogle Scholar
- Pushko P, Geisbert J, Parker M, Jahrling P, Smith J: Individual and bivalent vaccines based on alphavirus replicons protect guinea pigs against infection with Lassa and Ebola viruses. J Virol 2001, 75: 11677-11685. 10.1128/JVI.75.23.11677-11685.2001PubMedPubMed CentralView ArticleGoogle Scholar
- Schultz-Cherry S, Dybing JK, Davis NL, Williamson C, Suarez DL, Johnston R, Perdue ML: Influenza virus (a/HK/156/97) hemagglutinin expressed by an alphavirus replicon system protects chickens against lethal infection with Hong Kong-origin H5N1 viruses. Virology 2000, 278: 55-59. 10.1006/viro.2000.0635PubMedView ArticleGoogle Scholar
- Hooper JW, Ferro AM, Golden JW, Sivera P, Dudek JM, Alterson KD, Custer M, Rivers B, Morris J, Owens G, et al.: Molecular smallpox vaccine delivered by alphavirus replicons elicits protective immunity in mice and Non-human primates. Vaccine 2009,28(2):494-511. 10.1016/j.vaccine.2009.09.133PubMedPubMed CentralView ArticleGoogle Scholar
- Vander Veen RL, Loynachan AT, Mogler MA, Russell BJ, Harris DL, Kamrud KI: Safety, immunogenicity, and efficacy of an alphavirus replicon-based swine influenza virus hemagglutinin vaccine. Vaccine 2012, 30: 1944-1950. 10.1016/j.vaccine.2012.01.030PubMedView ArticleGoogle Scholar
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