Bovine Viral Diarrhea Virus
Vaccination

Evolution of BVDV Vaccines
Modified Live and Killed Vaccines
Subunit Vaccines
DNA Vaccines


The abilities and efficacy of a vaccine

Vaccines are an important tool used to help control and prevent viral and bacterial infections. By inducing a protective immune response within the host before infection, vaccines prime the immune system so it is better equipped to contain and eliminate the insulting agent. There are many types of vaccines however those that are commercially available at this time seem to be restricted to modified live and inactivated vaccines. The efficacy of these commercially available vaccines is in question and thus has lead to the development of new vaccine designs such as subunit and DNA vaccines.

To explore the efficacy of currently available vaccines, we first need to define the abilities, the constraints as well as the risks of vaccination. Kvasnicka et al. define three criteria for when vaccination is appropriate. Firstly, vaccination should only be considered when the immune system is capable of protecting against the disease in question. Since vaccination protection is designed around inducing a protective immune response, if that immune response in itself is not effective at protecting the host then there is no point in vaccinating. For example, inactivated vaccines contain killed virus and therefore do not follow the natural viral infection process as it cannot enter the cell. The immune response that inactivated vaccines induce may therefore not be appropriate and thus ineffective in disease protection. Secondly, the risks accompanying vaccination should not exceed the risks associated with contracting the disease. Modified live and attenuated vaccines contain live virus that has been altered to decrease its virulence and ability to cause disease. It is possible for live viruses to retain their residual virulence generating the risk of inducing disease in vaccinated animals. Lastly, Kvasnicka et al explain that vaccination is based on the control of disease in a population of animals rather than individual animals. Due to individual animal variability and their abilities to mount immune responses, not all of the population vaccinated will be fully protected. The concept of herd immunity reduces the probability of exposure, in turn reducing the chance that those in the population who cannot mount an acceptable protective immune response will contract the disease. Vaccination must therefore be a cost-effective method for controlling disease within the entire herd.


BVDV Diagnosis

Vaccination is a tool that is used for prevention of disease. The efficacy of that vaccination may be measured as it's ability to prevent disease. One must therefore have the knowledge and tools to diagnose the disease that one is trying to prevent. Below is a summary table of the recommended diagnostic tests for BVDV and their uses as described by the World Organization for Animal Health. More information may be found at our useful links page.

1.

Overview of diagnosis for types of BVD infection

a)

Diagnosis of persistent viremia in healthy animals

 

Biotyping

Noncytopathogenic virus

 

Viremia

Different tests:

1.  Culture susceptible cells with potentially infected blood or serum. Followed by identification of infected cells with immunocytochemistry for virus

2. Immune-labelling for viral antigen or RT-PCR in leukocytes.

 

Persistency

Confirmed by re-testing for virus / viral antigen/ viral RNA after a minimum 3 week interval.

 

Serology

·    Seronegative with concurrent viremia is a definitive test for persistent infection.  False positives in calves up to 3 months of age may be due to presence of maternal antibodies.

·    Older animals with persistent viremia may seroconvert (low BVD-specific antibody titers) due to development of antigenically unique subpopulation or  vaccination with heterologous modified live virus vaccine.  

 

Samples

1. Semen – Transient virus.  Test all bulls used for natural service or artificial insemination. May be indicated by reduced fertility.

2. Blood

3. Blood-associated organs (liver, lungs, spleen)

4. Fetal fluids, placenta

5. Skin

b)   

Diagnosis of acute infections


Biotyping

Cytopathogenic virus

 

Virus isolation, identification

Difficult because of transient virus.

Immunohistochemistry for increases sensitivity in affected tissues.

 

Serology

Seropositivity is a definitive test for acute infection.

 

Post-mortem

Erosion of intestinal mucosa. Lysis of Peyer’s patches, colonic tonsils;replacement with cellular debris and inflammation.

 

Samples

1. Blood, nasal swab – transient virus only

2. Intestinal tissue, ileal and jejunal Peyer’s patches, colonic tonsils

3. Spleen, tonsils, lymph nodes

3. Fetal tissue, fetal fluids, fetal serum

2.

Virus isolation, identification

 

·       All tests  require controls consisting of known non-infected and infected populations of cattle.

·       There are two designated OIE Reference Laboratories for BVD.  

 

Immuno-histochemistry

·    Cryostat sections of intestinal mucosa for suspected mucosal disease; skin for persistent infection.

     

Virus isolation   

·    Prescribed test for international trade.

·    Samples are applied to susceptible bovine cell cultures (kidney, lung, testis, turbinate cultures).

·    Live sample: buffy coat, whole blood, washed leukocytes, serum, (semen).

·    Post-mortem samples – tissue.

·    Infection of cultures verified by:

·    Monoclonal antibody-labelling of cultures.

·    Microscopic verification of cytotoxicity.

·    Variations of above protocol are as follows:

·    Microplate immunoperoxidase ,ethod for mass screening of virus in serum

·    2. Tube method for tissue, buffy coat suspensions or semen samples

     

Antigen-capture ELISA

·    The test is suitable for detection of persistently infected animals. Traditional tests  measure BVD antigen in lysates of peripheral blood leukocytes. Newer tests detect BVD antigen in blood, plasma and serum.

·    Depending on the commercial test, sensitivity can be as high as virus isolation.

·    Not recommended in suckling calves.

·    Less sensitive for detection of acute infection.

     

RT-PCR

·    Highest sensitivity if BVD-specific antibodies are present. Can be used to detect for viral genome somatic cells from a bulk milk tank sample. A positive result indicates at least one animal is infected with BVD.

3. 

Serological tests

     

Virus neutralization assay

·  Requires access to cytopathogenic laboratory-adapted strains of BVDV.

·  Most commonly used strains are 'Oregon C24V' and 'NADL'.

·  Use of only strain 1 or only strain 2 virus results in decreased sensitivity and specificity.

     

ELISA     

·  A number of commercial kits are available.

·  Serum from an individual animal should be tested against a wide range of different virus strains.

·  Future tests are being designed to differentiate between natural infection and strains of marker viruses used for vaccination.

Reference: http://www.oie.int/eng/normes/mmanual/A_00132.htm

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The evolution of BVDV vaccines

Bovine viral diarrhea virus (BVDV) is of the genus Pestivirus within the Flaviviridae family (Merk Veterinary Manual). BVDV infects cattle of all ages however clinical disease is seen most commonly in young cattle between 6 and 24 months of age. Intestinal mucosal damage from BVDV infection leads to diarrhea that is often fatal to young cattle. Transplacental infection occuring within the first 4 months of gestation results in persistently infected (PI) calves. These calves constantly shed large quantities of virus in their excretions and secretions (Merck Veterinary Manual) and are thus a very important source of infection for the rest of the herd (van Oirschot et al 1999). In utero infection occurring later in gestation however, results in abortion, congenital malformations or normal calves containing antibodies against BVDV (Merck Veterinary Manual). Many producers suffer large economic loss due to transplacental infections and therefore vaccination is commonly used as a tool to minimize these losses.

The limited abilities of conventional modified live and inactivated vaccines to meet the three criteria of Kvasnicka et al have pushed vaccine design towards new methods that are more effective, cheaper and safer. Newly developed vaccines often contain only parts of the virus in an attempt to minimize the risk of disease that can result from vaccination. Subunit vaccines contain viral protein antigens while DNA vaccines use pieces of viral genomes that can be expressed in host cells and cause protective immune responses without the necessity of using whole virus.


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Modified Live and Killed Vaccines

There is a large line of modified live and killed vaccines that are available to producers for their vaccination programs (IVIS Veterinary Drug Database 2004). Most vaccines contain a combination of viruses within one vaccine to help make vaccination more efficient and less labour intensive. Modified live and killed vaccines are aimed at preventing BVDV-induced disease however they are unsuccessful at preventing transplacental fetal infection (Carter et al 2005) and contain shortcomings within their designs.

Modified live vaccines contain BVDV particles that have been passaged multiple times through cell cultures of bovine and non bovine origin (van Oirschot et al 1999). Viral passaging is performed in cells and conditions that the virus is not normally adapted too (Kvasnicka et al) in an attempt to convert the viral population through selection pressure. As a result, we acheive the selection of BVDV particles that are not adapted to efficiently grow within the host that is to be vaccinated. Viral particles of modified live vaccines thereforeretain their ability infect host cells however viral replication is suboptimal. This induces an immune response that closely mimics natural infection. The primary disadvantage of modified live vaccines is that they have been known to cause mucosal disease (van Oirschot et al 1999) most likely due to retained residual virulence with the virus after passaging (Kvasnicka et al). Modified live vaccines can also cause immunosuppression (Kelling 2004 and van Oirschot et al 1999) and carry the risk of vaccine contamination with adventitious viruses (Kelling 2004 and van Oirschot et al 1999). Vaccination of pregnant cows with modified live vaccines can cause in utero infection as it is capable of crossing the placental barrier irrespective of time of gestation (van Oirschot et al 1999). Vaccination programs with modified live vaccines are therefore risky and tedious.

Killed vaccines contain viral particles that have been chemically treated to inactivate the virus but not disturb its surface antigenicity (van Oirschot et al 1999). Killed vaccines act outside the host cell and do not mimic the events of natural infection (Kvasnicka et al). Killed vaccines are therefore considered safer. However, because killed vaccines do not enter the cell it is believed that they do not stimulate an immune response that is as protective as one stimulated by a modified live vaccine (Kvasnicka et al). For this reason, killed vaccines are often accompanied by adjuvants in an attempt to induce a more adequate and longer immune response (Kelling 2004). Killed vaccine adjuvants have however been reported to cause local injection site reactions and are thus undersirable (van Oirschot et al 1999).

In addition to the above disadvantages, both modified live and killed vaccines have been shown to provide only incomplete and partial protection against fetal infection respectfully (Kelling 2004). Frey et al (2002) suggest a two-step vaccination protocol that may overcome this disadvantage by using both a modified live and a killed vaccine. Nine seronegative BVDV heifers were vaccinated with a killed vaccine and then given a booster four weeks later with a modified live vaccine. The nine vaccinated and six control heifers were then challenged with BVDV between 30 and 120 days of gestation. All calves from vaccinated heifers were born clinically healthy, seronegative and BVDV-free while calves of the control group were either viremic or stillborn. Frey et al (2002) demonstrated that the combination of a killed and modified live vaccination program has the potential to prevent in utero infection. However, implementing this vaccination program does not overcome the shortcomings of the vaccines themselves such as local injection site reaction, potential vaccine contamination and residual virulence. New strategies need to be explored to produce an effective, safe and economically viable vaccine.

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Subunit Vaccines

Host neutralizing antibodies of BVDV are directed against three viral envelope proteins Ems, E1 and E2 as well as the non structural proteins NS2/3 (van Oirschot et al 1999). The E2 structural protein is a potent immunogen and therefore is a good candidate for a subunit vaccine (van Oirschot et al 1999).


                                                                                                                                                    BVDV Genome
                                                                                                            http://www.fli.bund.de/569+M52087573ab0.html


Subunit vaccines contain antigenic viral proteins that are key to viral stimulation of protective immunity in the host. Subunit vaccines that contain the E2 BVDV protein have been reported to induce immune protection by stimulating the production of neutralizing antibodies (Harpin et al 1999). Bruschke et al (1997) reported that their E2 subunit vaccine prevented fetal infection in sheep when challenged with homologous BVDV strains after 2 vaccinations. Seven weeks after vaccination, served ewes were challenged with homologous BVDV strains. Three weeks post-challenge, the fetuses were removed and processed for viral detection. Fetal protection was observed in vaccinated animals however, Bruschke et al (1997) also reported a possible correlation between the amount of E2 glycoprotein found in the vaccine and the amount of fetal protection the vaccine achieved. Bruschke et al (1997) also noted a difference in immunogenicity of the E2 glycoprotein across BVDV strains. This suggests that the efficiency of E2 subunit vaccines for BVDV is dependent on both the dose of protein in the vaccine and the presence of multiple antigenically variant E2 glycoproteins.

Subunit vaccines are difficult and expensive to produce and therefore do not seem to be practical and cost effective for veterinary application (Wang et al 2004). However, Wang et al were successful in modifying the E2 subunit vaccine so that low dose antigen subunits could produce high levels of immune responses. Wang et al (2004) tagged the E2 gene with three copies of the cleavage fragment of the C3 complement protein (C3d). In theory, the tagging of C3d would enhance follicular trapping of BVDV antigens through C3d-CD19 interaction and thus enhance antigen presentation to activated antigen-specific B cells (Wang et al 2004). In result, B cells would be rescued from apoptosis thus promoting the development of the B cell memory population. Wang et al (2004) vaccinated mice with this E2-C3d tagged vaccine and monitored their immune response via ELISA. The results indicated that E2-C3d tagged vaccines were 10,000 time more immunogenic than immunization with E2 glycoproteins. Low antigen concentrations also had the ability to elicit high titers of neutralizing antibodies successfully enhancing the immune response. Although this study was performed in mice, Wang et al have demonstrated the potential to which this subunit vaccine could entail in the future.

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DNA Vaccines

DNA vaccines are amongst the newest design for vaccine development. Scientists have used the E2 glycoprotein gene of BVDV and inserted it into plasmid DNA. The plasmid is then injected directly into the host and BVDV antigens are synthesized endogenously mimicking a pattern of natural intracellular infection (Nobiron et al 2003). There exist many advantages DNA vaccines possess over conventional vaccines. Among the most important are the rapidity, simplicity and relative inexpensiveness required to produce plasmid DNA (Harpin et al 1999). Thermal stability of plasmid DNA also provides a long shelf life for DNA vaccines (Harpin et al 1999) as well these vaccines do not require virus propagation in complex and expensive cell culture systems as modified live vaccines do (Nobiron et al 2003). DNA vaccines are therefore relatively easy, safe and cost-effective to produce. The question remains however, as to their efficacy in inducing protective immune responses and preventing transplacental infection.

Once it was established that DNA vaccines could produce an immune response, scientists began to explore ways in which the immune response could be augmented and tailored to the diseases and viruses in question. Harpin et al (1999) explored the possibility of augmenting the immunogenicity of vaccines by using DNA entrapped in cationic liposomes. Harpin et al (1999) immunized cattle with plasmids containing the E2 BVDV glycoprotein in either naked DNA form or DNA entrapped in cationic liposomes. The naked DNA vaccinates produced varying levels of protection upon viral challenge while the cationic liposome entrapped DNA vaccinates did not produce any protective signs. Harpin et al (1999) was able to conclude through further investigation in their study that DNA vaccines encoding the E2 glycoprotein induced both neutralizing antibody production and lymphocyte proliferation. This indicates that the protective responses to DNA vaccines involve both humoral and cellular mediated immunity. This in turn reinforces the promising potential of DNA vaccines.

In an attempt to further optimize DNA vaccines, Nobiron et al (2001 and 2003) introduced cytokine adjuvancy to E2 glycoprotein DNA vaccines. Eventhough DNA vaccines induce both humoral and cell mediated immunities, these responses are weak and thus offering limited protection against disease and in utero infection (Nobiron et al 2003). In an attempt to increase the immunogenicity of the E2 plasmid vaccine, Nobiron et al co-administered plasmids encoding for interleukin 2 (IL-2) or granulocyte macrophage colony stimulating factor (GM-CSF) in an attempt to increase the production of neutralizing antibodies and enhance lymphocyte proliferation. Nobiron et al were successful at demonstrating that co-administration with IL-2 or GM-CSF significantly enhanced the priming of the T-cell response however, post-challenge responses of vaccinated cattle with DNA adjuvants did not show any benefit compared to E2 DNA vaccinated animals in the respect of increased neutralizing antibody titres or the number of individuals that responded compared to the E2 vaccinated group. In other words, IL-2 and GM-CSF did not augment the hosts control over viral replication upon challenge with BVDV. Nobiron et al however do suggest that the E2 glycoprotein is not a major T-cell target and control of viremia may require priming of responses to additional BVDV antigens. Further studies need to be performed to explore this concept.


  Diagrammatic representation of E2 BVD DNA vaccine                                 
             http://www.genomenewsnetwork.org/articles/07_00/fusinggenes_vaccines.shtml                                                         

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Conclusion

Conventional vaccines are not the most ideal design for safe, cost-effective and efficient protection against BVDV, however their effectiveness can be manipulated through vaccination programs such as the two step program described above. Subunit and DNA vaccines offer their own new light on the protection against BVDV infection and disease. These relatively new vaccine designs offer great potential on many levels for inducing protective immunity in vaccinated animals.

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