S. van Drunen Littel-van den Hurk

Induction of immune responses by DNA vaccines in large animals

It is generally recognized that DNA vaccines are often less effective in large animals than in mice. One possible reason for this reduced effectiveness may be transfection deficiency and the low level of expression elicited by plasmid vectors in large animals. In our attempt to enhance transfection efficiency and, thereby, enhance immune responses, we employed a variety of methods inducing gene gun delivery or suppositories as delivery vehicles to mucosal surfaces, as well as electroporation for systemic immunization. To test these different systems, we used two different antigens-a membrane antigen from bovine herpesvirus glycoprotein (BHV-1) gD and a particulate antigen from hepatitis virus B. Gene gun and suppository delivery of BHV-1 gD to the vagina resulted in the induction of mucosal immunity not only in the vagina, but also at other mucosal surfaces. These data support the contention of a common mucosal immune system. In the case of electroporation, we were able to develop significant enhancement of gene expression following electroporation with surface electrodes (non-invasive electroporation) as well as invasive electroporation using single or six-needle electrodes. Various delivery systems such as bioject or needle delivery also influenced the immune response in both the presence and absence of electroporation. These studies also demonstrated that co-administration of plasmids coding for two different antigens (BHV-1 gD and hepatitis B surface antigen (HbsAg)) did not result in significant interference between the plasmids. These studies suggest that various combinations of delivery systems can enhance immunity to DNA-based vaccines and make them practical for administration of these vaccines in large animals.

Protection of cattle from bovine herpesvirus type I (BHV-1) infection by immunization with individual viral glycoproteins.

The major glycoproteins gI, gIII, and gIV of bovine herpesvirus-1 (BHV-1) were found to induce high levels of antibody in cattle which could neutralize virus and participate in antibody-dependent cell cytotoxicity of BHV-1-infected cells. Immunized animals were fully protected from disease, using a BHV-1/Pasteurella haemolytica aerosol challenge model but not from infection with the virus. Thus, virus could still replicate in the nasal passages of immunized animals, although to a lesser extent than in placebo-treated animals or animals immunized with a commercial killed whole virus vaccine. Systemic spread of the virus in immunized animals did not appear to occur since there was not a dramatic alteration of leukocyte function following challenge. These results suggest that any one of the three major BHV-1 glycoproteins may be useful as a subunit vaccine either individually or in combination.

Intradermal immunization with a bovine herpesvirus-1 DNA vaccine induces protective immunity in cattle.

Although intramuscular (i.m.) injection of DNA encoding glycoprotein D (gD) of bovine herpesvirus-1 (BHV-1) induces immune responses in cattle, this route of delivery is inefficient. Here we assessed three parameters that may enhance the efficacy of a gD DNA vaccine in cattle. First, the immune response generated by i.m. injected plasmid expressing a secreted form of gD (tgD) was determined and found to be very similar in magnitude to the response induced by gD-expressing plasmid. Secondly, gD- and tgD-expressing plasmids were administered by intradermal (i.d.) immunization, which resulted in a superior immune response to the secreted form, but no improvement in the response to the membrane-associated form. However, the form of gD used for immunization did not influence the immunoglobulin subtype, the ratio of antigen-specific IgG1 to IgG2 being approximately 4:1. Finally, the effect of promoter strength was assessed by replacing the Rous sarcoma virus (RSV) promoter, which was used in the original experiments, with the human cytomegalovirus immediate early promoter and first intron A (HCMV/IA). Although upon transfection in vitro the HCMV/IA promoter appeared to be stronger than the RSV promoter, there was only a 2-fold higher antibody response in vivo upon i.d. injection of cattle. Protection against virus challenge was obtained in the calves immunized i.d. with tgD-encoding plasmid, as shown by a significant reduction in weight loss, virus excretion, temperature response and clinical disease. No significant protection was observed in the animals vaccinated i.d. with the gD-expressing plasmid, which correlates with the lower level of immunity pre-challenge.

CpG-containing oligodeoxynucleotides, in combination with conventional adjuvants, enhance the magnitude and change the bias of the immune responses to a herpesvirus glycoprotein.

Vaccine adjuvants must have the capacity to increase protective immune responses with minimal side effects. Conventional adjuvants not only cause undesirable tissue site reactions, but often induce T-helper type 2 (Th2)-biased responses which may be undesirable in certain disease scenarios. Oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG ODN) are novel adjuvants known to promote Th1-type immune responses. In this study, we compared various mineral oil, metabolizable oil and non-oil adjuvants alone and in combination with CpG ODN for their ability to augment immune responses to a truncated secreted form of bovine herpesvirus (BHV) glycoprotein D (tgD). All adjuvants tested induced Th2-biased immune responses characterized by a predominance of serum IgG1 as well as interleukin-4 (IL-4) production by in vitro stimulated splenocytes. The inclusion of CpG ODN in these formulations not only increased immune responses, but more importantly enhanced serum IgG2a levels and production of interferon-gamma (IFN-gamma) by splenocytes, indicating a more balanced or Th1-type response. The use of a mineral oil-based adjuvant at reduced doses in combination with CpG ODN attenuated the tissue damage while not compromising the magnitude of the immune response in both mice and sheep. In addition, reduced amounts of mineral oil combined with CpG ODN induced a more balanced Th1/Th2 immune response than the mineral oil used alone. Our results clearly demonstrate that CpG ODN can be used to enhance magnitude and balance of an immune response while reducing the amount of mineral oil and hence undesirable side effects of vaccine adjuvants.

The immunogenicity and protective efficacy of bovine herpesvirus 1 glycoprotein D plus Emulsigen are increased by formulation with CpG oligodeoxynucleotides

ABSTRACT The immunogenicity and protective efficacy of a bovine herpesvirus 1 (BHV-1) subunit vaccine formulated with Emulsigen (Em) and a synthetic oligodeoxynucleotide containing unmethylated CpG dinucleotides (CpG ODN) was determined in cattle. A truncated, secreted version of BHV-1 glycoprotein D (tgD) formulated with Em and CpG ODN at concentrations of 25, 2.5, or 0.25 mg/dose produced a more balanced immune response, higher levels of virus neutralizing antibodies, and greater protection after BHV-1 challenge compared to tgD adjuvanted with either Em or CpG ODN alone. In contrast, tgD formulated with Em and either 25 mg of a non-CpG ODN or another immunostimulatory compound, dimethyl dioctadecyl ammonium bromide, induced similar immunity and protection compared to tgD formulated with Em alone, a finding which confirms the immunostimulatory effect of ODN to be CpG motif mediated. Our results demonstrate the ability of CpG ODN to induce a strong and balanced immune response in a target species.

Protection of cattle from BHV-1 infection by immunization with recombinant glycoprotein gIV

High levels of recombinant bovine herpesvirus-1 (BHV-1) glycoprotein IV were produced in baculovirus, adenovirus, vaccinia virus and Escherichia coli expression systems. The different recombinant forms as well as authentic gIV were injected intramuscularly into seronegative calves. With the exception of E. coli-produced gIV, all forms of gIV induced high levels of neutralizing antibodies both in the serum and in the nasal superficial mucosa. Animals immunized with gIV produced in insect or mammalian cells were completely protected from infection with BHV-1, as demonstrated by the absence of temperature responses, clinical signs or detectable virus in the nasal secretions after challenge exposure. The E. coli-derived gIV induced partial protection from clinical disease, even though it was not glycosylated and did not induce appreciable levels of neutralizing antibodies. This study demonstrated that all forms of glycosylated gIV, whether authentic or recombinant, confer protection from BHV-1 infection and thus may be useful as an effective subunit vaccine.

Gene Gun-Mediated DNA Immunization Primes Development of Mucosal Immunity against Bovine Herpesvirus 1 in Cattle

ABSTRACT Vaccination by a mucosal route is an excellent approach to the control of mucosally acquired infections. Several reports on rodents suggest that DNA vaccines can be used to achieve mucosal immunity when applied to mucosal tissues. However, with the exception of one study with pigs and another with horses, there is no information on mucosal DNA immunization of the natural host. In this study, the potential of inducing mucosal immunity in cattle by immunization with a DNA vaccine was demonstrated. Cattle were immunized with a plasmid encoding bovine herpesvirus 1 (BHV-1) glycoprotein B, which was delivered with a gene gun either intradermally or intravulvomucosally. Intravulvomucosal DNA immunization induced strong cellular immune responses and primed humoral immune responses. This was evident after BHV-1 challenge when high levels of both immunoglobulin G (IgG) and IgA were detected. Intradermal delivery resulted in lower levels of immunity than mucosal immunization. To determine whether the differences between the immune responses induced by intravulvomucosal and intradermal immunizations might be due to the efficacy of antigen presentation, the distributions of antigen and Langerhans cells in the skin and mucosa were compared. After intravulvomucosal delivery, antigen was expressed early and throughout the mucosa, but after intradermal administration, antigen expression occurred later and superficially in the skin. Furthermore, Langerhans cells were widely distributed in the mucosal epithelium but found primarily in the basal layers of the epidermis of the skin. Collectively, these observations may account for the stronger immune response induced by mucosal administration.

Epitope specificity of the protective immune response induced by individual bovine herpesvirus-1 glycoproteins.

Affinity-purified bovine herpesvirus-1 (BHV-1) glycoproteins gI, gIII and gIV, as well as a virus-free BHV-1-infected cell lysate were injected intramuscularly into seronegative calves. All immunized animals developed specific serum-neutralizing antibodies and they were fully protected from disease, using a BHV-1/Pasteurella haemolytica challenge model. After challenge, viral replication in the nasal passages was significantly reduced in animals vaccinated with gIV (10,000-fold) or BHV-1-infected cell lysate (450,000-fold) but just slightly reduced in animals immunized with gI (500-fold) or gIII (25-fold). All of the known epitopes of the glycoproteins were retained during the affinity-purification or preparation of the cell lysate. The high level of protection induced by gIV and the virus-infected cell lysate in particular indicates the potential of glycoprotein gIV as a subunit vaccine, ideally in combination with component(s) from the cell lysate, which may mediate cellular immune responses.

A subunit gIV vaccine, produced by transfected mammalian cells in culture, induces mucosal immunity against bovine herpesvirus-1 in cattle.

A truncated version of bovine herpesvirus-1 (BHV-1) glycoprotein IV (tgIV) was produced in a novel, non-destructive expression system based upon regulation of gene expression by the bovine heat-shock protein 70A (hsp70) gene promoter in Madin Darby bovine kidney (MDBK) cells. In this system, up to 20 micrograms ml-1 of secreted tgIV, which is equivalent to the yield from 4 x 10(6) cells, was produced daily over a period of up to 18 days. Different doses of tgIV were injected intramuscularly into seronegative calves. Virus-neutralizing antibodies were induced by all doses of tgIV, both in the serum and in the nasal superficial mucosa. However, the low dose (2.3 micrograms) induced significantly (p < 0.05) lower antibody titres than the medium (7 micrograms) and high (21 micrograms) doses. The medium and high doses of tgIV conferred protection from BHV-1 infection, as demonstrated by a significant (p < 0.05) reduction in clinical signs of respiratory disease and virus shedding in the nasal secretions postchallenge. However, the 2.3 micrograms group, although partially protected, was not significantly (p > 0.05) different from the placebo group. This study demonstrated the potential of an intramuscularly administered tgIV subunit vaccine to induce mucosal immunity to BHV-1 using an economic protein production system and an acceptable vaccine formulation. In addition, a strong correlation was observed between neutralizing antibodies in the serum and nasal superficial mucosa, virus shedding and clinical disease. Thus, serum neutralizing antibody levels in tgIV-immunized animals may be a good prognosticator of protection from BHV-1 infection and disease.

CpG-containing oligodeoxynucleotides augment and switch the immune responses of cattle to bovine herpesvirus-1 glycoprotein D☆

The adjuvanticity of a synthetic oligodeoxynucleotide containing unmethylated CpG motifs (CpG ODN) was determined in cattle. Calves were immunized with a truncated secreted version of glycoprotein D (tgD) of bovine herpes virus-1 (BHV-1) formulated with alum, CpG ODN, or a combination of both. BHV-1 tgD formulated with CpG ODN or with alum and CpG ODN induced a stronger and more balanced immune response than tgD in alum. This level of immunity was of sufficient magnitude to minimize weight loss and significantly reduce the duration of virus shedding after intranasal viral challenge. Local tissue reactions generated by CpG ODN were very mild and transient, whereas reactions induced by alum or a combination of CpG ODN and alum were moderate in severity and duration. These data demonstrate that CpG ODN causes minimal injection site reactions and yet acts as an effective adjuvant in cattle.