Luana Alves Dummer

Bovine herpesvirus glycoprotein D: a review of its structural characteristics and applications in vaccinology

The viral envelope glycoprotein D from bovine herpesviruses 1 and 5 (BoHV-1 and -5), two important pathogens of cattle, is a major component of the virion and plays a critical role in the pathogenesis of herpesviruses. Glycoprotein D is essential for virus penetration into permissive cells and thus is a major target for virus neutralizing antibodies during infection. In view of its role in the induction of protective immunity, gD has been tested in new vaccine development strategies against both viruses. Subunit, DNA and vectored vaccine candidates have been developed using this glycoprotein as the primary antigen, demonstrating that gD has the capacity to induce robust virus neutralizing antibodies and strong cell-mediated immune responses, as well as protection from clinical symptoms, in target species. This review highlights the structural and functional characteristics of BoHV-1, BoHV-5 and where appropriate, Human herpesvirus gD, as well as its role in viral entry and interactions with host cell receptors. Furthermore, the interactions of gD with the host immune system are discussed. Finally, the application of this glycoprotein in new vaccine design is reviewed, taking its structural and functional characteristics into consideration.

Cloning and expression of a truncated form of envelope glycoprotein D of Bovine herpesvirus type 5 in methylotrophic yeast Pichia pastoris.

Meningoencephalitis caused by Bovine herpesvirus type 5 (BoHV-5) is responsible for heavy economic losses in the cattle industry. As in other Alphaherpesviruses, the envelope glycoprotein IV (gD), which mediates penetration into host cells, is one of the major candidate antigens for a recombinant vaccine, since it induces a strong and persistent immune response. The DNA coding for a truncated form of BoHV-5 gD (tgD) has been cloned into the Pichia pastoris expression vector pPICZalphaB to allow protein secretion into the medium. After induction with methanol, a approximately 55kDa protein was obtained. Enzyme deglycosylation with Endo H showed a smaller size band in SDS-PGAE, with approximately 50kDa, suggesting that tgD has N-linked oligosaccharides and that it is not hyperglycosylated. The approximately 55kDa protein was recognized by several polyclonal antibodies, including polyclonal antibody anti-tgD and polyclonal antibodies of different animal species immunized with BoHV-5 and BoHV-1. This is the first report of BoHV-5 gD expression in yeast. It was shown that the recombinant truncated form of BoHV-5 gD has antigenic and immunogenic properties similar to the native BoHV-5 gD. Expression of tgD as a secreted protein allows simple and inexpensive purification methods that can be used for further studies to evaluate its immunogenicity in cattle.

The immune modulation of Bacillus cereus var. Toyoi in mice immunized with experimental inactivated Bovine Herpesvirus Type 5 vaccine

In recent years, there been significant progress toward develop more efficient vaccines. Different compounds with adjuvant capacity have been tested; however, no compound has emerged that suitable for universal use. Several efforts have been made to produce effective vaccines against Bovine herpesvirus 5 (BoHV-5), an important cattle pathogen. In this study we examine the use the probiotic Bacillus cereus var. Toyoi as a potential adjuvant to improve BoHV-5 vaccine efficacy. We observed in the supplemented animals a systemic enhanced IgG antibody response toward Th1, and increased IFN-γ, IL-12 and IL-10 cytokines mRNA levels. These results suggest that this probiotic could provide a promising means of improving vaccine efficacy, particularly those vaccines that rely on a cell-mediated immune response.

Immune responses of mice against recombinant bovine herpesvirus 5 glycoprotein D.

Glycoprotein D (gD) is essential for attachment and penetration of Bovine herpesvirus 5 (BoHV-5) into permissive cells, and is a major target of the host immune system, inducing strong humoral and cellular immune responses. The aim of this study was to evaluate in mice the immunogenicity of recombinant BoHV-5 gD (rgD5) expressed in Pichia pastoris. Vaccines formulated with rgD5 alone or adjuvanted with Montanide 50 ISA V2; Emulsigen or Emulsigen-DDA was administered intramuscularly or subcutaneously. Almost all formulations stimulated a humoral immune response after the first inoculation. The only exception was observed when the rgD5 was administered subcutaneously without adjuvant, in this case, the antibodies were observed after three doses. Higher titers of neutralizing antibodies were obtained with the three oil-based adjuvant formulations when compared to non-adjuvanted vaccine formulations. The rgD5 vaccine stimulated high mRNA expression levels of Th1 (INF-γ) and pro-inflammatory cytokines (IL-17, GM-CSF). The results demonstrated that the recombinant gD from BoHV-5 conserved important epitopes for viral neutralization from native BoHV-5 gD and was able to elicit mixed Th1/Th2 immune response in mice.

The use of quorum sensing to improve vaccine immune response

Enterotoxigenic Escherichia coli (ETEC) infection is an important cause of diarrhea in both newborn and post-weaning pigs, it is also responsible for economic losses on farms worldwide. Vaccines that use ETEC virulence factors have been well documented, and several vaccines containing inactivated bacteria with protective antigens, or purified (isolated) antigens are available on the market. Vaccination of pregnant sows is widely seen as an effective strategy for the control of the disease. Yet these vaccines very often do not lead to efficient protection. In this study, we produced an ETEC bacterin with the use of quorum sensing (QS), and observed a significant expression of F4 adhesin, and heat-labile toxin (LT) in the cultures when compared to the controls. Mice, and pigs vaccinated with the QS bacterin demonstrated higher antibody titers against these antigens when compared with commercial and control bacterin. Our results suggest that the system might bring promising improvements in ETEC bacterin efficacy.

PEROXIDASE-LINKED ASSAY FOR DETECTION OF ANTIBODIES AGAINST BOVINE LEUKOSIS VIRUS

A peroxidase linked assay (PLA) was designed to screen bovine sera for the presence of specific antibodies against bovine leukosis virus (BLV). Out of 201 samples of bovine sera analyzed, 52.2% were considered positive by PLA, 26.4% by AGID, and 38.9% by ELISA. Western blotting analyses excluded 27 samples found to be positive by PLA. PLA showed 100% of sensitivity when compared with AGID and ELISA. Specificity was 64.8% and 78%, respectively (kappa coefficients were 0.70 and 0.83). These findings indicate that PLA can be used as an alternative method for the diagnosis of BLV infection in cattle.

Immune responses in bovines to recombinant glycoprotein D of bovine herpesvirus type 5 as vaccine antigen

Bovine herpesvirus 5 (BoHV-5) is responsible for outbreaks of meningoencephalitis that cause important economic losses in young cattle. BoHV-5 glycoprotein D (gD5) is essential for attachment and penetration into permissive cells and targeting of host immune systems, inducing strong humoral and cellular immune responses. The aim of this study was to evaluate the vaccinal immune response of vaccines formulated with the recombinant BoHV-5 gD (rgD5) in bovines. For the experiment, 72 heifers were randomly allotted into 6 different groups with 12 animals each. Group 1: vaccine formulated using inactivated BoHV-5 (iBoHV-5) adjuvanted with ISA50V2; Group 2: iBoHV-5 associated with 100 µg of rgD5 adjuvanted with ISA50V2; Group 3: 100 µg of rgD5 adjuvanted with ISA50V2; Group 4: 100 µg of rgD5 adjuvanted with Al(OH)3; Group 5: commercial vaccine; and Group 6: control group. Two doses were administered in a 26-day interval and the third after 357 days from primo vaccination. Cattle vaccinated with the vaccines formulated with iBoHV-5 plus rgD5 showed a significant (p < 0.01) five-fold increase in total immunoglobulin G (IgG) for BoHV-5, BoHV-1, and rgD5 as compared with the commercial and control groups. Also, a significant (p < 0.05) increase in IgG1 and IgG2a levels was induced in serum for rgD5. In addition, these same vaccines showed significant (p < 0.01) four-fold higher titers of BoHV-1 and -5 neutralizing antibodies. The results demonstrated that the rgD5 conserved important epitopes that were able to stimulate bovine humoral immunity response capable of viral neutralization of BoHV-1 and -5, suggesting it as a promising vaccine antigen to be used in vaccine for BoHV-1 and -5 endemic areas.

Probiotics Bacillus toyonensis and Saccharomyces boulardii improve the vaccine immune response to Bovine herpesvirus type 5 in sheep

There have been significant efforts toward the development of more efficient vaccines for animal health. A strategy that may be used to improve vaccine efficacy is the use of probiotics. Bovine herpesvirus 5 (BoHV-5) is an example of an important animal pathogen for which vaccines have provided only limited protection. In this study, we examined the use of the probiotics Bacillus toyonensis and Saccharomyces boulardii as a potential immune modulator to improve vaccine efficiency. Thirty, 5-month-old lambs were randomly grouped in three lots of 10 each and vaccinated at days 0, 21 and 42 of the experiment. They grazed on the same pasture and were fed ad libitum twice a day with commercial sheep feed supplemented with either B. toyonensis (1×106CFU/g of feed) or S. boulardii (1×107CFU/g of feed), or non-supplemented feed. The probiotic supplementation was suspended day 28; thereafter, the next 35days, they were fed with the same commercial feed as control group. Animals supplemented with probiotics showed a significant (p>0.001) increased seroconversions against BoHV-5, and higher neutralizing antibodies titres (p>0.05) to BoHV-5 than non-supplemented animals. At 63days of experiment, splenocytes from the supplemented sheep had higher mRNA transcription levels of cytokines IL-10 and IL-17A. These results suggest that these probiotics could provide a promising means of improving vaccine efficacy.

Development of an Indirect ELISA for Serological Diagnosis of Bovine herpesvirus 5.

Bovine herpesviruses 1 and 5 (BoHV-1 and BoHV-5) are economically important pathogens, associated with a variety of clinical syndromes, including respiratory and genital disease, reproductive failure and meningoencephalitis. The standard serological assay to diagnose BoHV-1 and BoHV-5 infections is the virus neutralization test (VNT), a time consuming procedure that requires manipulation of infectious virus. In the present study a highly sensitive and specific single dilution indirect ELISA was developed using recombinant glycoprotein D from BoHV-5 as antigen (rgD5ELISA). Bovine serum samples (n = 450) were screened by VNT against BoHV-5a and by rgD5ELISA. Compared with the VNT, the rgD5ELISA demonstrated accuracy of 99.8%, with 100% sensitivity, 96.7% specificity and coefficient of agreement between the tests of 0.954. The rgD5ELISA described here shows excellent agreement with the VNT and is shown to be a simple, convenient, specific and highly sensitive virus-free assay for detection of serum antibodies to BoHV-5.