Suresh K. Tikoo

Bovine herpesvirus 1 (BHV-1): biology, pathogenesis, and control

Publisher Summary The past decade has seen very significant progress in the identification and characterization of the proteins of vovine herpesvirus 1 (BHV-1), and in understanding their role in the initiation of infection at the receptor level and in virus replication. Furthermore, advances in immunology have allowed dissection of the host immune response and identification of the specific protein/glycoproteins and epitopes on these glycoproteins involved in the modulation of the infection. This progress has provided an opportunity to better understand the events involved in the pathogenesis of BHV-1 as well as to develop more effective methods of controlling this disease through vaccine development. The ability to study these virus-cell interactions in the natural host not only aids in controlling BHV-1, but lays the foundation for a better understanding of herpesvirus infection in general. This chapter summarizes the recent advances regarding the proteins/glycoproteins present in the virus, their characterization, and function in infection and virus replication, as well as their interaction with various cells of the immune system.

In vivo cutaneous interferon‐γ gene delivery using novel dicationic (gemini) surfactant–plasmid complexes

Localized scleroderma (morphea and linear scleroderma) is a connective tissue disease, accompanied by excessive proliferation and deposition of collagen within the skin, inflammation, vasculopathy and a deranged immune system. Interferon γ (IFNγ), an inhibitor of collagen synthesis and an immunomodulator, could be a potential therapeutic agent if it could be delivered into or expressed locally in affected skin in a non‐invasive manner. In this study, the feasibility of topical delivery of the IFNγ gene and expression of IFNγ were investigated in mice.

Immunology of bovine herpesvirus 1 infection

Immune responses to bovine herpesvirus 1 (BHV-1) have been studied following exposure of animals to virulent virus, conventional live or killed vaccines, genetically engineered live virus vaccines, subunit vaccines and, more recently, following immunization with plasmids encoding putative protective antigens. In all cases reported to date, exposure to BHV-1 or its glycoproteins induced specific responses to the virus which are capable of neutralizing virus and killing virus infected cells. These studies clearly indicate that the responses to BHV-1 are broad based, including both Th1 and Th2. In addition to inducing neutralizing antibodies, which can prevent virus attachment and penetration, these antibodies can also participate in antibody complement lysis of infected cells or in antibody dependent cell cytotoxicity. The virus also induces a myriad of specific cellular responses including the induction of cytokines, which either directly or indirectly inhibit virus replication by activation of effector cells. These activities have been associated with lymphocytes, NK-like cells, macrophages and polymorphonuclear neutrophils. These effector cells can kill virus infected cells either directly or by interacting with antibody to induce cell death by antibody dependent cell cytotoxicity. Killing of virus infected cells occurs after the expression of viral antigens on the cell surface of infected cells. Since the relationship between the time of cell killing and completion of virus assembly will influence whether the infectious cycle is aborted or results in productive viral replication any enhancement in viral killing will dramatically reduce the virus load. Based on these studies, many people conclude that antibody is critical in preventing infection and spread to susceptible contacts. In contrast, cell mediated immunity is involved in recovery from infection. However, none of these events occur in isolation in a body and a defect in one will dramatically influence the other. Furthermore, the relative importance of each effector mechanism will clearly depend on whether the animal is exposed to the virus for the first time (primary infection) or it is a secondary exposure following vaccination or infection with the field virus. Following a primary infection, where there is no antibody to interfere with the initial virus-cell interaction at the receptor level, the virus initiates an infection. These initial interactions are mediated primarily by the viral glycoproteins. Following the initial infection, viral protein synthesis induces a series of events which stimulate the nonspecific immune responses of the host. Therefore, the nonspecific immune responses (mediated primarily by viral products which induce early cytokines) are amongst the first line of defense in helping clear the infection both directly as well as indirectly by stimulating the specific immune response. The macrophage is instrumental in focusing the specific immune response by producing various cytokines and subsequently responding to cytokines produced by T-cells to kill to virus infected cells. This activity is detectable within 2 days after infection in lung parenchymal cells and 5-7 days in peripheral blood leukocytes. Interactions between various effector functions in limiting virus replication are described.

Development of porcine adenovirus-3 as an expression vector.

Porcine adenovirus-3 (PAV-3) was developed as an expression vector using homologous recombination in Escherichia coli BJ 5183. As a prerequisite, the complete genome of PAV-3 was first introduced as a PacI restriction fragment into a bacterial plasmid. The plasmid, when PacI restricted and transfected into swine testicular cells, produces an infectious virus. The potential of this procedure was demonstrated by the construction of several PAV-3 recombinants. Part of the E3 region, which is nonessential for virus replication under cell culture conditions, was identified and deleted from the virus genome. The gene for glycoprotein D (gD) of pseudorabies virus (PRV), which elicits PRV-neutralizing antibodies in pigs, was cloned and expressed from the E3 region of PAV-3. A 50 kDa polypeptide was identified in recombinant PAV-3-infected cell lysates by immunoprecipitation assays using gD-specific monoclonal antibodies. In another experiment, a region between the right inverted terminal repeat and the promoter of the E4 region was used to clone and express the chloramphenicol acetyltransferase (CAT) gene under the control of SV40 immediate early promoter. CAT gene expression was observed irrespective of the orientation of the CAT gene. These results indicate that the helper-independent recombinant PAV-3 could be used as an expression vector and has potential as a recombinant vaccine vector in pigs.

Mucosal immunization of calves with recombinant bovine adenovirus-3: induction of protective immunity to bovine herpesvirus-1

To determine the potential of replication-competent (E3-deleted) bovine adenovirus-3 (BAV-3) as a delivery system for vaccine antigens in calves, we evaluated the ability of recombinant BAV-3 expressing different forms of of bovine herpesvirus-1 (BHV-1) glycoprotein gD to protect against BHV-1 infection in calves that had pre-existing BAV-3 specific antibodies. Three- to four-month-old calves, vaccinated intranasally with recombinant BAV-3 expressing full-length gD (BAV3.E3gD) or a truncated version of gD (gDt) (BAV3.E3gDt), or with E3-deleted BAV-3 (BAV3.E3d; control), were challenged with BHV-1 strain 108. Vaccination with BAV3.E3gD or BAV3.E3gDt induced gD-specific antibody responses in serum and nasal secretions, and primed calves for gD-specific lymphoproliferative responses. In addition, all calves developed complement-independent neutralizing antibodies against BHV-1. Protection against viral challenge was observed in calves vaccinated with recombinant BAV3.E3gD or BAV3.E3gDt as shown by a significant reduction in body temperature and clinical disease, and a partial reduction in the amount and duration of virus excretion in nasal secretions. These results indicate that replication-competent BAV-3-based vectors can induce protective immune responses in calves (the natural host) that have pre-existing BAV-3-specific antibodies.

Adenoviruses as vectors for delivering vaccines to mucosal surfaces

Abstract Immunization of mucosal surfaces has become an attractive route of vaccine delivery because of its ability to induce mucosal immunity. Although various methods of inducing mucosal immunity are being developed, our laboratory has focused on developing adenoviruses as replication–competent and replication–incompetent vectors. The present report will summarize our progress in sequencing the entire bovine adenovirus-3 genome and identifying regions which can be deleted and subsequently used as insertion sites for foreign genes in developing recombinant viral vaccines. Using these recombinant viruses, we demonstrated the ‘proof-of-principle’ in developing mucosal immunity and, more importantly, inducing protection against bovine herpes virus in a natural host–cattle. Finally, we demonstrated that immunity and protection occurred even in animals that had pre-existing antibodies to the vector.

Porcine adenovirus-3 as a helper-dependent expression vector

Porcine adenovirus has been proposed as a potential vector for generating novel and effective vaccines for pigs. As a prerequisite for the generation of helper-dependent porcine adenovirus-3 (PAV-3) vectors, two E1-complementing porcine cell lines expressing E1 proteins of human adenovirus-5 (HAV-5) were made. These cell lines could be efficiently transfected with DNA and allowed the rescue and propagation of a PAV-3 recombinant, PAV201, containing a 0.597 kb E3 deletion and a 0.803 kb E1A deletion. Our data demonstrate that E1A proteins of HAV-5 have the capacity to transform foetal porcine retina cells and complement for the E1A proteins of PAV-3. The green fluorescent protein (GFP) gene placed under the control of a cytomegalovirus immediate early promoter was inserted into the E1A region of the PAV201 genome. Using these cell lines, a helper-dependent PAV-3 recombinant expressing GFP, PAV202, was constructed and characterized. The wild-type PAV-3 and the recombinant PAV202 expressing GFP were used to determine the ability of the virus to enter and replicate in cells of human and animal origin under cell culture conditions. Our results suggest that PAV-3 enters but does not replicate in dog, sheep, bovine and human cells.

Oligodeoxynucleotides containing CpG motifs (CpG-ODN) predominantly induce Th1-type immune response in neonatal chicks

Earlier, we demonstrated that intramuscular administration of oligodeoxynucleotides containing CpG motifs (CpG-ODN) induces protection in neonatal chicks against a lethal challenge of Escherichia coli. However, the mechanism of induction of the protection was not clear. In an attempt to elucidate the mechanism of induced protection, we determined the kinetics of expression of cytokines/chemokines in the spleen and bursa of Fabricius of newly hatched chicks that had received intramuscular administration of CpG-ODN or non-CpG ODN compared to saline-treated controls. SyBr green, real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis of the RNA demonstrated increased expression of IL-1beta, IL-6, IL-8, IL-10, IL-18, IFN-gamma and MIP-3alpha mRNAs in the spleen and; IL-10 and IFN-alpha in bursa of Fabricious of chicks that had received CpG-ODN. However, non-CpG ODN failed to induce any of the cytokine. The increased level of IL-18 and IFN-gamma but not IL-4 mRNA suggests that the administration of CpG-ODN elicits a Th1 biased immune response, which may be important in inducing protection against infections in neonatal chicks. To our knowledge, this is the first report evaluating the induction of cytokines/chemokines in neonatal chicks following administration of CpG-ODN.

Bovine herpesvirus-1 vaccines

Vaccination has been important in controlling a wide variety of viral and bacterial infections of man and animals. Vaccines to herpesvirus infection of cattle are no exception. The present review describes the different types of conventional vaccines that have been used to date and furthermore describes the novel approaches which are presently being implemented to develop more effective vaccines. These include subunit vaccines as well as genetically engineered modified live deletion mutants. Both these novel vaccine approaches appear to be more efficacious than conventional vaccines. Furthermore, these vaccines provide an additional dimension for control and eradication of infection by providing an opportunity to develop companion diagnostic tests to differentiate infected animals from vaccinated animals. This review summarizes these developments as well as present knowledge regarding the important host defence mechanisms required for preventing infection and aiding recovery from infection.

CpG Oligodeoxynucleotides Activate Innate Immune Response that Suppresses Infectious Bronchitis Virus Replication in Chicken Embryos

Abstract The understanding of innate immune modulation by pathogens and immune-modulating agents, including synthetic oligodeoxynucleotides (CpG ODNs), has offered several new approaches to improve prophylactic and therapeutic strategies against infectious diseases in humans and animals. However, in this regard not much work has been done in avian medicine. In the present study, we analyzed the kinetics of interferon (IFN), cytokine, and chemokine mRNA expression in chicken embryonic spleen at 6 hr, 24 hr, 48 hr, and 72 hr after administration of CpG ODN 2007 (B-class) in 18-day-old chicken embryos. Our data showed enhanced expression of IFN-γ; interleukin (IL)-1β, IL-6, and IL-8; and oligoadenyl synthetase A mRNA after CpG ODN administration. In addition, CpG ODN administration to chicken embryos 24 hr before the challenge with infectious bronchitis virus (IBV) was capable of limiting IBV propagation in different embryonic tissues. Based on the kinetics and type of cytokines induced after in ovo administration of CpG ODN, it may be speculated that in ovo administration of CpG ODNs may enhance resistance from viral infection in neonatal chicks and that CpG ODNs may contribute toward the development of more effective and safer poultry vaccines including in ovo vaccines.