Abstract
New technologies in the field of vaccinology arise as necessity for treatment and control of many diseases. Currently modified live virus and inactivated vaccines used for Bovine Herpesvirus-1 (BoHV-1) have several disadvantages. Previous works for preventive treatment of BoHV-1 with DNA based vaccines have demonstrated the capability to induce humoral and cellular immune response. Nevertheless, it is well known that “naked” DNA induces low immunogenic response. Thus, loading of antigen encoding DNA sequences in liposomal formulations targeting dendritic cell receptors could be a promising strategy to better activate these antigen presenting cells (APC). In this work, DNA based vaccine encoding the truncated version of gD glycoprotein (pCIgD) of BoHV-1 was investigated alone and upon encapsulation on liposomal formulation coated with MANα1-2MAN-PEG-DOPE and LPS from Brucella ovis (pCIgD-Man-L) in mice and cattle assay. Results showed that the use of pCIgD-Man-L was capable to enhance the immune response in both animal models. Significant levels of humoral immunity were achieved when total antibody titers and isotypes were detected in sera and mucosa. For cellular immunity, specific viral lymphoproliferation was detected in the animals inoculated with pCIgD-Man-L. In addition, positively modulation of CD40 molecules on the surface of bovine dendritic cells (DCs) was observed when cells were stimulated and activated with vaccine formulations. When challenge assay was performed, bovines inoculated with pCIgD and liposome decorated with MANα1-2MAN-PEG-DOPE elicited better protection and diminished viral excretion.
The results demonstrate the targeting of the MANα1-2MAN coated liposomes toward dendritic cells and their ability to boost the immunogenicity according to an adjuvant effect that results in long-lasting immunity.
Liposome decorated with MANα1-2MAN-PEG-DOPE were for the first time tested as DNA based vaccine in cattle as preventive treatment of BoHV-1. These results open up new perspectives for the design of vaccine for the control of bovine rhinotracheitis.
Keywords: adjuvant, BoHV-1, DNA, DC targeting vaccine, liposome
Introduction
Bovine herpesvirus 1 (BoHV-1), an enveloped virus belonging to the alphaherpesvirus subfamily, infects cattle of all ages and breeds worldwide (Babiuk et al., 1987; Tikoo et al., 1995). This virus is pathogen whose infection can severely impact cattle production. It causes a variety of symptoms in cattle including infectious bovine rhinotracheitis (IBR), conjunctivitis, abortions and shipping fever, which is a complicated infection of the upper respiratory tract (Jones, 2003). The pathogenesis is responsible for considerable economic losses due to decreased milk production, weight loss and abortion and has been recognized as an important component of the bovine respiratory complex.
BoHV-1 initiates the disorder through immunosuppression that could render the animals more susceptible to secondary bacterial infections, leading to pneumonia and occasionally to death. BoHV-1 establishes latency. Latently infected animals should always be considered a potential source of infection (Bitsch, 1973), although vaccination can considerably reduce the amount of virus excreted following reactivation (Mars et al., 2001).
In general, there are control programs with the use of conventional modified and killed vaccines inactivate but there are no specific international programs to eradicate BoHV-1. In Europe, only a small number of countries has achieved the goal of IBR-eradication (Blickenstorfer et al., 2010), by the destruction of a great number of healthy, seropositive animals because they are persistently infected with BoHV-1. In others European countries, control strategy against this virus has been vaccination with live and killed gE-deleted marker (van Drunen Littel-van den Hurk, 2006; Romera et al., 2014). In endemic countries like Argentina, Brazil and Spain, voluntary intensive vaccination programs are implemented to reduce the prevalence of infected animals.
Vaccination remains one of the most cost-effective strategies to prevent and control the clinical signs and transmission of these viruses. Nevertheless, these vaccines have a series of disadvantages for BoHV-1. In the case of conventional vaccines, they may protect individual animals against clinical disease, but they cannot prevent either the efficient transmission of the virus or the establishment of latency. Inactivated vaccines do not provide complete protection because they are generally poor inducers of cellular immunity, while attenuated vaccines despite giving good levels of protection are not completely safe (Deshpande et al., 2002; Blome et al., 2013; Quattrocchi et al., 2017).
BoHV-1 uses a variety of mechanisms to elude the host’s immune system. By spreading intracellularly, it can exist in the presence of anti-viral specific antibodies (Fuller and Lee, 1992; Miethke et al., 1995). For this reason, cytotoxic T-lymphocytes (CTL) are critical for the elimination of the virus (Langellotti et al., 2011).
DNA vaccines emerge as an alternative to conventional vaccines. These vaccines have the potential to induce both cellular and humoral immune responses against antigen encoded by recombinant DNA (Kanthesh et al., 2018) and are highly specific; the expressed immunizing antigen is subject to the same modifications as natural infection (Lee et al., 2015) without the risk of trace pathogenicity due to incompletely inactivated virus or due to reverted attenuated virus (Rajčáni et al., 2005). It is well known that the use of naked DNA vaccines in small animal models function as an adjuvant or immunomodulator itself, (Yamamoto et al., 1992; Krieg et al., 1995) but in some cases and especially in large animals, the protection conferred against disease is inefficient. For this reason, there are many trials in the use of plasmids with adjuvants and delivery systems to improve and enhance the immune response (Quattrocchi et al., 2017).
The aim of this study was to evaluate the immune response elicited in mice model and in cattle by a plasmid encoding BoHV-1 secreted gD (pCIgD) with the use of a liposome engineered through their coating with a ligand that specifically target dendritic cells (Man-L) and the inclusion of an adjuvant that helps stimulate the immune response. The liposomes were decorated with a patented synthetic molecule (Pappalardo et al., 2013), whose specific α1,2Mannobiose ligand has an affinity for DC-SIGN (Feinberg et al., 2007; Yamakawa et al., 2008). The MANα1-2MAN-PEG-DOPE derivative was selected because it can target dendritic cells (DCs) of different species (Pappalardo et al., 2013) and thus provide selective delivery of different antigens and nucleic acids that can then be processed for presentation in the contexts of MHC-I and II. Brucella ovis  HS antigen (LPS), which contains LPS and some OMP proteins, is associated to this nanovaccine as adjuvant. The formulation of DNA loaded nanovaccine confers advantages such as increased antigen uptake by antigen presenting cells (APCs), cytokine secretion stimulation by APC or lymphocytes, increased antigen stability and decreased antigen degradation (Zaman et al., 2013). Previous studies have demonstrated the ability to better deliver an antigen to DC by anchoring this di-mannose molecule on liposome surface in nanovaccine formulation. It has been proven effective in vitro on murine and human DCs. There are also in vivo results of the effectiveness of this mannose ligand used in nanovaccines against Brucella ovis in sheep (Pappalardo et al., 2016).
To our knowledge, BoHV-1 DNA based vaccine was for the first time formulated with liposome targeted to DC with MANα1-2MAN-PEG-DOPE and tested in cattle. These results could be useful to design a vaccine for the control of bovine rhinotracheitis.
Materials and Methods