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