3.7 pCIgD and pCIgD-Man-L presented similar clinical score
Clinical score was obtained by measuring the symptoms acquired by the
cattle after the viral challenge.
At different days post challenge, calves were clinically examined, and
rectal temperature was recorded. Clinical score after viral challenge
was established according to: grade 0 = normal; 1 = slight rhinitis with
serous mucus with or without mild serous conjunctivitis; 2 =
moderate/heavy rhinitis with fibrinous serous mucus with or without
moderate serous conjunctivitis; 3 = fibrinopurulent mucus with moderate.
Therefore, animal vaccinated with pCIgD and pCIgD-Man-L had a lower
clinical score and there were significant differences compared with
pCIneo group (p<0.05) at 6 dpc for pCIgD and at 7 dpc for
pCIgD-Man-L (p<0.05) but there were no significant differences
between them (Fig. 7 ). Clinical score AUC was analyzed and
again pCIgD-Man-L treated animals had the lowest level respect the other
groups.
Discussion
DNA vaccines have taken great interest in the field of vaccinology
because of their simplicity, safety, stability, and versatility for
development (Kanthesh et al., 2018). Previous studies with these
vaccines have shown to induce good levels of humoral and cellular
immunity in different species model depending on the type of encoded
antigen, route of administration and promoter efficiency (Huang et al.,
2005). Glycoprotein D is involved in virus penetration and has been
considered the major target in vaccine development against bovine and
human herpesvirus. A major limit of DNA vaccine is the low
immunogenicity due to difficulties in delivering DNA plasmid into the
host cell for further synthesis of the encoded antigenic protein
(Suschak et al., 2017). Here, the use of suitable delivery systems and
specific molecular activators can be combined to generate adjuvants that
help to improve the efficiency of DNA based vaccines. In the search to
set up a vaccine that generates both types of immunity against BoHV-1,
we investigated DNA based vaccines using a DNA sequence encoding for the
truncated version of gD glycoprotein (pCIgD). Our previous studies using
the same DNA plasmid combined with chemical adjuvants in mice provided
good protection evidences (Zamorano et al., 2002; Langellotti et al.,
2011; Di Giacomo et al., 2015) while studies in cattle with a subsequent
viral challenge showed the protection was only partial (Quattrocchi et
al., 2017) which prompted us to further search for new formulation
strategies.
In this context, we have designed here a complex DNA based nanovaccines
based on liposomes decorated with mannose molecules for DC targeting and
including LPS as molecular adjuvant which is expected to enhance the
transfection efficiency in DCs and thus promote immunity. This will
result in virtue of the ability of the liposomes to selectively target
the DC-SIGN receptor in DCs. Liposomes were selected because they
represent a self-assembling carrier that allow to combine different
functional components on their surface resulting in a cooperative
adjuvant effect. In addition, liposomes protect DNA from
deoxyribonuclease attack (Gregoriadis et al., 2002).
The intradermal inoculation is one of the best performing administration
strategy to deliver a DNA based vaccine (Zonouzi et al., 2016). The
rational for this is that the skin possess associated lymphoid tissue,
including Langerhans cells, dendritic cells, keratinocytes and other
immune cells. Here, we inoculated animals by intradermal route, and we
observed remarkable humoral and cellular immune response. In agreement
with evidences that truncated glycoprotein D can induce good levels of
humoral immune response (Van Drunen Littel-van den Hurk et al., 1998;
Lewis et al., 1999; Huang et al., 2005) we obtained and increasing
antibody titers upon administration of pCIgD which was significantly
increased when the plasmid DNA encoding for truncated glycoprotein D was
formulated with DC targeted liposome including LPS adjuvant
(pCIgD-Man-L). It is interesting to note that although no new
inoculation was made after 40 dpv in mice, there were high levels of
antibody even at 120 dpv with pCIgD-Man-L, but this phenomenon was not
observed with pCIgD alone. This can be attributed to the local depot
effect, and the selective delivery and activation capacities of the
liposomes combined with LPS from B. ovis (Fredriksen and Grip,
2012) thus enhancing and prolonging the exposure of the immune cells to
the antigen which results in specific immune response and long-lived
immunity. When isotype antibody was analyzed, the specific effect of the
liposome-based vaccine resulted in the increase of the levels of IgG1
and the profile of IgG2a, IgG2b when compared with the pCIgD treated
group where the levels of antibodies were significantly lower. Notably
IgG2a is an isotype that is crucial for the defense against viruses.
Previous works have shown that IgG2a antibodies are produced in mice
upon viral infections (Coutelier et al., 1991) and there are also
triggered when DNA based vaccines are administered for immunizations
(Raz et al., 1996) as a result of T helper 1 cells involvement. This
isotype is efficient at complement fixation and acts as opsonizing
agent. It is reported that IFNγ can stimulate the production of IgG2a
both by in vitro and in vivo activated B lymphocytes. This
increase of IgG2a in the animals treated with our free DNA plasmid
suggest that the gD plasmid could be internalized by DCs and that
interaction between DCs-B cells occurred according to CD40:CD40L
dependent manner (Bao et al., 2014). The B cell switching was confirmed
by the presence of the IgG2a isotype in sera of mice and by the positive
modulation of the T CD4+ cells response to a Th1/Th2 profile.
The immune system quick response to a viral infection confirm a cellular
memory to the viral antigens. This would prove that the viral antigens
were previously processed by the host and CD4+/CD8+ memory lymphocytes
were generated. In this work, we observed that splenocytes from mice
immunized with gD plasmids were able to lymphoproliferate against
inactivated BoHV-1 in cell culture. Indeed, it is important to highlight
that these animals were last immunized 100 days before their splenocytes
were exposed to inactivated BoHV-1. This ability to lymphoproliferate in
the pCIgD-Man-L group was certainly provided by the adjuvant effect of
the targeted liposome because this was not observed with the DNA plasmid
administered alone.
We then tested the novel liposome based nanovaccine in a field trial in
cattle. When we assessed BoHV-1 specific antibodies in blood the
difference was significant between the pCIgD-Man-L immunized group
compared to the control group. This increase was also observed in the
isotype antibody titres. While in the murine model, the transcription
factors in T helper lymphocytes such as GATA-3 associated with enhanced
type 2 cytokine gene transcription (IL-4) or T-bet associated to type 1
cytokine (IFNγ) have been widely shown, these factors are not evident in
bovine (Estes and Brown, 2002). Bovine IL-4 could induce IgG1 while IFNγ
may enhance IgG2 over IgG1 (Estes et al., 1995). The results obtained in
our study for bovine isotypes elicited by the vaccines, showed a
relationship IgG1 and IgG2 increases as it was observed in previous
tests with other DNA based vaccines or adjuvants (Arulkanthan et al.,
1999; Brown et al., 1999; Quattrocchi et al., 2017). However, it could
be observed that there is a greater IgG2 titer in the groups of animals
that received pCIgD-Man-L than the DNA vaccine alone.
IgA was assessed from nasal swabs and evaluated 6 days post challenge
resulting in higher titre in the pCIgD-Man-L treated group with respect
to the control pCIgD treated group. Reasonably, the persistence and
enhanced exposure to glycoprotein D post challenge can be responsible
for the expansion of DC-primed cell and IgGs and IgA secreting B cell
populations as hypothized by Manoj (Manoj et al., 2004).
CD40 is a co-stimulatory cytokine for various types of cells including B
cells, macrophages, non-hematopoietic cells and DCs, for which it has an
crucial role in immunity development and in activation of the latter
cell types (Elgueta et al., 2009). DCs are the major antigen presenting
cells (APC) and they are crucial mediators between immune innate and
adaptive response (Vázquez et al., 2012). When DCs are inactivated, CD40
is expressed constitutively at relatively low levels on their surface
(Banchereau et al., 1994). Upon DC cells encounter pathogens, antigens
(Kawai and Akira, 2007) or apoptotic cells (Ip and Lau, 2004), CD40
expression is up-regulated on their surface and is activated. In this
work, ALDCs were stimulated and activated after incubation with free DNA
plasmid and DNA loaded targeted liposomes embedding LPS fromBrucella ovis . It is well known that DNA possess CpG domain which
can be recognize by APC and trigger their response (Kuwajima et al.,
2006). Here, we observed that
ALDCs incubated with free DNA
plasmid showed positive up regulation of CD40 molecule. Also, targeted
liposome alone or associated with DNA and functionalized with LPS fromBrucella ovis are capable to enhance the expression of CD40 on
the surface of DCs. This result proves that the nanovaccines represent a
promising platform that comply the idea that a performing vaccine must
addressed APC. Notably, the liposome obtained without functionalization
with LPS were not able to activate DCs. In this way, the MANα1-2MAN
targeting provides a crucial function to selectively target the
dendritic cells while addressing the LPS as adjuvant of the cell
response.
Since it is well known that cell-mediated immunity is very important for
virus clearance upon BoHV-1 and other viruses infection, a better
protection induced after challenge in animals immunized with targeted
DNA loaded liposome was observed. Animals from pCIgD-Man-L treated group
had less viral excretion with significant decrease of viral titer at 5
dpc respect to control group. Furthermore, induction of cellular immune
response was observed at 12 dpc in pCIgD-Man-L treated group with
positive response to viral-specific PBMCs proliferation. This could be
correlated with the enhancement of IgG1 isotype as an indicator of
activation of cellular immune response (Sin et al., 1999). The increased
of the lymphoproliferative effect in pCIgD-Man-L treated group shows a
better armed immune response, in which memory cells became established
and were able to recognize the viral antigen more quickly than in pCIgD
treated and control groups.
Although, the clinical score was similar for the animals vaccinated with
free DNA plasmid and those immunized with the targeted DNA loaded
liposomes, these animals presented less clinical symptoms than the
control animals during viremia.
The development of performing vaccines for BoHV-1 based on DNA selective
delivery to DC cells using new carriers including liposomes may allow
better control of virus circulation in livestock. Although this trial as
a proof of concept demonstrates that animals vaccinated with the
engineered liposomal formulation, we set up improved humoral and
cellular response levels, the impact of formulation parameters including
vaccine dose and booster/lipid ratio still need to be systematically
elucidated. Nevertheless, this study opens up perspectives for the
generation of a new generation vaccine for robust protection of the
animals against viral exposure.
To our knowledge, this is the first time that DNA based vaccine for
BoHV-1 immunization is generated with liposomes decorated with
MANα1-2MAN-PEG-DOPE as immune targeting agent and tested in cattle.
Acknowledgments
The authors wish to thank Osvaldo Zabal and Marta D´Angelo for technical
assistance in tissue culture, Pamela Angeletti for laboratory technical
assistance, Dr. Gabriel Pinto, Fátima Torales and Sandra Rivarola for
technical assistance in biotherium, Mgter. Carlos Robles for the LPS ofB. ovis , Dr. S. Srikumaran for providing mouse monoclonal
antibodies, INTA Balcarce for kindly supplying the animals, Martín
Mayoral and Walter Bagazette for the care of the bovines.