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.