The immune response to pathogens is an amalgamation of the host’s innate and adaptive immunity. The innate immune response exists as a broad range of non-specific primary responses to infection, while the adaptive immune response represents acquired immunity. Part of the work of the innate immune response allows for the priming of adaptive immunity within a host. A branch of the innate immune response, thought to have a role in bridging the innate and adaptive immune responses [17, 18], is represented by the complement activation pathways which acts to recognize pathogens and then help eliminate them through proteolytic chain of reactions that lead to proinflammatory stimulation, lysis, or opsonization that “complements” the activity of the innate immune cells increasing the host’s defensive response to pathogens. There are several complement pathways that bolster the innate immune response through sequential events that lead to activation of C3 and C5 convertase; the classical pathway which is initiated by antigen-antibody recognition, the lectin pathway initiated by pathogen recognition by lectins, and the alternative pathway . Within this study, the classical complement pathway was shown to be active in the MLV vaccinated cattle. This pathway bolsters the innate and adaptive immune response and is initiated by the recognition of pathogen surfaces and /or antibody-antigen complexes formed as part of the adaptive immune response by the gene C1q. The Gene C1q is the first step in the classical complement pathway cascade. Structurally, functional C1q is composed of several genes; C1QA, C1QB, and C1QC, which contain globular domains that allow for binding to the antigen-antibody complexes. Initiation of the classical pathway through C1q leads to a bolstering of the immune response through pro-inflammatory peptides and phagocytosis of pathogens [18, 20]. The components of C1q; C1QA, C1QB and C1QC were observed to be overexpressed (FDR ≤ 0.15) within the MLV group in this study, but not the KV group. This shows a potential difference in the how each vaccine reinforces innate and adaptive immune functions. The ability of the classical complement pathway to recognize the antigen-antibody complexes allows it to act as a bridge between the innate and adaptive responses that furthers the effectiveness of the combined immune response.
The results of the transcriptomic analysis of the sub-clinical BVDV infection uncovered multiple genes and pathways related to differences in the immune response and cross-protection related to BRD vaccination. This study allowed for inroads into the immune expression that may accompany sub-clinical BVDV infections, however larger sample sizes will be needed to fully explore this phenomenon. The challenge presented by BVDV and BRD infections portends a need for identifying ways for cattle producers to find the best level of protection balanced against the cost of treatment. While the KV may be considered cheaper and safer, the MLV has the advantage of not needing a booster and appears to lead to immune system differences at the genomic level.
Increased complement activation in MLV group may bolster better cross-protective immune response
It is possible that within the MLV vaccinated cattle, the classical pathway of the complement system is being stimulated by the over-expression of C1QA, C1QB and C1QC, which make up functional C1q, to maintain primed phagocytic cells that can bridge the innate and adaptive immune response to the viral cells. This overexpression may allow for quicker pathogen recognition and immunoregulatory activation  through the complement cascade not initiated in the KV vaccinated cattle, in which C1QC was downregulated. It could also be feasible that the upregulation of the complement cascade system is a side effect of the immunosuppressive activity exerted by BVDV during infections and may be related to negative regulation by C1QC and C1QB of host macrophages and granulocytes [7, 8, 20, 21]. Cai et al.  showed that significantly upregulated C1q can be a potential biomarker of active tuberculosis infection . It may be that the slight upregulation in the samples marks a slow return to the baseline expression levels after adaptive immunity initiation at 14 dpi, which may be useful as a marker of sub-clinical infection. The MLV group may also be experiencing a better response through the added complement pathway mediated pathogen recognition and neutralization.
Overexpression of key genes related to promoting adaptive immunity were unique to the MLV vaccinated group
The immune related genes were overexpressed in the MLV treated group included the complement cascade genes; C1QA, C1QB, and C1QC, discussed previously and other immune genes such as MHC gene BOLA-DQA5, EBD, CD1d, and a cytokine sequestration gene in IL23R.
The gene IL23R may be the key to the MLV group having a better response to the BVDV challenge due to being necessary to stimulate CD4+ maturation into Th17 pro-inflammatory molecules . Because no statistically significant differential expression of the interleukin 23 gene was observed, it is possible that IL23R overexpression at 14 dpi is a remnant of the biological processes involved in Th17 production that helped drive the MLV group toward adaptive immunity by creating a more robust innate immune response. Additionally, IL23R is a key player in intestinal immune responses and has been dubbed an inflammatory bowel disease (IBD) susceptibility gene .
The gene BOLA-DQA5 is one of the major bovine MHC-II genes  and indicated that the MLV group had transitioned into adaptive immune functions despite lower antibody titers. Besides antigen presentation, the gene also takes part in Th1, 2, and 17 differentiation which may help bolster MLV efficacy in concert with some of the other genes observed within the study. The gene is also a component of the intestinal immune network for IgA production and its over expression could help limit mucosal disease prevalence in MLV vaccinated cattle.
The gene EBD is a bovine beta defensin gene involved in IL-17 signaling. In general, the beta-defensins function as antimicrobial peptides and chemo-attractants in cattle [26, 27]. However, EBD is considered an enteric beta defensin involved in intestinal immunity and may serve as an indication of the importance of long-term neutrophil potentiation in the protection against BVDV. The overexpression of this gene in intestinal tissue of cattle could possibly serve as a marker of adaptive protection unique to MLV vaccination.
The gene CD1d functions as an antigen presenting glycoprotein that helps drive innate immune functions against viral pathogens through presentation to natural killer T-cells and the MHC. The gene is considered to be MHC-like and involved in the immunoregulatory action of lymphoid cells. It is possible that the overexpression of CD1d is linked to higher mean levels of lymphocytes observed in the MLV group at 14 dpi, which may have allowed for better viral clearance by the innate immune system effectively reducing the volume of neutralizing antibodies needed by adaptive immune system. Another possibility is that CD1d overexpression led to increased antigen presentation that effectively increased the adaptive response to react earlier and led to less antibody production at later timepoints. Interestingly, studies in mice have shown viruses such as the herpes simplex virus (HSV) can inhibit CD1d antigen presentation  that could assist viral evasion of the host immune system. However, it seems that in the MLV treated group CD1d has the ability to escape the immunosuppressive effects of BVDV.
The difference in immune class potentiation between MLV (adaptive) and KV (innate) could explain the difference in anamnestic antibody response
Downey-Slinker et al.  showed evidence that the increased antibody response observed within the KV group compared to the MLV was not a reflection of protection, but rather a reflection of lower MLV titres, which may be more indicative of reduced viral replication within the MLV treated group. Downey-Slinker et al.  also suggested that it is possible that this difference is linked to more than just a humoral response. This was observable within our study as the MLV group displayed a higher number of cell-mediated immune response genes being differentially expressed in response to challenge. Although the KV group had higher antibody titres than the MLV group, it did not have many upregulated adaptive immune response genes being expressed when contrasted against the MLV group. Also observable in the MLV vs. KV contrast was that the KV group showed more innate immune gene upregulation at 14 dpi, suggesting the KV group was still actively battling the virus. In the KV treatment group one of the most upregulated genes is the LOC618416 leukocyte immunoglobulin-like receptor subfamily A member 6 gene, which is the bovine homolog for the human LILRB5 gene. These receptors are found on the surface of antigen-presenting cells. The human homolog of LOC618416 is considered to have immunoregulatory functions and may elucidate the lack of adaptive immune gene expression in the KV group. In humans these receptors can bind to MHC genes through immunoreceptor tyrosine-based inhibitory motifs that effectively inhibit signaling of adaptive immune responses [7, 11, 29].
It is possible that upregulation of LOC618416 is preventing the antigen-presenting cells in the KV group from properly signaling the MHC, leading to little or no adaptive immunity being triggered during sub-clinical BVDV infections. This could be the result of the immunosuppressive ability of BVDV. Although, the KV group has been shown to have higher antibody titers at 14 dpi for all challenge pathogens, the differential expression analysis showed that many of the innate immune function genes were still being upregulated. This provides additional evidence that the KV group had not fully transitioned away from the innate response by 14 dpi in contrast to the MLV group.
Interestingly, some of the human inhibitory leukocyte immunoglobulin-like receptors have been shown to interact with the gene CD1d , which was only seen as being upregulated in the MLV vs. KV and MLV vs. NON comparisons. It may be possible that part of the difference in vaccine efficacy is linked to upregulated leukocyte immunoglobulin-like receptors impairing the antigen presenting activity of CD1d that could possibly delay onset of adaptive immunity .
What may also contribute to the difference between MLV and KV cross-protective efficacy is the downregulation of CD101 observed in the MLV-treated group. In humans, expression of CD101 inhibits normal T-cell proliferation effecting both lymphocyte activation and IL2 pro-inflammatory signaling [12, 31]. The study provides evidence that part of the difference in vaccine efficacy of the MLV vs. KV treated groups can be represented by the differential expression of genes involved in lymphatic activity and antigen presentation to T-cells. The upregulation of CD1d, EBD, and IL23R along with the downregulation of CD101 within the MLV group appears to allow for a more efficient activation of the adaptive immunity possibly through better T-cell immunomodulation. This was observable through the expressed MHC and MHC-like genes at 14 dpi that were only seen to be upregulated in the MLV contrasts. This activity at 14 dpi within the MLV group is juxtaposed to the upregulation of genes with immune inhibition functions witnessed in the KV group.