Depletion of key antigen-presenting cells including macrophages, monocytes, and/or DCs transiently occurs in animals during infection with ASFV, PRRSV, BTV, FMDV, LSDV, and CSFV (135–138). Depletion of these immune cell subsets during acute infection ultimately hinders the development of protective immunity. Furthermore, downregulation of MHC class I and/or MHC class II expression during FMDV (139), ASFV (140), and PRRSV (141) infection hampers antigen presentation and impairs T-cell responses. Therefore, measuring the number, viability, and MHC expression of these cells could act as an early indicator of vaccine success or failure ( Figures 1C-E ). However, such changes in cell counts can vary with strain and virulence, as seen in ASFV infection of pigs (142).

Natural killer (NK) cells elicit cytotoxic functions through secretion of cytotoxic molecules (perforin and granzyme) and cytokines, including interferon gamma (IFNγ) and tissue necrosis factor alpha (TNFα) (143). NK cells mediate cytotoxicity directly via cytotoxic molecules or indirectly via antibodies (see Section 5.2) ( Figure 1E ). NK responses are suppressed following infection with ASFV, FMDV, and PRRSV; however, vaccination can retain NK cell activity (35, 90, 144). Notably, protected pigs showed elevated NK cell cytotoxicity and cytokine secretion compared to non-protected pigs following PRRSV LAV vaccination ( Table 1 ) (35). Similarly, enhanced NK cell activity has also been observed in pigs immunized with a low virulent strain of ASFV upon subsequent challenge with a highly virulent strain, compared to unimmunized pigs ( Table 1 ) (90). Finally, NK cells have shown high levels of cytotoxicity in vitro in bovine and porcine cells infected with FMDV (144, 145). Collectively, the literature suggests that the functional response and frequency of NK cells could be assessed as an indicator of vaccine-mediated protection in the host for ASFV, FMDV, and PRRSV. However, the contribution of NK cells in protective immunity remains to be assessed for CSFV, LSDV, and BTV ( Table 1 ).

Viral proteins frequently inhibit the host’s Type I interferon (IFN) production during natural infection via various mechanisms (146–151). Type I IFNs, including IFNα and IFNβ, are anti-viral cytokines secreted by monocytes, macrophages, DCs, and NK cells. In vitro studies where virus-infected cells were treated with Type I IFNs have highlighted the potential for these cytokines to inhibit viral replication of BTV (152), ASFV (153), PRRSV (154), CSFV (155), and FMDV (156). Similarly, elevated Type I IFNs have been implicated in protection from homologous challenge following vaccination for BTV (13, 23) and PRRSV (157). Type I IFN adjuvanted vaccines have protected animals from clinical signs and viremia early post-vaccination in the absence of humoral immunity for FMDV (71, 72) and CSFV (116), providing strong evidence for the contribution of Type I IFN in early protective immunity. Interestingly, the contribution of Type I IFN to protection against FMDV appears to be host specific. Elevated levels of protection have been demonstrated in pigs in contrast to cattle using Adenovirus (Ad5) vectored vaccination adjuvanted with host-specific Type I IFN (71, 72). Notably, elevated levels of Type I IFNs following vaccination—either generated by the vaccine or the adjuvant—can enhance the antibody response, which can subsequently promote a long-lived adaptive immune response (116, 158). The potential role of Type I IFNs in early protection from infection with BTV, PRRSV, CSFV, and FMDV is well established ( Table 1 ). As for ASFV, IFNα may play a role in vaccine-induced recall response to challenge (87), although the definitive role of IFN and other cytokines in protection from ASFV requires further investigation in protected animals. Similarly, the role of Type I IFN in protection from LSDV has not been assessed and should be investigated further.

Clinical signs associated with systemic disease caused by ASFV (159–161), BTV (30, 162, 163), PRRSV (146, 164), LSDV (165), and CSV (166, 167) are commonly associated with a “cytokine storm” where overproduction of pro-inflammatory cytokines occurs, leading to extensive immunopathology. The “cytokine storm” has not been reported for FMDV (168). Infection of natural hosts with virulent strains of these viruses have been associated with high levels of cytokines including IL-6, TNFα, IL-1β, IL-8, IFNγ, and/or Type I IFNs (169–178). However, when pro-inflammatory cytokines are generated in a regulated manner to these viruses, they have anti-viral potential (13, 30, 73, 117, 173, 179, 180) and are essential to orchestrating the adaptive protective immune response (87, 181) ( Figure 1E ) ( Table 1 ). Measurement of cytokines, such as interleukin-2 (IL-2) or IFN gamma (IFNγ), are commonly used as a surrogate measure of CMI and vaccine immunogenicity (182–184). Most notably, protection from FMDV and CSFV has been correlated to IFNγ secretion by T cells (58, 73, 109). Similarly, IFNγ secretion by T cells has also been linked to protection from PRRSV, but does not fully predict protection (36). Furthermore, elevated levels of these cytokines post-vaccination are not always a reliable indicator of protection. Several candidate ASFV vaccines are considered immunogenic and induce a robust IFNγ response but are not protective (88, 160, 185). Owing to secretion of IFNγ by multiple immune cell subsets (various T cells, macrophages, NK cells, etc.), caution must be taken when making conclusions about the role of specific subsets in protection based on such data. Importantly, protection elicited by the innate immune response is not strain specific and, thus, may generate cross-protection to viruses with high antigenic diversity, including BTV, ASFV, and FMDV, and should be studied using vaccinated protected natural hosts (14, 87). The role of cytokines in protection from LSDV remains a large gap in knowledge and, thus, should be studied further (7–9). It is important to note that many different cytokines have a role in protective immunity for these diseases, and therefore, research should aim to investigate cytokines beyond IFNγ as CoPs (30, 37, 44, 59, 60, 118).

The most-utilized CoPs are neutralizing antibody (Nab) titers, with higher antibody titers commonly linked to increased protection ( Figures 1E, F ). Reduced viral loads and the absence of clinical disease have consistently been associated with higher Nab titers following passive immunization or vaccination against FMDV (sheep, cattle, and pigs) (62–64, 81), PRRSV (15, 32, 51, 54, 55), BTV (15, 26, 27, 31), and CSFV (107, 110, 111, 117, 120) ( Table 1 ). Sterilizing immunity is associated with high Nab titers for PRRSV (54, 186), CSFV (187), and BTV (188, 189). Notably, the longevity of the vaccine-induced Nab response can vary among different vaccines for different diseases (189, 190). While strong Nab responses to PRRSV, FMDV, and BTV can induce strong protection to homologous strains, vaccine-induced protection mediated by Nabs is often strain/genotype specific, due to high levels of antigenic diversity. However, induction of broadly reactive Nabs following active or passive immunization can confer high levels of cross-protection against different strains of PRRSV and CSFV (55, 120, 191). It is also important to note that vaccination against PRRSV, FMDV, and BTV in several instances can elicit protection in the absence of Nabs (192–197).

The role of Nabs in protection from ASFV is a topic of contention. While protection from lethal ASFV infection has been correlated to Nab titers by Silva et al. (2022) (91) following LAV vaccination, many other studies have reported that Nabs were not sufficient for protection following vaccination, for example, with subunit vaccines ( Table 1 ) (198, 199). Similarly, while Nabs can protect cattle against LSDV infection, Nabs are not a reliable indicator of protection following vaccination against LSDV as not all vaccinated animals that are fully protected against LSDV seroconvert (10, 11) ( Table 1 ). This highlights the risk of assessing Nab titers as the sole CoP in all contexts.

Antibodies bound to viral particles or viral antigens expressed on infected cells engage with Fc receptors (FcRs) on immune cells (e.g., macrophages, DCs, monocytes, and NK cells), resulting in the activation of Fc effector functions. Fc effector functions include antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC), which clear viral particles and kill infected cells to reduce disease severity ( Figure 1E ) (200, 201). Furthermore, Fc effector functions can enhance cross-protection due to the combined breadth of antibody targets recognized by neutralizing and non-Nabs (202, 203). While the mechanism of protection mediated by non-Nabs is poorly understood, phagocytosis has been shown to play a role. Phagocytosis of FMDV by macrophages was observed in mice that received FMDV non-neutralizing monoclonal antibody treatment and were protected from FMDV challenge, compared to untreated mice that succumbed to infection (65, 82, 83). ADCC has been identified as a potential CoP for ASFV. Elevated levels of ADCC were observed in surviving pigs following passive transfer of anti-ASFV immune sera and following serial immunization with attenuated ASFV compared to non-immunized susceptible pigs (99, 104, 105). However, the contribution of ASFV-specific ADCC to protection is likely vaccine dependent (204).

In rare instances, vaccine-induced antibodies may play a role in antibody-dependent enhancement (ADE) of infections where viral immune complexes are phagocytosed by immune cells and lead to a productive infection (205). ADE in livestock viral infections has been speculated for ASFV, PRRSV, and FMDV (206–208). Most literature surrounding ADE stems from in vitro assays where enhanced levels of viral replication have been detected in the presence of serum antibodies (206). Although well described for dengue virus (209), ADE remains controversial for these livestock diseases as the small number of in vivo studies reported have shown limited evidence of ADE occurring (206, 210).

Research on Fc effector functions such as CoPs has been limited or absent for ASFV, CSFV, BTV, LSDV, PRRSV, and, to a lesser extent, FMDV ( Table 1 ). Thus, there is a need to characterize the role of vaccine-induced Fc effector functions in protection using in vivo models for these livestock diseases to make meaningful conclusions.

CD4+ T cells have a key role in generating strong protective humoral immunity through the development of T cell-dependent antibody responses against BTV (211), ASFV (212), and FMDV (213) and should be investigated for CSFV, PRRSV, and LSDV ( Table 1 ). Notably, the depletion of CD4+ helper T cells in sheep enhanced clinical signs of BTV primary infection and impaired the development of Nabs, thus highlighting the importance of these cells in the development of protective antibody-mediated immunity (31). The level of T-cell dependency for generating an antibody response is likely antigen dependent (31). However, while BTV-specific CD4+ helper T-cell responses appear to be crucial in the development of a primary antibody response, CD4+ helper T cells were not activated/expanded in vitro following stimulation (180), and therefore may play a limited role in the memory response to challenge. Measurement of CD4+ T cells could provide a strong indicator of vaccine failure during vaccine development.

Both CD4+ T cells and/or CD8+ T cells can perform antiviral functions through the release of cytotoxic molecules (perforin and granzymes) and cytokines (IFNγ, TNFα, and IL-2), which trigger apoptosis of the infected cell (214). Double-positive CD4+CD8+ T cells are considered a porcine effector memory subset of CD4+ T cells and are strong inducers of cytotoxicity and secretion of IFNγ, and are a major feature in the memory/recall response to vaccination (215). Polyfunctional T-cell responses have been described as important for protective immunity and cross-protection post-vaccination, including proliferation, cytokine secretion, and cytotoxicity for BTV (14, 31), ASFV (87, 92, 93, 182), FMDV (58, 61, 66–68, 216), CSFV (112–114), and PRRSV (42, 47, 217) ( Table 1 ). Intriguingly, unlike the other viruses discussed in this review where Nabs—to homologous strains—are likely to be the primary CoP, CMI appears to be the primary CoP for ASFV ( Table 1 ) (89, 218, 219). Following vaccination against LSDV, T-cell responses increase; however, the functional role of these cells is yet to be studied (9, 11). The importance of CD8+ T cells in protection from ASFV was highlighted through depletion of CD8+ cells (T cells and NK cells) from pigs immunized against ASFV, resulting in complete loss of protection in these pigs to ASFV challenge, compared to total protection observed in pigs immunized with CD8+ cells (92). Finally, early protection elicited post-vaccination is likely to be cell mediated as observed with the CSFV C-strain LAV, which induced protective immunity in the absence of Nabs, associated with CD4+ T-cell proliferation and IFNγ production (220). While total T-cell responses are commonly assessed, antigen-specific T-cell responses have been rarely characterized against high-priority viral diseases of livestock discussed in this review, and the protective potential of other T-cell subsets remains poorly characterized; for example, γδ (gamma delta) T cells and NK T cells.

It is important to note that some subsets of T cells elicit regulatory functions (221). Known as Tregs, these cells secrete high concentrations of the immunoregulatory IL-10, and excess induction of these responses has been associated with vaccine failure for ASFV (218) and poor clinical outcomes during infection with PRRSV (222, 223). Upregulation of IL-10 following PRRSV vaccination also resulted in reduced induction of an immune response to CSFV vaccination (223). Therefore, secretion of IL-10 by Tregs may act as an indicator of vaccine failure for ASFV and PRRSV. This relationship has not been observed or well characterized following vaccination against FMDV, CSFV, BTV, or LSDV. Further research is needed to elucidate the role of Tregs in protective immunity for disease discussed in this review.

With the emergence, re-emergence, and global movement of high-priority viral diseases of mammalian livestock, understanding CoPs and their applications to biosecurity programs is critical. Identification of CoPs can provide an evidence-based scientific framework to guide next-generation vaccine development and immune surveillance and maintain biosecurity in the livestock industry. Furthermore, a comprehensive understanding of CoPs can facilitate the identification of sub-optimal vaccines or vaccine failures, thus ensuring resilience of livestock populations, promoting global trade, protecting public health, and contributing to sustainable agricultural practices. However, for many important livestock viral diseases, an in-depth understanding of CoPs is lacking, making it difficult to develop improved next-generation vaccines and implement effective vaccination programs for biosecurity. Livestock research needs to take a holistic and comprehensive approach to identifying CoPs using current and novel technologies to drive vaccine development for successful implementation of vaccines in biosecurity strategies.