NF-κB as an Important Factor in Optimizing Poxvirus-Based Vaccines against Viral Infections

Poxviruses are large dsDNA viruses that are regarded as good candidates for vaccine vectors. Because the members of the Poxviridae family encode numerous immunomodulatory proteins in their genomes, it is necessary to carry out certain modifications in poxviral candidates for vaccine vectors to improve the vaccine. Currently, several poxvirus-based vaccines targeted at viral infections are under development. One of the important aspects of the influence of poxviruses on the immune system is that they encode a large array of inhibitors of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which is the key element of both innate and adaptive immunity. Importantly, the NF-κB transcription factor induces the mechanisms associated with adaptive immunological memory involving the activation of effector and memory T cells upon vaccination. Since poxviruses encode various NF-κB inhibitor proteins, before the use of poxviral vaccine vectors, modifications that influence NF-κB activation and consequently affect the immunogenicity of the vaccine should be carried out. This review focuses on NF-κB as an essential factor in the optimization of poxviral vaccines against viral infections.


Introduction
Poxviridae is a family of dsDNA viruses. It is divided into two subfamilies: Chordopoxvirinae, the viruses of vertebrates, and Entomopoxvirinae, the viruses of insects. The Chordopoxvirinae subfamily includes 18 [1]. Poxviruses are represented by numerous human and animal pathogens. Among them, variola virus (VARV) orthopoxvirus, a human pathogen, is the causative agent of smallpox, a disease that had caused over 300 million deaths worldwide by the late 1970s before the global smallpox eradication program was completed. In the global smallpox eradication program, vaccinia virus (VACV), a zoonotic pathogen belonging to the Orthopoxvirus genus, was used [2,3]. Other members of the Poxviridae family, such as orf virus (ORFV) and goatpoxvirus (GTPV), which represent the Parapoxvirus and Capripoxvirus genera, respectively, may also serve as vaccines and are described in this review.
With the exception of parapoxviruses, poxvirus virions have a brick shape. The virions of parapoxviruses are cocoon-shaped. The virions of parapoxvirus and other members of the Poxviridae family have dimensions of 260 × 160 nm and 350 × 250 nm, respectively [4,5]. Depending on the number of membranes surrounding the virion, two infectious forms of poxviruses are observed. Mature virus (MV), which contains a tubular nucleocapsid surrounded by a biconcave core wall and proteinaceous lateral bodies, is enclosed by a single proteolipid membrane bilayer. In turn, extracellular One of the advantages that make poxviruses good vaccine vectors is that the cytoplasmic replication cycle of these vectors eliminates the risk of integration into the host genome and persistence within the host. Importantly, poxviral vaccines are easy to store, especially when freeze-dried. The thermostability of these vaccines can also be ensured by using sugar-glass technology. Additionally, the cost of poxviral vaccines is low and their administration is needle-free [12][13][14].
Although poxviruses are regarded as promising vaccine tools, certain challenges limit the design of poxviral vaccines. When using VACV and other poxvirus-based vaccines, it is desirable to achieve enhanced immunogenicity and/or virus attenuation. This is particularly important for improving the safety profile of the vaccine. Due to the abundance of immunomodulatory genes and cellular targets of the poxviruses, which remain unrevealed, there are still many opportunities for virus modification in order to improve the vaccine efficacy by inducing stronger immunological memory. In addition, reduction of dosage and administration regimes would be beneficial as well [7]. One of the strategies employing poxvirus vaccines is prime-boost vaccination, in which poxviral vectors that enhance T cell responses as boosters are combined with other vectors. On the other hand, when used as primers with protein and adjuvant, poxviruses improve the B cell responses. Furthermore, the optimization of the antigen expression is based on mosaic immunogen sequences [13].
When modifying poxviral vaccine vectors, the immunomodulatory genes should be removed in order to enhance immunogenicity [13]. Poxviruses, which express a wide range of host response modifiers influencing cellular signaling pathways involved in immunity and inflammation, share multiple mechanisms of host evasion. Since Poxviridae family members encode a number of cellular signaling inhibitors, this review describes the influence of poxvirus-based vaccines on the NF-κB transcription factor [9]. Several data indicate the importance of NF-κB in the development of poxvirus-derived vaccines. These are based on both veterinary and human antiviral vaccines. Therefore, in this review, we focus on the benefits of certain modifications of poxviral vaccine vectors and how these modifications can affect NF-κB signaling in different cells and hosts and the possible mechanisms of immune response modulation that can be shared by individual poxvirus genera. We describe the VACV-, ORFV-, and GTPV-based vaccines, which can be used against viral infections.

Poxviral Vectors for Vaccine Applications
The vaccine used in the global smallpox eradication program was based on several strains of VACV. For instance, in the United States, the New York City Board of Health (known as NYCBH) and Lister strains were used for vaccination against smallpox, while in Europe, Lister, Bern, Paris, and Copenhagen (called VACV-COP) strains were applied. The first-generation antismallpox vaccines were propagated in the skin of calf and other animals, while the second-generation VACV-based vaccines were grown in tissue culture and chicken embryos instead of live animals. Unfortunately, the vaccines generated in cell cultures are not sufficiently safe. Therefore, the use of second-generation antismallpox vaccines is limited [3]. Recently, it has been shown that chicken embryonic stem cells (cESCs) may serve as an alternative source for the propagation of poxvirus vaccine vectors. The idea behind the use of cESCs rather than mammalian ESCs is linked to ethical issues and safety concerns, the most important of which is that cESCs lack transforming oncogenes or adventitious agents [26].
Despite the eradication of smallpox, the risk of reoccurring VARV infections remains to be eliminated because of the bioterrorist threat or the possible de novo synthesis of the virus. Therefore, the pathogenesis of orthopoxvirus diseases is still of interest to researchers [27,28]. Although VACV-based vaccines used in the global smallpox eradication program were effective, some adverse effects of vaccination, including postvaccinal encephalitis, generalized vaccinia, progressive vaccinia, and eczema vaccination, were observed in immunocompromised individuals and patients with skin conditions. To overcome these, modified VACV Ankara (MVA)-Bavarian Nordic (MVA-BN), a third-generation attenuated antismallpox vaccine-has been introduced. This vaccine is approved in Canada (Imvamune) and the European Union (Imvanex) [29,30].
The currently used VACV-based vaccines, which are nonreplicating and attenuated, such as MVA-BN-vectored encephalitic alphavirus vaccine, target the biothreat viruses. Another VACV-derived vaccine, RABORAL V-RG, an antirabies vaccine expressing the rabies virus (RABV) glycoprotein gene (V-RG), has been used in Europe and North America to vaccinate foxes and raccoons [30]. Additionally, VACV-based vaccine candidates have been demonstrated to protect against emerging viral diseases, such as chikungunya virus (CHIKV) disease [41,42] and yellow fever [43] in preclinical animal models. The Sementis Copenhagen Vector (SCV) is a new multiplication-defective VACV-COP-derived vaccine vector with targeted deletion of D13L gene encoding D13 protein that is essential for viral assembly. This new vector has recently been successfully tested in nonhuman primates as a vaccine against Zika and chikungunya [41].

NF-κB Signaling
One of the key factors involved in the proper induction of antiviral immunity is NF-κB. It constitutes a family of dimeric transcription factors, which regulate the expression of numerous genes involved in the cell cycle, apoptosis, and immunity. The NF-κB family consists of five proteins: RelA/p65, RelB, c-Rel, NFκB1 p105/p50, and NFκB2 p100/p52. The NF-κB dimer that is most commonly detected in the cytoplasm of unstimulated cells is composed of RelA and p50 subunits [50]. The RelA/p50 heterodimer remains in the cytoplasm due to the activity of IκBα, which masks the nuclear localization sequences (NLSs) of NF-κB [51]. The classical NF-κB signaling pathway is induced by proinflammatory cytokines such as interleukin-1β (IL-1β), IL-18, and TNF-α and various ligands of pattern recognition receptors (PRRs), which are represented by retinoic acid-inducible gene-I (RIG-I) and Toll-like receptors (TLRs). In the NF-κB signaling cascade, the cellular receptors cooperate with adapter molecules and induce the cellular pathways that activate the transcriptionally active dimers [52]. Upon the stimulation of NF-κB signaling, transforming growth factor (TGF)-β-activated kinase 1 (TAK1) activates IKK, the IKKβ subunit of which triggers IκBα phosphorylation at Ser32 and Ser36. This event results in the recognition of IκBα by the E3 ubiquitin ligase complex composed of β-transducin repeat-containing proteins: S-phase kinase-associated protein 1 (Skp1)-Cullin 1-F-box (SCF β−TrCP ). Conjugation of phosphorylated IκBα with K48-linked polyubiquitin chains of Lys 48 of ubiquitin by SCF β−TrCP results in 26S proteasome-mediated IκBα degradation and the release of RelA/p50 dimers. These dimers translocate to the nucleus, where they bind DNA and initiate the transcription of target genes. E3 ubiquitin ligase complex is also involved in p105 proteasomal processing to p50 [50,53,54] ( Figure 1). On the other hand, the noncanonical NF-κB signaling triggered by the members of the TNF superfamily leads to the activation of NF-κB-inducing kinase (NIK), which then activates IKKα. IKKα, in turn, phosphorylates the C-terminal portion of p100 precursor protein, which retains RelB in the cytoplasm due to its IκB activity. Following the phosphorylation of p100 at Ser866 and Ser870, IκB-like C-terminal portions of this protein are ubiquitinated, leading to the generation of a p52 active NF-κB subunit. RelB/p52 dimers translocate to the nucleus and initiate the transcription of target genes [55]. In general, the canonical NF-κB signaling is responsible for the regulation of innate immunity [56], whereas the noncanonical NF-κB activation pathway regulates the adaptive immune responses. However, there exist regulatory mechanisms for these two signaling pathways as well as for the crosstalk between them [55,57]. The modulation of NF-κB signaling is attributed to viral pathogens, one excellent example of which is the viruses belonging to the Poxviridae family encoding multiple immunomodulatory proteins; these proteins affect the components of NF-κB signaling and therefore disrupt the antiviral innate response [52,58]. Selected NF-κB inhibitors of VACV, ORFV, and GTPV, which may be relevant to the efficacy of poxviral vaccines, are shown in Figure 1.

Vaccinia Virus
VACV is a pathogen whose origin or natural host has not been identified so far. It was believed that VACV infections occur due to the spread of vaccine strains into new wild hosts. However, it is now obvious that VACV is transmitted via peridomestic rodents, which infect wild animals and cows. In humans, VACV can be transmitted via infected animals and is observed in milkers in areas where the virus circulates, especially in Asian and South American countries, such as Brazil. Furthermore, VACV infection of humans occurs via their direct contact with the crusts containing viral particles, which results in the formation of skin focal lesions on the hands and forearms [18,37,59]. After a few days, the focal lesions form pustules leading to edema and erythema. Ulcerated and necrotic lesions appear after a maximum of 12 days of VACV infection, following which crusts develop. In 4 weeks, the lesions disappear, but local lymphadenopathy can be observed for 20 days. Alternatively, a systemic infection manifested by fever, headache, and muscle ache develops immediately after the appearance of lesions [60].

Modified VACV Ankara
VACV is regarded as a universal vaccine carrier. However, the use of replicating VACV as an antismallpox vaccine has led to severe adverse effects in individuals with an immunocompromised immune system or skin disorders. Currently, MVA obtained by 570 passages of chorioallantois VACV Ankara (CVA) strain in chicken embryo fibroblasts remains an excellent alternative to traditional antismallpox vaccines [61]. MVA does not replicate in human cells but displays good immunogenicity as well as a good safety profile in vivo. In addition, MVA can be successfully used in immunocompromised humans [12,15,32,[62][63][64][65][66]. The ongoing clinical trials on MVA-vectored vaccines targeted at viral diseases are shown in Table 1.
MVA is a good candidate for an effective and safe vaccine vector. Nevertheless, its immunogenicity can be enhanced to improve the efficacy of the vaccine, for which the introduction of immune-stimulating genes into MVA and reinsertion of certain VACV genes to obtain a replication-competent virus are considered beneficial [67].
Despite the expression of A46 (Toll/IL-1-receptor signaling interference protein), B16 (IL-1-binding protein (IL-1BP)), and K7 (B cell lymphoma 2 (Bcl-2)-like protein), which inhibit NF-κB signaling, MVA stimulates NF-κB [61,68,69]. Studies on NF-κB signaling in MVA-infected cells have shown that early MVA protein expression in human 293T fibroblasts activates the phosphorylation of extracellular signal-regulated kinase 2 (ERK2), which, in turn, mediates the activation of NF-κB [70]. Further research revealed that MVA triggers NF-κB activation via VACV growth factor (VGF), which interacts with the epidermal growth factor receptor (EGFR). Moreover, in 293T cells and Hacat keratinocytes infected by MVA deprived of an early C11R gene encoding VGF, reduced activation of ERK2 and NF-κB was observed. Since keratinocytes are the immediate target of the vaccine, the prosurvival role of NF-κB would be beneficial to stimulate cells for a long duration for optimal induction of immune response before cell lysis occurs [71].
Other studies on MVA revealed that, during the early phase of viral replication in human embryonic kidney 293 cells that were transformed with large T antigen (HEK 293T), IκBα degradation occurs before the initiation of viral replication. It has been shown that in Chinese hamster ovary (CHO), HEK 293T, and rabbit kidney 13 (RK13) cells, IκBα degradation can be inhibited by the expression of CP77, a CPXV Brighton Red (CPXV-BR) early host-range gene [72].
CP77-encoded protein is one of the ankyrin (ANK) repeat proteins [73]. In general, ANK repeats, consisting of 30-34 amino acid residues, are involved in protein-protein, protein-sugar, or protein-lipid interactions. ANK repeat proteins take part in cellular signaling, vesicular trafficking, cell cycle control, and inflammation, are responsible for cytoskeleton integrity, and regulate transcription. They are present in both eukaryotic and prokaryotic cells, such as intracellular bacteria [74]. ANK repeat proteins are not commonly expressed by viruses. However, within the Chordopoxvirinae subfamily of poxviruses, only three species of different genera lack ANK proteins. In poxviruses, these proteins are composed of multiple ANK motifs, which start from the N-terminus. The ANK motifs are followed by non-ANK linker sequence. In turn, the C-terminus has the homolog of cellular F-box sequence. The cellular F-box sequence interacts with ubiquitin ligase E3 complexes, thus allowing ubiquitination and subsequently 26S proteasome-mediated protein degradation. ANK proteins of poxviruses belonging to Avipoxvirus, Parapoxvirus, Orthopoxvirus, and the Leporipoxvirus supergroup genera may either bind to Skp1 or interact with cellular proteins, thus acting as inhibitors of cellular signaling, such as NF-κB [75]. Some poxviral ANK repeat proteins, including VACV K1 [76] and myxoma virus (MYXV) M150 [77], may act as nuclear NF-κB inhibitors.
Furthermore, certain poxviral ANK repeat proteins, in which the F-box domain is absent, serve as host-range proteins [75]. These are represented by VACV K1 protein encoded by K1L gene [78]. It has been shown that MVA expressing the VACV Western Reserve (VACV-WR) gene K1L, which prevents IκBα degradation [79] and p65 acetylation [76], potentially inhibits IκBα degradation in HEK 293T cells. Importantly, in mouse embryonic fibroblasts (MEFs), human or mouse dsRNA-activated protein kinase R (PKR) is crucial for IκBα degradation. It can be assumed that the induction of PKR by MVA may stimulate immune response and impair virus replication, which is critical for the safety and efficacy of the vaccine [72].
Furthermore, stimulation of immune response is necessary for the prolonged presentation of antigens and delayed viral clearance. The insertion of a VACV-CVA 5.2-kb region containing apoptosis inhibitor and ANK repeat protein gene M1L [80] and NF-κB inhibitors encoded by M2L (mitogen-activated protein kinase kinase (MEK)/ERK and NF-κB inhibitor) and K1L (PKR and NF-κB inhibitor) genes into MVA decreases both apoptosis and virus-mediated NF-κB activation in antigen-presenting cells (APCs) in vivo. Moreover, VACV-specific CD8 + T cell response is diminished in vivo after treatment with MVA/5.2 kb compared to MVA. These results contradict the findings observed in vitro, in which MVA/5.2 kb displays an immunostimulatory effect [67].

New York VACV
Another VACV-based vaccine vector, the New York VACV strain (NYVAC), derived from the VACV-COP strain, is deprived of 18 ORFs, including the host-range K1L gene, encoding an IFN antagonist protein and NF-κB inhibitor. It has been shown that both MVA and NYVAC-infected HeLa cells display enhanced expression of NF-κB protein, degradation of IκBα, and secretion of IL-6. Furthermore, NYVAC upregulates certain NF-κB-responsive genes, including activating transcription factor 3 (ATF3). It can be concluded that ATF3 may act pro-apoptotically during NYVAC infection. However, the ectopic expression of VACV-WR K1L gene in NYVAC-infected cells induced apoptosis but inhibited NF-κB. This indicates that K1 does not prevent apoptosis in NYVAC-infected cells. Both induction of apoptosis and inhibition of NF-κB may not be favorable for the NYVAC replication cycle; however, apoptosis occurs at the late stage of the viral lifecycle when the replication is completed. These events are important for the generation of immune response and vector clearance [81]. Interestingly, although both MVA and NYVAC stimulate NF-κB, A52R gene is present in NYVAC, but not in MVA. The absence of A52, a Bcl-2-like protein, which binds IL-1 receptor (IL-1R)-associated kinase 2 (IRAK2) and thus inhibits NF-κB, is likely to upregulate the TLR3 pathway in MVA-infected cells. Since A52 binds to IRAK2 and TNFR-associated factor 6 (TRAF6), which are important for TLR signaling, upregulation of TRAF6 and downregulation of IL-1α and IL-1β were observed in MVA-infected immature human monocyte-derived dendritic cells (MDDCs), but not in NYVAC-infected cells [82].
NYVAC has been proposed as a candidate for a human immunodeficiency virus (HIV) vaccine. NYVAC-C ∆A52R ∆B15R ∆K7R, and NYVAC deletion mutant lacking A52R, B15R (encoding B14 protein that binds IKKβ), and K7R (encoding TRAF6 and IRAK2-binding protein) has been used to express HIV type 1 (HIV-1) envelope (Env) glycoprotein 120 (gp120) and GPN (Gag-Pol-Nef) clade C antigens. In mice, NYVAC-C ∆A52R ∆B15R ∆K7R induced the activation of chemokines/cytokines and migration of Nα and Nβ neutrophils to the infection site. This effect was accompanied by an increase in T cell response toward HIV antigens. The activation of virus-specific CD8 + T cells was triggered by Nβ neutrophils displaying an APC-like phenotype [83]. Further analyses of mice models infected with NYVAC mutants (NYVAC-C ∆A52R, NYVAC-C ∆A52R ∆K7R, or NYVAC-C ∆A52R ∆B15R) showed that the infection increased the number of CD11c + major histocompatibility complex (MHCII) + -positive DCs in mice. Deletion of A52R gene influenced the migration of DCs, whereas double-gene deletion affected the migration of both DCs and neutrophils. Finally, the deletion of all A52R, B15R, and K7R genes not only enhanced the migration of DCs, neutrophils, and natural killer (NK) cells but also influenced chemokine release. In addition, NYVAC-C ∆A52R ∆B15R, NYVAC-C ∆A52R ∆K7R, and NYVAC-C ∆3 (triple-deletion) mutants induced CTLs. Among the double-deletion mutants, NYVAC-C ∆A52R ∆B15R not only induced a strong T CD8 + response but was also effective in the induction of IgG. These studies demonstrate that double or triple deletion of NF-κB inhibitors from NYVAC enhances both T cell-specific and humoral anti-HIV responses. The induction of Gagand Pol-specific CD8 + T lymphocytes by NYVAC mutants showed that NYVAC-based vectors are promising anti-HIV vaccine candidates [84].

VACV Western Reserve
Another VACV strain that can be used as a vaccine vector is VACV-WR. When modifying the VACV-WR genome, single-gene deletions are more beneficial than the deletions of multiple genes, which may decrease the immunogenicity of the vaccine [85]. One of the candidate genes that can be deleted from VACV-WR is N1L, encoding a Bcl-like inhibitor of NF-κB, which prevents NF-κB activation by proinflammatory cytokines including TNF-α or IL-1β [86]. Studies on intradermal murine model infection with VACV-WR devoid of N1L gene have shown that NF-κB is essential for CD8 + T cell memory and, consequently, for the efficacy of vaccines. N1 is an early VACV protein that inhibits apoptosis. Therefore, its mutation or deletion reduces the virulence of VACV and, at the same time, enhances CD8 + T cell response, which is desirable for antiviral protection induced by the vaccine [87]. VACV-WR vaccine can also be modified by the deletion of K1L NF-κB inhibitor. In mice models, K1-deficient virus induced VACV-specific CD8 + T cell response and prevented lethal VACV infections, despite silencing the innate immune response. Above all, at day one postinfection, the deletion mutant did not induce the expression of NF-κB-regulated genes, such as Nfkbia and Tnf. However, Ifna4, Il7, and Nfkb2, which are only partially controlled by NF-κB, were downregulated [88].
Recently, the importance of VACV-WR BTB-BACK-Kelch (BBK)-like protein, A55, in NF-κB modulation has been described. A55 is an NF-κB inhibitor that disturbs the p65-importin interaction and thus prevents the transcription of NF-κB-regulated genes and impairs inflammatory response. Especially, NF-κB-regulated cytokines and inflammation influence the proliferation and development of effector and memory T cells. As expected, the deletion of the A55R gene from VACV-WR resulted in the enhancement of CD8 + T cell memory, and the vaccine displayed increased immunogenicity and protected the mice challenged with VACV intranasally [89].

Orf Virus
ORFV, a virus belonging to the Parapoxvirus genus, is the causative agent of orf disease-highly contagious ecthyma. In humans, orf is an enzootic and self-limiting disease manifested as pustular dermatitis, which spontaneously resolves within 3 to 6 weeks. The ORFV infections caused in humans are frequently observed in Asia and Africa. In sheep and goats, these infections cause scabby mouth disease, which is characterized by high morbidity in infected sheep worldwide. ORFV may also infect cats, reindeers, camels, serows, and musk oxen. Mortality due to ORFV infections is rarely associated with secondary infections and aspiration pneumonia. In general, orf disease is a threat to kids and lambs and may cause farms to suffer economic losses [90][91][92][93][94].
The use of ORFV as a vaccine vector may constitute a novel strategy to vaccinate both permissive and nonpermissive hosts against orf, which is an alternative to the current attenuated vaccines that are inefficient or insufficiently safe. The immunomodulatory properties of ORFV, as well as its ability to replicate in various hosts, make it a good vaccine candidate. Moreover, preclinical studies have confirmed these properties of inactivated ORFV [91]. Due to the fact that ORFV does not spread systemically or neutralize antibody production, it can be successfully used as a vaccine vector for repeat immunizations [95]. The highly attenuated anticontagious ecthyma vaccine strain D1701 derived from ORFV protects sheep for 4-6 months [96,97]. When adapted to Vero cells, D1701-V can be used to deliver target genes into the vegf-e gene for the construction of a vaccine against pseudorabies virus (PRV), the causative agent of Aujeszky's disease [98][99][100]. D1701-V-VP1 expressing capsid protein VP1 has been used to immunize rabbits against rabbit hemorrhagic disease virus (RHDV) [101]. Another recombinant D1701-V vector, D1701-V-RabG expressing the RABV glycoprotein, has been designed as a new antirabies vaccine for companion animals and tested on murine, dog, and cat models [97]. D1701-V-HAh5n expressing H5 hemagglutinin has also been proposed as a vaccine against avian influenza virus H5N1 and tested on mice models [102]. In addition, DNA vaccines expressing ORFV011 EEV envelope phospholipase and ORFV059 immunodominant envelope antigen F1L protein have shown enhanced immunogenicity and triggered lasting immunity in mouse models [103].

ORFV-IA82
Thus far, several ORFV-encoded proteins capable of inhibiting NF-κB signaling have been identified, including'ORFV024 [104], ORFV002 [105,106], ORFV121 [107], ORFV073 [108], and ORFV119 [109]. Recently, ORFV020, a dsRNA-binding IFN resistance protein, displaying dsRNA adenosine deaminase activity, has been described. ORFV020 is a counterpart of VACV-WR E3, which inhibits the activation of PKR and NF-κB. Moreover, it belongs to viral IFN (VIR) resistance proteins that inhibit IFN-mediated antiviral response. Therefore, ORFV expressing ORFV020 is resistant to the activity of IFN type I and type II. Considering the conservative nature of ORFV isolates, the deletion or mutation of the E3L counterpart may be ideal for vaccine construction [110].
ORFV strain ORFV-IA82 is used as a vaccine against porcine epidemic diarrhea. The ORFV-PEDV-S virus expressing spike (S) proteins of porcine epidemic diarrhea virus (PEDV) was constructed using the ORFV121 locus insertion site. This site encodes a unique parapoxviral NF-κB inhibitor, which blocks the phosphorylation of p65 and its nuclear translocation [107]. Immunization of pigs with ORFV-PEDV-S induced the production of neutralizing antibodies and PEDV-specific serum IgA and IgG. Importantly, ORFV-PEDV-S ensured protection from the clinical outcomes of the infection. Reduced virus shedding was also found upon the immunization of infected animals [111]. Furthermore, ORFV-PEDV-S vaccine has been shown to induce passive immunity in newborn piglets [112].
In addition, ORFV-IA82 can be used as an antirabies vaccine. The gene ORFV024 encoding a unique parapoxviral inhibitor of IKK kinase and IκBα degradation [89] was used as an insertion site for the RABV glycoprotein (G) gene. Similarly, ORFV121 gene encoding NF-κB inhibitor has been used as an insertion site for G-encoding gene. Immunization of pigs and cattle with ORFV ∆024 RABV-G or ORFV ∆121 RABV-G resulted in the induction of neutralizing antibodies. Of these, the ORFV∆121 mutant was more immunogenic [113].

Goatpox Virus
GTPV, a member of the Capripoxvirus genus, is a sheep pathogen and the causative agent of the goatpox disease. Goatpox is transmitted via aerosols and insects and causes systemic infections in goats and sheep, which manifest as fever, enlargement of lymph nodes and skin, and respiratory and gastrointestinal lesions. Goatpox disease is also a source of economic loss to domestic ruminant farms. In general, GTPV is an economically important capripoxvirus in central Asia, North Africa, the Middle East, and India [114][115][116]. At present, only attenuated vaccines are available for GTPV and other capripoxviruses [117]. The capripoxvirus-based vaccines, which are obtained by serial passages, confer protective immunity for 1 year after vaccination. Capripoxviruses can also be used as vectors for vaccines against diseases caused by ruminant pathogens, such as bluetongue, Rift Valley fever, peste des petits ruminants, or rinderpest. Certain GTPV strains, such as Isiolo and Kedong, which infect goats, sheep, and cattle, are used as a universal vaccine against capripox diseases [46]. Gorgan strain-based vaccines protect cattle against lumpy skin disease. Caprivac (Jordan Bio-Industries Centre, JOVAC) is one of the vaccines used against goatpox in cattle in the Middle East [118].

GTPV-AV41
The existing GTPV vaccine, GTPV-AV41, contains an attenuated strain obtained by passages of GTPV-AV40 strain in the testis cells of goats and sheep. Unfortunately, it may cause generalized skin lesions and miscarriages in vaccinated animals, and thus, its use hinders distinguishing between vaccinated and infected ones [119]. Therefore, modifications of the virus are needed to improve the vaccine. It is worth noticing that the inactivation of NF-κB-related genes has been observed among capripoxvirus vaccine strains. For instance, in the GTPV Gorgan vaccine, a 1.6-kbp deletion led to the inactivation of GTPV_144 and GTPV_145 genes. GTPV_144 is a counterpart of VACV-COP A55R, encoding Kelch repeat and BTB domain-containing protein 1, while GTPV_145 is related to VACV-COP B4R, encoding ANK repeat protein [117]. Mutations in these two genes are common in capripoxviruses and are typical for vaccine strains. Since A55 inhibits CD8 + T cell memory, the deletion of A55R gene or its GTPV counterpart may improve the immunogenicity of the vaccine [89]. GTPV_145 encodes an ANK repeat protein, a counterpart of B4 VACV-COP and EVM154 proteasome inhibitor of ectromelia virus, which interacts with Skp1 and conjugated ubiquitin and subsequently inhibits IκBα degradation. It is believed that that EVM154 may be involved in virus spread and its depletion may cause attenuation of the virus [120,121].
One of the insertion site candidates for GTPV-based vaccines is the 135 ORF containing an early gene, which is not essential for the viral replication in vitro and in vivo. The 135 gene encodes an 18-kDa protein, which inhibits NF-κB and apoptosis. The GTPV135 protein is a counterpart of a Bcl-like VACV-WR N1 protein. The 135 gene is a host innate immune response inhibitor and may therefore serve as an insertion site for live attenuated dual vaccines instead of the tk locus insertion site. Interestingly, the GTPV AV41 vaccine strain expressing the hemagglutinin protein of peste des petis ruminants virus (PPRV), whose gene was inserted in the ORF135 insertion site, displayed a stronger antibody neutralization response than the strain with a tk insertion site [122]. To improve GTPV-AV41 and prevent the side effects of the vaccine, it may be necessary to perform modifications based on the deletion of the nonessential gene. For instance, deletion of the viral tk gene and ORF8-18 may be beneficial for the vaccine. Among ORF8-18 homologs of VACV, the following NF-κB inhibitors can be found: ORF12, which encodes ANK repeat protein and a counterpart of B4, and ORF15, encoding a homolog of VACV IL-18BP, C12. ORF16, in turn, encodes an EGF-liked growth factor, a C11 VACV counterpart, which may activate NF-κB. In vaccinated animals, the attenuated vaccine GTPV-TK-ORF vector allowed maintaining immunogenicity and increased safety compared to wild-type GTPV-AV41. The vaccine also induced the production of neutralizing antibodies and GTPV-specific antibodies, as well as the release of IFN-γ in goats. Hence, removal of nonessential genes that are linked to apoptosis inhibition and immune modulation is considered as a factor that may improve the efficacy of the vaccine [123].

Conclusions
Generation of effective immune response and immunological memory, as well as safety, is the main concern in vaccine development. When employing virus-based vaccines, it is necessary to ensure both the complete replication cycle of the virus and proper induction of immunological memory for determining the vaccine efficiency. The loss of viral immunomodulatory proteins may affect these parameters, thus influencing efficiency. Since poxviruses modulate the activation of immune cells by affecting the NF-κB-mediated apoptosis regulation, inflammation, and immunological memory, discovering new mechanisms of NF-κB inhibition and cellular targets of poxviruses may help modify vaccine candidates to improve the efficacy of poxvirus-based vaccines and the immunological memory generated by them.
Author Contributions: J.S. contributed to conceptualization and writing (original draft preparation, review, and editing). L.S.-D. contributed to conceptualization and writing (figure preparation, review, and editing). All authors have read and agree to the published version of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.