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BoHV-4-Based Vector Single Heterologous Antigen Delivery Protects STAT1(-/-) Mice from Monkeypoxvirus Lethal Challenge

Monkeypox virus (MPXV) is the etiological agent of human (MPX). It is an emerging orthopoxvirus zoonosis in the tropical rain forest of Africa and is endemic in the Congo-basin and sporadic in West Africa; it remains a tropical neglected disease of persons in impoverished rural areas. Interaction of the human population with wildlife increases human infection with MPX virus (MPXV), and infection from human to human is possible. Smallpox vaccination provides good cross-protection against MPX; however, the vaccination campaign ended in Africa in 1980, meaning that a large proportion of the population is currently unprotected against MPXV infection. Disease control hinges on deterring zoonotic exposure to the virus and, barring that, interrupting person-to-person spread. However, there are no FDA-approved therapies against MPX, and current vaccines are limited due to safety concerns. For this reason, new studies on pathogenesis, prophylaxis and therapeutics are still of great interest, not only for the scientific community but also for the governments concerned that MPXV could be used as a bioterror agent. In the present study, a new vaccination strategy approach based on three recombinant bovine herpesvirus 4 (BoHV-4) vectors, each expressing different MPXV glycoproteins, A29L, M1R and B6R were investigated in terms of protection from a lethal MPXV challenge in STAT1 knockout mice. BoHV-4-A-CMV-A29LgD106ΔTK, BoHV-4-A-EF1α-M1RgD106ΔTK and BoHV-4-A-EF1α-B6RgD106ΔTK were successfully constructed by recombineering, and their capacity to express their transgene was demonstrated. A small challenge study was performed, and all three recombinant BoHV-4 appeared safe (no weight-loss or obvious adverse events) following intraperitoneal administration. Further, BoHV-4-A-EF1α-M1RgD106ΔTK alone or in combination with BoHV-4-A-CMV-A29LgD106ΔTK and BoHV-4-A-EF1α-B6RgD106ΔTK, was shown to be able to protect, 100% alone and 80% in combination, STAT1(-/-) mice against mortality and morbidity. This work demonstrated the efficacy of BoHV-4 based vectors and the use of BoHV-4 as a vaccine-vector platform.


Introduction
Monkeypox virus (MPXV) is an orthopoxvirus with a broad-host range capable of infecting many animal species [1]. In humans, MPXV causes a disease very similar to the closely related variola virus, the etiological agent of smallpox: a rash (a 2-4 week period where macules develop and form papules, vesicles and pustules) which is preceded by a 10-14 day incubation, followed by a 1-3 day interval characterized by a prodromal fever, malaise and severe lymphadenopathy of the neck, inguinal and axillary regions [2][3][4]. The more virulent strains of MPXV (from the Congo Basin region) can induce mortality rates of~10% [5][6][7][8]. The main clinical difference between human MPX and smallpox is the presentation of lymphadenopathy in the former [9,10]. MPXV is endemic to Central and West Africa with increasing numbers of human infections being reported. In 2003, the transmissibility of MPXV became acutely obvious when an outbreak occurred in the United States and MPXV was transmitted to humans from prairie dogs [11,12]. The global vaccination campaign that eradicated smallpox utilized vaccines that are also efficacious against MPXV and was successful because there is no animal reservoir for variola [13]. Indeed, prior to the eradication of smallpox, the existence of MPXV was unknown and it is likely that many cases of human MPX were reported as smallpox. Cases of human MPX are increasing in Africa, this may be due to several factors: 1) increasing interactions between humans and infected animals through environmental degradation; 2) the cessation of routine vaccination against smallpox; 3) an increase in human susceptibility to the virus; 4) an increase in animal-human and human-human transmissibility of the virus; and 5) adaption of the virus to new host species that co-exist in, or close to, human geographical regions [2,[14][15][16][17][18]. The high mortality rate and morbidity rate of MPXV in humans makes it an important emerging disease for study. To date, no antiviral has been FDA-approved for the treatment of human MPX. Vaccines against smallpox have demonstrated good protection against MPXV in animal models and anecdotally in humans [1]; however, most first-and second-generation smallpox vaccines are associated with at least some level of morbidity and a large proportion of the population are contraindicated to vaccination [19]. Third-generation vaccines, such as MVA, have been shown to be safer in non-human primates but require 2

Constructs generation
The A29L, M1R and B6R ORFs, were amplified from Monkeypox DNA (strain V79-I-005; Accession: HQ857562.1) using primer pairs listed in Table 1. The sense primer contains at its 5 0 -end the NheI restriction site along with the Kozak's sequence preceding the ATG, for a better translation initiation, whereas the antisense primer contains at its 5 0 -end a SalI restriction site for the in-frame cloning of the ORF with a gD106 tag peptide present in pCMV-IgK-VP2-gD 106 plasmid vector. pCMV-IgK-VP2-gD 106 has the human cytomegalovirus Immediate Early gene (CMV) promoter, the Immunoglobulin kappa light chain leader sequence (IgK), the Bluetongue virus VP2 ORF, the gD 106 epitope of Bovine herpesvirus 1 glycoprotein D, successfully used as a tag during the cloning, and the bovine growth hormone polyadenylation site [37].
The 3 ORFs were cut with NheI/SalI and cloned into pCMV-IgK-VP2-gD 106 cut with the same enzymes. This strategy allowed the substitution of the IgK-VP2 sequence with those of the 3 ORFs to generate pCMV-A29LgD 106 , pCMV-M1RgD 106 and pCMV-B6RgD 106 .
To generate pTK-CMV-A29LgD 106 -TK, pCMV-A29LgD 106 was cut with BamHI, treated with T4 DNA polymerase for blunt ending and cut with NheI; A29LgD 106 (442 bp) was excised from the pCMV-A29LgD 106 and inserted into the shuttle vector pINT2EGFP [25] cut with NheI/SmaI to replace GFP ORF with the chimeric protein. pINT2EGFP contains two BoHV-4 Thymidine kinase gene homology regions flanking the Green Fluorescent Protein (GFP) expression cassette driven by the CMV promoter, cutting with NheI/SmaI the GFP ORF was replaced with that of A29LgD 106 .

Transient transfection
Confluent HEK293T cells were seeded into 6 well plates (3x10 5 cells/well) and incubated at 37°C with 5% CO 2 ; when the cells were sub-confluent the culture medium was removed and the cells were transfected with pTK-CMV-A29LgD 106 -TK, pTK-EF1α-M1RgD 106 -TK or pTK-EF1α-B6RgD 106 -TK using Polyethylenimine (PEI) transfection reagent (Polysciences, Inc.). Briefly, 3 μg of DNA were mixed with 7.5 μg PEI (1mg/mL) (ratio 1:2.5 DNA-Pei) in 200 μL of Dulbecco's modified essential medium (DMEM) at high glucose percentage (Euroclone) without serum. After 15 min at RT, 800 μL of medium without serum was added and the transfection solution was transferred to the well and left on the cells for 6 h at 37°C with 5% CO 2 in air, in a humidified incubator. The transfection mixture was then replaced with fresh medium (EMEM, with 10% FBS, 50 IU/ml of penicillin, 50 μg/ml of streptomycin and 2.5 μg/ml of Amphotericin B) and incubated for 24 h at 37°C with 5% CO 2 .

Viruses and viral replication
BoHV-4-A, BoHV-4-A-CMV-A29LgD 106 ΔTK, BoHV-4-A-EF1α-M1RgD 106 ΔTK and BoHV-4-A-EF1α-B6RgD 106 ΔTK were propagated by infecting confluent monolayers of BEK and VERO cells at a multiplicity of infection (MOI) of 0.5 50% tissue culture infectious doses (TCID 50 ) per cell and maintained in medium with only 2% FBS for 2 h. The medium was then removed and replaced with fresh EMEM containing 10% FBS. When the cytopathic effect (CPE) occurred in the majority of the cell monolayer (*72 h post infection), the virus was prepared by freezing and thawing cells three times and pelleting the virions through a 30% sucrose cushion, as described previously [39]. Virus pellets were then resuspended in cold EMEM without FBS. TCID 50 were determined with BEK cells by limiting dilution. A plaque-purified isolate of the MPXV strain ZAI-79 [40] was purified through a sucrose cushion [41] and propagated in BS-C-1 cells [42]. Virus infectivity was estimated as described previously [43]. Briefly, virus suspensions were serially diluted in PBS +1% FCS (Fetal Clone II, HyClone), absorbed to monolayers for 1 hour at 37°C, and overlaid with a suspension of 1% carboxyl methyl cellulose in DMEM +5% FCS. After 2 days at 37°C, virus plaques were visualized and virus inactivated by the addition to each well of a 0.3% crystal violet/10% formalin solution.

BAC recombineering and selection
Recombineering was performed as previously described [44] with some modifications. Five hundred microliters of a 32°C overnight culture of SW102 containing BAC-BoHV-4-A-Kana- GalKΔTK, were diluted in 25 ml Luria-Bertani (LB) medium with or without chloramphenicol (SIGMA) selection (12.5 mg/ml) in a 50 ml baffled conical flask and grown at 32°C in a shaking water bath to an OD 600 of 0.6. Then, 10 ml were transferred to another baffled 50 mL conical flask and heat-shocked at 42°C for exactly 15 min in a shaking water bath. The remaining culture was left at 32°C as the uninduced control. After 15 min the two samples, induced and uninduced, were briefly cooled in ice/water bath slurry and then transferred to two 15mL Falcon tubes and pelleted using 5000 r.p.m. (eppendorf centrifuge) at 0°C for 5 min. The supernatant was poured off and the pellet was resuspended in 1mL ice-cold ddH 2 O by gently swirling the tubes in ice/water bath slurry. Subsequently, 9 ml ice-cold ddH 2 O were added and the samples pelleted again. This step was repeated once more, the supernatant was removed and the pellet (50 μL each) was kept on ice until electroporated with gel-purified fragments (*3.3, *4.4 and *4.6 kb respectively for TK-CMV-A29LgD 106 -TK, TK-EF1α-M1RgD 106 -TK and TK-EF1α-B6RgD 106 -TK) obtained by cutting pTK-CMV-A29LgD 106 -TK, pTK-EF1α-M1RgD 106 -TK and pTK-EF1α-B6RgD 106 -TK with ClaI/PvuII (Thermo Scientific). An aliquot of 25 μl (~200 ng) was used for each electroporation in a 0.1 cm cuvette at 25 μF, 2.5 kV and 201O. After electroporation, for the counter selection step, the bacteria were recovered in 10 mL LB in a 50 mL baffled conical flask and incubated for 4.5h in a 32°C shaking water bath. Bacteria serial dilutions were plated on M63 minimal medium plates containing 15 g/L agar, 0.2% glycerol, 1mg/L D-biotin, 45 mg/L L-leucine, 0.2% 2-deoxy-galactose and 12.5 mg/mL chloramphenicol. All the complements for M63 medium were purchased from SIGMA. Plates were incubated 3-5 days at 32°C; then several selected colonies were picked up, streaked on McConkey agar indicator plates (DIFCO, BD Biosciences) containing 12.5 g/mL of chloramphenicol and incubated at 32°C for 3 days until white colonies appeared. White colonies were grown in duplicate for 5-8h in 1mL of LB containing 50 mg/mL of kanamycin (SIGMA) or LB containing 12.5 mg/mL of chloramphenicol. Only those colonies that were kanamycin negative and chloramphenicol positive were kept and grown overnight in 5mL of LB containing 12.5 mg/mL of chloramphenicol. BAC DNA was purified and analyzed through HindIII restriction enzyme digestion for TK-CMV-A29LgD 106 -TK, TK-EF1α-M1RgD 106 -TK and TK-EF1α-B6RgD 106 -TK fragment targeted integration, was separated by electrophoresis overnight in a 1% agarose gel, stained with ethidium bromide, capillary transferred to a positively charged nylon membrane (Roche), and cross-linked by UV irradiation by standard procedures [28]. Hybridization with digoxigenin-labeled probes confirmed the identity of specific restriction fragments.

Cell culture electroporation and recombinant virus reconstitution
BEK or BEKcre cells were maintained as a monolayer with complete DMEM growth medium with 10% FBS, 2 mM L-glutamine, 100 IU/mL penicillin and 10 mg/mL streptomycin. When cells were sub-confluent (70-90%) they were split to a fresh culture vessel (i.e., every 3-5 days) and were incubated at 37°C in a humidified atmosphere of 95% air-5% CO2. BAC DNA (5 μg) was electroporated in 600 μl DMEM without serum (Equibio apparatus, 270 V, 960 mF, 4-mm gap cuvettes) into BEK and BEKcre cells from a confluent 25-cm 2 flask. Electroporated cells were returned to the flask, after 24h the medium was replaced with fresh medium, and cells were split 1:2 when they reached confluence at 2 days post-electroporation. Cells were left to grow until the appearance of CPE.

Animals
Eight-week old female 129 stat1 -/mice were bred in-house and housed in filter-top microisolator cages and fed commercial mouse chow and water ad libitum. The randomized mice were housed in an animal biosafety level 3 containment area, with 5 mice/group. Animal husbandry and experimental procedures were approved by the Institutional Animal Care and Use Committee. Mice were monitored every day until the termination of the experiment.

Vaccines and vectors
BoHV-4s vectors were injected intraperitoneally (IP) in a total volume of 300 μl with DMEM used as a vehicle. For vaccinations with one vector, injections were comprised of 100 #x03BC;l of vector + 200 μl of vehicle. For the combination injections, 100 #x03BC;l of each vector was included for a total of 300 #x03BC;l Injections were given as a primary vaccination at T = 0 days and as a booster vaccination at T = 23 days (see Table 2). Each vector was injected at a Modified Vaccinia Ankara (MVA) (a gift from the NIAID-NIH, Bethesda, MD) was provided at a dose of 2x10 8 plaques forming units (PFU)/ml and was injected in 0.1 ml between the skin and underlying layers of tissue in the scapular region on the backs of mice.

MPXV challenge
Mice were anesthetized with 0.1 ml/10 g body weight of ketamine HCl (6 mg/ml) and xylazine (0.5 mg/ml) by intraperitoneal injections. Anesthetized mice were laid on their dorsal side with their bodies angled so that the anterior end was raised 45°from the surface; a plastic mouse holder was used to ensure conformity [45]. MPXV was diluted in PBS without Ca 2+ and Mg 2+ to the required concentration and slowly loaded into each nare (5 #x03BC;l/nare). Mice were subsequently left in situ for 2-3 minutes before being returned to their cages.

Statistics
Paired T-tests (tailed) were used to compare means between groups of mice. Mortality rates were compared using the Fisher's exact test. Blinded lesion pictures were measured qualitatively using a scoring system ranging 0-4 in severity. P values <0.05 were considered statistically significant.

Design and expression of monkeypox virus tagged peptides
Among the approximately 200 genes that comprise the Monkeypox virus genome, only few genes encoding antigenic proteins-that are known-are able to elicit a neutralizing antibody response in vaccinated animals. Among these antigens, A29L, M1R and B6R orthologs were selected as candidate antigens to be delivered by BoHV-4 based-vector. A29L, M1R and B6R ORFs were amplified by PCR from a Cosmid library and sub-cloned in frame with a tag peptide, gD106 [46] (Fig 1A, 1E and 1H), which was contained in a mammalian expression vector plasmid construct. M1R and B6RgD 106 tagged ORFs were placed under the transcriptional control of the EF1α promoter (Fig 1F and 1I), whereas A29LgD 106 tagged ORF under the transcriptional control of the CMV promoter ( Fig 1B). So generated expression cassettes (CMV-A29LgD 106 , EF1α-M1RgD 106 and EF1α-B6RgD 106 ) were validated, in terms of protein expression, by transient transfection in 293T cells and Western-immunoblotting with a monoclonal antibody directed against the gD 106 tag peptide. A29L, M1R and B6RgD 106 tagged antigens were all well expressed in the whole cell extracts of the transiently transfected cells (Fig 1C, 1G and 1J), further A29LgD 106 was also secreted (Fig 1D) in the supernatant of the transiently transfected cells.
In vivo efficacy testing of BoHV-4-A-CMV-A29LgD 106 ΔTK, BoHV-4-A-EF1α-M1RgD 106 ΔTK and BoHV-4-A-EF1α-B6RgD 106 ΔTK To test the efficacy of the vectors in vivo, we sought to determine if they could protect mice against a lethal challenge with MPXV. Several murine strains have been developed as models of MPXV, in this study we utilized the 129 stat1 -/strain. Thirteen cages were prepared with 5 mice/cage ( Table 2). Cages 1 and 2 were un-vaccinated. Cage 3 was vaccinated with vehicle (DMEM without FBS). Cages 4 and 5 were vaccinated with MVA where cage 4 received a primary injection of vaccine at T = 0 days and a vehicle booster at T = 23 days, and cage 5   Cages 12 and 13 were also vaccinated following the above regimen; however, these mice received a combination (combo) of the 3 BoHV-4 vectors. BoHV-4 vectors were injected IP in a total volume of 0.3 ml. There was no apparent morbidity-as measured visually-or weightloss recorded in any of the mice (S2 Fig). At T = 50 days, mice in cages 2-13 were challenged with 2x10 5 PFU/mouse of MPXV. Mortality rates are shown in Fig 4. As expected, the MVA vaccinated mice in cages 4 (MVA/ Veh) and 5 (MVA/MVA) were 100% protected against challenge. Mice in cage 9 (M1R/M1R) were also 100% protected (P = 0.004); and although mice in cage 13 (Combo/Combo) experienced 1 death, they were still 80% protected against the MPXV challenge (P = 0.02). When comparing weight-change (Fig 5), we found that mice in cage 5 (MVA/MVA) did not lose weight compared to the PBS control (cage 1) and that mice in cage 4 (MVA/Veh) only lost weight (5%) on days 6, 7 and 8 post challenge. We also found that mice in cages 10 (B6R/ Veh), 11 (B6R/B6R), 12 (Combo/Veh), and 13 (Combo/Combo) had significantly reduced weight-loss from day 8 post challenge (15%, 15%, 11%, and 15% on day 8, respectively), even though mice in cage 13 were 80% protected against the challenge. Some protection from weight-loss was also afforded to mice in cages 8 (M1R/veh) and 9 (M1R/M1R) on days 13, 14 and 15 post challenge (15%).
These data indicate that the presence of M1R is required to protect the mice against challenge, and that a booster vaccination is required. It also indicates that a combination of the 3 vectors improves protection against weight-loss. Although M1R could provide protection, it was inferior to that provided by vaccination with MVA. The data also indicate that although B6R does not provide protection against mortality, it does provide protection against weightloss in surviving mice (see above).

Discussion
The aim of the current study was to ascertain the potential utility of Bovine herpesvirus 4 (BoHV-4)-based vectors as safe, potent, large-capacity vaccine vectors for category A agents-MPXV for this specific case. This study demonstrated protection in the STAT1 -/model and consideration should be given to also evaluating the vectors in CAST/EiJ mice which have also been established as a murine model of monkeypox [49,50]. Ultimately, this study could pave the way for further studies in other animal models such as prairie dogs (Cynomys ludovicianus) [51][52][53], the 13-lined ground squirrel (Spermophilus tridecemlineatus) [54,55] and non-human primates (reviewed in [1])-all of which have been extensively used as models of human monkeypox-and ultimately in human protection studies. In fact, since MPXV also infects humans and causes clinical-signs very similar to smallpox, it is classified as a category A select agent [56]. Primarily for ethical but also for cost reasons, C57BL/6 STAT1 knockout mice (STAT1 (-/-) ) were chosen as an in vivo model before progressing to gene delivery and immunogenicity studies in non-human primates. STAT1 (-/-) miceare highly sensitive to MPXV and the disease course in MPXV infected STAT1 (-/-) mice, characterized by weight loss and death by day 10 post infection-is similar to that observed in wild-type mice infected with mousepox/ectromelia virus (ECTV), the etiology agent of mousepox-and probably the best small animal model of smallpox [35,57]. Moreover it was revealed that antiviral therapy could protect mice to a degree similar to that of vaccination with Dryvax or MVA, supporting the use of STAT1 (-/-) mice as a reliable model to evaluate orthopoxvirus prophylactics and therapeutics [35]. STAT1 (-/-) mice were found to be sensitive to a wide number of pathogens due to the loss of STAT1, a key factor responsible for type 1 and 2 interferon (IFN) signaling [58][59][60][61][62][63]. BoHV-4-based vectors in wild type and immunocompromised mice behave as replicating incompetent viral vectors, showing absence of pathogenicity [31,32, [64][65][66] and BoHV-4  replication is strongly impaired in vitro after the treatment of BoHV-4 infected cells with IFNγ [67]. Considering that IFN-γ is an activator of STAT1, the potential pathogenicity of BoHV-4-based vectors in STAT1 (-/-) mice was a concern. However, intra peritoneal BoHV-4 inoculated STAT1 (-/-) mice did not show any overt clinical sign, detrimental effect or pathology correlated to challenge.
A29L, M1R, and B6R MPXV antigens were selected, as candidate antigens to be delivered by BoHV-4-based vectors, as they are orthologous to A27L, L1R and B5R Vaccinia Virus (VV) antigens respectively. A27L is a 14 kDa protein thought to be involved in VV entry events [68]. L1R is a 23-29 kDa myristoylated surface protein involved in a yet-to-be-identified post viral attachment and pre-fusion events [69]. Whereas B5R is a 42 kDa glycoprotein found on the surface of the virus [70,71] and is involved in cell surface glycosaminoglycan-mediated disruption of the viral outer membrane [72]. Further, they were shown to be able to elicit a protective immune response in mice and non-human primates when formulated in combination as a subunit vaccine consisted of purified proteins, plasmid DNA vaccines, recombinant adenovirus and alphavirus replicons [73][74][75][76][77][78][79]. Since the purpose of this study was to determine the capability of BoHV-4-based viral vectors to protect STAT1 (-/-) mice against a lethal MPXV infection, the first concern was the generation of optimized expression cassettes to be integrated into the BAC-BoHV-4-A genome that were able to efficiently express A29L, M1R and B6R antigens. Because no antibodies are available for A29L, M1R and B6R proteins, a short in-frame sequence coding for a tag peptide (gD 106 ) was provided at the 3 0 end of their ORFs and this allowed their expression to be monitored by western immunoblotting. Initially A29L, M1R and B6R tagged ORFs were customized under the transcriptional control of the CMV promoter; however, the only ORF to be efficiently expressed was A29L. For this reason, the CMV promoter of the M1R and B6R expression cassette was substituted with the human EF1α promoter which induced expression. The reason why the CMV promoter did not work with the ORFs of M1R and B6R has not been determined. Another interesting observation was the presence of A29L protein into the supernatant of the transfected cells despite the absence of a canonical signal peptide within the primary sequence of the protein as deduced by different signal peptide prediction software (http://www.csbio.sjtu.edu.cn/bioinf/Signal-3L/; http:// www.cbs.dtu.dk/services/SignalP/; http://phobius.sbc.su.se/) [80,81]. In fact, the A29L protein analysis by SecretomeP (http://www.cbs.dtu.dk/services/SecretomeP), a sequence based method for the prediction of mammalian secretory proteins targeted to the non-classical secretory pathway, included A29L within the group of non-classical secreted proteins like fibroblast growth factors, some interleukins and galectins.
BoHV-4 is considered a virus without a clear disease association, the existence of a BoHV-4 potentially pathogenic biotype cannot be absolutely excluded when a virus is going to be exploited as a gene delivery vector. Bovine herpesvirus 4 (BoHV-4) has been most consistently associated with uterine disease in postpartum cattle and BoHV-4 infection is often identified concurrently with bacteria that cause uterine diseases [82,83]. The association between BoHV-4 infection and uterine disease has been difficult to establish. It was suggested that there may be an association with bacterial endometritis which leads to secretion of prostaglandin E2 (PGE2) and then stimulation of viral replication by PGE2, TNF-α and lipopolysaccharide (LPS)-which causes further endometrial tissue damage and inflammation [84][85][86]. For this reason, a putative non-pathogenic strain of BoHV-4 (BoHV-4-A) isolated from the cell milk fraction of a healthy cow whose genome was cloned as a bacterial artificial chromosome (pBAC-BoHV-4-A) [28] was used. CMV-A29LgD 106 , EF1α-M1RgD 106 and EF1α-B6RgD 106 were integrated into the TK locus of pBAC-BoHV-4-A, and proved to be stable through passages in E. coli SW102 and recombinant viable BoHV-4-A-CMV-A29LgD 106 ΔTK, BoHV-4-A-EF1α-M1RgD 106 ΔTK, BoHV-4-A-EF1α-B6RgD 106 ΔTK were successfully reconstituted in BEKcre cells. When BoHV-4-A-CMV-A29LgD 106 ΔTK, BoHV-4-A-EF1α-M1RgD 106 ΔTK and BoHV-4-A-EF1α-B6RgD 106 ΔTK were characterized in terms of replication kinetics, a reduction of replication was observed for BoHV-4-A-EF1α-B6RgD 106 ΔTK and this was attributed to a partial toxic effect induced by the abundant expression of B6RgD 106 -which was also observed in transfections of cells with EF1α-B6RgD 106 expression cassette. This latter observation excluded a potential detrimental effect induced by the topological location of the foreign DNA in the BoHV-4 genome. Despite their replication characteristics, all three recombinant BoHV-4s abundantly expressed their transgene in infected cells.
In vivo protection studies determined that M1R protected against a lethal MPXV challenge. One hundred % protection was achieved when the vectors were administered twice (prime followed by booster), although the M1R expression vector was not superior to vaccination with MVA as measured by weight-loss. Protection was also afforded to mice when the vectors were administered in combination (combo) as a prime and booster. These mice experienced less weight-loss than mice vaccinated with M1R alone. This finding is surprising as the other vectors included in the combo were not protective when administered individually, although we did find protection against weight-loss when mice were treated with B6R alone. Nevertheless, our studies reveal that protection can be afforded even when a small number of mice are used. Further studies should be considered that increase the dose of vector administered to the mice. Also, since the combination of all 3 vectors gave 80% protection against mortality and morbidity, various vector permutations should be considered to elucidate the most efficacious combination and ratio of vectors.
No overt clinical-signs were observed following vaccination with the prime or booster injection, suggesting low immuno-reactivity and therefore possibly low-levels of adverse events in NHPs and humans. Although second generation live vaccines, such as ACAM2000, provide the most robust immune response, they are quite reactogenic and induce some level of morbidity in all vaccines. Furthermore, a significant portion of the human population are contraindicated to vaccination with first-and second-generation vaccines [19]. MVA is a non-replicating vaccine that has demonstrated efficacy in many animal trials. The main draw-back to MVA is its relatively low immunogenicity, meaning that booster administrations are usually required for 100% protection against morbidity and mortality [87,88]. Future studies could reveal that vectors' studied here could be used as alternatives to MVA.
In summary, our findings have demonstrated that BoHV-4 based vectors can be used as vaccines to protect against a lethal MPXV challenge in mice. Our studies utilized STAT1 (-/-) mice; however, other strains have demonstrated sensitivity to MPXV, namely the CASTE/EiJ strain [49,50,89]. Future studies should consider evaluating the protection of these vectors in this strain also. This work provides a "proof-of-concept" for the BoHV-4-based vector as a potential vaccine for category A agents. Future and ongoing studies are focused on the design of BoHV-4-based vectors expressing antigens from other category A pathogens, as well as an assessment of protection in non-human primate.