Improvement of the Capripoxvirus, lumpy skin disease virus for use as a vaccine vector

Lumpy skin disease (LSD) is a notifiable viral infection due both to its morbidity in cattle and its severe economic burden. The disease was confined to Sub-Saharan Africa but has in recent years spread to the Middle East and Europe. Vaccination is the only way of preventing LSD. Live attenuated lumpy skin disease virus (LSDV) has been used as a vaccine against LSD. The most successful LSD vaccine is the Neethling vaccine strain (nLSDV) from South Africa. There are however, reports of nLSDV being too attenuated or too virulent in different breeds of cattle. A South African produced vaccine strain of LSDV, Herbivac, was said to be more immunogenic than nLSDV (personal communication, Deltamune). Whole genomic sequence comparison of Herbivac with nLSDV revealed a single potentially significant change in open reading frame (ORF) 131. This ORF encodes a superoxide dismutase (SOD) homologue. The mutation identified in Herbivac is a 2bp deletion which causes a frameshift mutation that restores the SOD homologue to resemble the full-length SOD homolog encoded by the virulent field strain. This SOD homologue gene is truncated in nLSDV. Protein structural alignment of SOD homologues from LSDV and other characterised SOD homologues was done. Both the truncated and full-length SOD homologues lacked the catalytic arginine at position 142 which is involved in SOD activity. Some similarities with known SOD homologues which act as SOD decoys, such as the Leporipoxviruses myxoma and Shope Fibroma virus, were observed. Like Leporipoxvirus’ SOD homologues, the full-length Herbivac SOD homologue contained regions of homology with the copper chaperone for SOD (CCS). The putative SOD protein from Herbivac, and not nLSDV, contained 6 out of 8 metal binding residues. Unlike the SOD decoy from myxoma virus, the full-length SOD homolog from Herbivac did not include a cysteine molecule at position 56 that stabilizes the SOD-CCS heterodimer. The alignment suggests that all SOD homologs from LSDV are inactive as enzymes. Transcriptome analysis of messenger RNA from spleens of mice infected with nLSDV or Herbivac for 24hrs was carried out to determine the effect of Herbivac or nLSDV infection on host gene expression. Compared to the PBS control, nLSDV and Herbivac induced the differential expression of 98 genes in common, largely related to response to viral infection. nLSDV differed in the unique expression of 6 genes and Herbivac differed in the unique expression of 36 genes, including granzyme A and Poly (ADP-ribose) polymerase (Parp 9). Herbivac upregulated genes associated with pathogen pattern recognition, interferon response, immune response and cell death. More Gene ontology (GO) processes were enriched after Herbivac infection than nLSDV infection. Amongst these processes were immune response processes, the interferon and cell death related responses. To characterise the SOD homolog with respect to SOD activity, cell death and whether it plays a role in growth of viruses expressing it, recombinant viruses were constructed. The first set of recombinants included a SOD knock-out virus where the SOD gene from nLSDV was replaced with reporter gene GFP. A SOD knock-in virus was constructed with the SOD homologue gene altered to improve the stability of the gene. A reporter gene mCherry was also inserted. Histological examination of CAMs infected with nLSDVdSOD-M showed vacuolation and oedema to a greater degree than nLSDV, Herbivac and nLSDVSODis-M. Herbivac showed greater immune cell infiltration. SOD knock-in, nLSDVSODis-M showed increased epithelial hyperplasia and fibroplasia. Another set of recombinants without marker genes was made, nLSDVdSOD-UCT (SOD knock-out), and nLSDVSODis-UCT (SOD knock-in). Deleting the SOD homolog reduced virus yield by approximately ten-fold in MDBK cells. Histologically, the presence of the SOD homolog in nLSDVSODis-UCT caused greater inflammatory changes in the mesoderm after 5 days of infection compared to nLSDVdSOD-UCT. In vitro functional studies were done in MDBK cells which are permissible to LSDV infection. No difference in SOD activity could be detected amongst the different viruses. Differences could, however, be detected in induction and inhibition of apoptosis. There was increased induction of apoptosis following infection by all viruses containing full-length SOD compared to infection with truncated or SOD knock-out virus. Viruses expressing a full-length SOD showed greater inhibition of camptothecin induced apoptosis than nLSDV or the SOD knockout. Similarly, full-length SOD induced cell death by necrosis to a greater extent than the SOD knock-out or nLSDV; and all viruses inhibited camptothecin induced necrosis. These results suggest that the presence of a SOD homolog in a vaccine could be advantageous with respect to growth of the vaccine to higher titres and to improved immunogenicity of the vaccine. Further work is required to test this hypothesis in a bovine animal model

The immune response to lumpy skin disease virus in cattle is influenced by inoculation route

Lumpy skin disease virus (LSDV) causes severe disease in cattle and water buffalo and is transmitted by hematophagous arthropod vectors. Detailed information of the adaptive and innate immune response to LSDV is limited, hampering the development of tools to control the disease. This study provides an in-depth analysis of the immune responses of calves experimentally inoculated with LSDV via either needle-inoculation or arthropod-inoculation using virus-positive Stomoxys calcitrans and Aedes aegypti vectors. Seven out of seventeen needle-inoculated calves (41%) developed clinical disease characterised by multifocal necrotic cutaneous nodules. In comparison 8/10 (80%) of the arthropod-inoculated calves developed clinical disease. A variable LSDV-specific IFN-γ immune response was detected in the needle-inoculated calves from 5 days post inoculation (dpi) onwards, with no difference between clinical calves (developed cutaneous lesions) and nonclinical calves (did not develop cutaneous lesions). In contrast a robust and uniform cell-mediated immune response was detected in all eight clinical arthropod-inoculated calves, with little response detected in the two nonclinical arthropod-inoculated calves. Neutralising antibodies against LSDV were detected in all inoculated cattle from 5-7 dpi. Comparison of the production of anti-LSDV IgM and IgG antibodies revealed no difference between clinical and nonclinical needle-inoculated calves, however a strong IgM response was evident in the nonclinical arthropod-inoculated calves but absent in the clinical arthropod-inoculated calves. This suggests that early IgM production is a correlate of protection in LSD. This study presents the first evidence of differences in the immune response between clinical and nonclinical cattle and highlights the importance of using a relevant transmission model when studying LSD

The Development of Dual Vaccines against Lumpy Skin Disease (LSD) and Bovine Ephemeral Fever (BEF)

Dual vaccines (n = 6) against both lumpy skin disease (LSD) and bovine ephemeral fever (BEF) were constructed, based on the BEFV glycoprotein (G) gene, with or without the BEFV matrix (M) protein gene, inserted into one of two different LSDV backbones, nLSDV∆SOD-UCT or nLSDVSODis-UCT. The inserted gene cassettes were confirmed by PCR; and BEFV protein was shown to be expressed by immunofluorescence. The candidate dual vaccines were initially tested in a rabbit model; neutralization assays using the South African BEFV vaccine (B-Phemeral) strain showed an African consensus G protein gene (Gb) to give superior neutralization compared to the Australian (Ga) gene. The two LSDV backbones expressing both Gb and M BEFV genes were tested in cattle and shown to elicit neutralizing responses to LSDV as well as BEFV after two inoculations 4 weeks apart. The vaccines were safe in cattle and all vaccinated animals were protected against virulent LSDV challenge, unlike a group of control naïve animals, which developed clinical LSD. Both neutralizing and T cell responses to LSDV were stimulated upon challenge. After two inoculations, all vaccinated animals produced BEFV neutralizing antibodies ≥ 1/20, which is considered protective for BEF

The Isolated in Utero Environment Is Conducive to the Emergence of RNA and DNA Virus Variants

The host’s immune status may affect virus evolution. Little is known about how developing fetal and placental immune milieus affect virus heterogeneity. This knowledge will help us better understand intra-host virus evolution and how new virus variants emerge. The goal of our study was to find out whether the isolated in utero environment—an environment with specialized placental immunity and developing fetal immunity—supports the emergence of RNA and DNA virus variants. We used well-established porcine models for isolated Zika virus (RNA virus) and porcine circovirus 2 (DNA virus) fetal infections. We found that the isolated in utero environment was conducive to the emergence of RNA and DNA virus variants. Next-generation sequencing of nearly whole virus genomes and validated bioinformatics pipelines identified both unique and convergent single nucleotide variations in virus genomes isolated from different fetuses. Zika virus and PCV2 in utero evolution also resulted in single nucleotide variations previously reported in the human and porcine field samples. These findings should encourage further studies on virus evolution in placenta and fetuses, to better understand how virus variants emerge and how in utero viral evolution affects congenital virus transmission and pathogenicity

Capripoxvirus Infections in Ruminants: A Review

Lumpy skin disease, sheeppox, and goatpox are notifiable diseases of cattle, sheep, and goats, respectively, caused by viruses of the Capripoxvirus genus. They are responsible for both direct and indirect financial losses. These losses arise through animal mortality, morbidity cost of vaccinations, and constraints to animals and animal products’ trade. Control and eradication of capripoxviruses depend on early detection of outbreaks, vector control, strict animal movement, and vaccination which remains the most effective means of control. To date, live attenuated vaccines are widely used; however, conferred protection remains controversial. Many vaccines have been associated with adverse reactions and incomplete protection in sheep, goats, and cattle. Many combination- and recombinant-based vaccines have also been developed. Here, we review capripoxvirus infections and the immunity conferred against capripoxviruses by their respective vaccines for each ruminant species. We also review their related cross protection to heterologous infections

Influence of the Viral Superoxide Dismutase (SOD) Homologue on Lumpy Skin Disease Virus (LSDV) Growth, Histopathology and Pathogenicity

Lumpy skin disease is an important economic disease of cattle that is controlled by vaccination. This paper presents an investigation into the role of the lumpy skin disease virus (LSDV) superoxide dismutase (SOD) homologue on growth and histopathology of the virus both in vitro and in vivo. SOD homologue knock-out and knock-in recombinants (nLSDV∆SOD-UCT and nLSDVSODis-UCT, respectively) were constructed and compared to the Neethling vaccine (nLSDV) for growth in a permissive bovine cell line as well as on fertilized chick chorioallantoic membranes (CAMs). The infected CAMs were scored for histological changes. Deletion of the SOD homologue from LSDV reduced virus growth both in Madin-Darby bovine kidney (MDBK) cells as well as on CAMs. Furthermore, the knockout virus showed reduced inflammation in CAMs and more ballooning degeneration. A pilot experiment was performed in cattle to compare the lesions produced by the different LSDV constructs in the same animal. One animal developed a larger lesion to nLSDV∆SOD-UCT compared to both nLSDVSODis-UCT and nLSDV. Histological analysis of biopsies of these lesions shows less inflammation and necrosis associated with nLSDVSODis-UCT compared to nLSDV and nLSDV∆SOD-UCT. None of the vaccinated animals showed disseminated LSDV disease, indicating that the candidate vaccines are safe for further testing. Our results suggest that the SOD homologue may improve immunogenicity and reduce virulence

The Development of Dual Vaccines against Lumpy Skin Disease (LSD) and Bovine Ephemeral Fever (BEF)

Dual vaccines (n = 6) against both lumpy skin disease (LSD) and bovine ephemeral fever (BEF) were constructed, based on the BEFV glycoprotein (G) gene, with or without the BEFV matrix (M) protein gene, inserted into one of two different LSDV backbones, nLSDV∆SOD-UCT or nLSDVSODis-UCT. The inserted gene cassettes were confirmed by PCR; and BEFV protein was shown to be expressed by immunofluorescence. The candidate dual vaccines were initially tested in a rabbit model; neutralization assays using the South African BEFV vaccine (B-Phemeral) strain showed an African consensus G protein gene (Gb) to give superior neutralization compared to the Australian (Ga) gene. The two LSDV backbones expressing both Gb and M BEFV genes were tested in cattle and shown to elicit neutralizing responses to LSDV as well as BEFV after two inoculations 4 weeks apart. The vaccines were safe in cattle and all vaccinated animals were protected against virulent LSDV challenge, unlike a group of control naïve animals, which developed clinical LSD. Both neutralizing and T cell responses to LSDV were stimulated upon challenge. After two inoculations, all vaccinated animals produced BEFV neutralizing antibodies ≥ 1/20, which is considered protective for BEF