Novel strategy for Poxviridae prevention: Thermostable combined subunit vaccine patch with intense immune response.

Poxviruses gained international attention due to the sharp rise in monkeypox cases in recent years, highlighting the urgent need for the development of a secure and reliable vaccine. This study involved the development of an innovative combined subunit vaccine (CSV) targeting poxviruses, with lumpy skin disease virus (LSDV) serving as the model virus. To this end, the potential sites for poxvirus vaccines were fully evaluated to develop and purify four recombinant proteins. These proteins were then successfully delivered to the dermis in a mouse model by utilizing dissolvable microneedle patches (DMPs). This approach simplified the vaccination procedure and significantly mitigated the associated risk. CSV-loaded DMPs contained four recombinant proteins and a novel adjuvant, CpG, which allowed DMPs to elicit the same intensity of humoral and cellular immunity as subcutaneous injection. Following immunization with SC and DMP, the mice exhibited notable levels of neutralizing antibodies, albeit at a low concentration. It is noteworthy that the CSV loaded into DMPs remained stable for at least 4 months at room temperature, effectively addressing the storage and transportation challenges. Based on the study findings, CSV-loaded DMPs are expected to be utilized worldwide as an innovative technique for poxvirus inoculation, especially in underdeveloped regions. This novel strategy is crucial for the development of future poxvirus vaccines.

Development and Evaluation of a Combined Contagious Bovine Pleuropneumonia (CBPP) and Lumpy Skin Disease (LSD) Live Vaccine.

Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia (CBPP). Lumpy skin disease (LSD) is a viral disease of cattle caused by lumpy skin disease virus (LSDV). LSD and CBPP are both transboundary diseases spreading in the same areas of Africa and Asia. A combination vaccine to control CBPP and LSD offers significant value to small-scale livestock keepers as a single administration. Access to a bivalent vaccine may improve vaccination rates for both pathogens. In the present study, we evaluated the LSDV/CBPP live combined vaccine by testing the generation of virus neutralizing antibodies, immunogenicity, and safety on target species. In-vitro assessment of the Mycoplasma effect on LSDV growth in cell culture was evaluated by infectious virus titration and qPCR during 3 serial passages, whereas in-vivo interference was assessed through the antibody response to vaccination. This combined Mmm/LSDV vaccine could be used to protect cattle against both diseases with a single vaccination in the endemic countries. There were no adverse reactions detected in this study and inoculated cattle produced high levels of specific antibodies starting from day 7 post-vaccination, suggesting that this combination vaccine is both safe and effective.

Development of an inactivated combined vaccine for protection of cattle against lumpy skin disease and bluetongue viruses.

Lumpy Skin Disease (LSD) and Bluetongue (BT) are the main ruminants viral vector-borne diseases. LSD is endemic in Africa and has recently emerged in Europe and central Asia as a major threat to cattle industry. BT caused great economic damage in Europe during the last decade with a continuous spread to other countries. To control these diseases, vaccination is the only economically viable tool. For LSD, only live-attenuated vaccines (LAVs) are commercially available, whilst for BT both LAVs and inactivated vaccines are available with a limited number of serotypes. In this study, we developed an inactivated, oil adjuvanted bivalent vaccine against both diseases based on LSDV Neethling strain and BTV4. The vaccine was tested for safety and immunogenicity on cattle during a one-year period. Post-vaccination monitoring was carried out by VNT and ELISA. The vaccine was completely safe and elicited high neutralizing antibodies starting from the first week following the second injection up to one year. Furthermore, a significant correlation (R = 0.9040) was observed when comparing VNT and competitive ELISA in BTV4 serological response. Following BTV4 challenge, none of vaccinated and unvaccinated cattle were registered clinical signs, however vaccinated cattle showed full protection from viraemia. In summary, this study highlights the effectiveness of this combined vaccine as a promising solution for both LSD and BT control. It also puts an emphasis on the need for the development of other multivalent inactivated vaccines, which could be greatly beneficial for improving vaccination coverage in endemic countries and prophylaxis of vector-borne diseases.

Goatpox virus (G20-LKV) vaccine strain elicits a protective response in cattle against lumpy skin disease at challenge with lumpy skin disease virulent field strain in a comparative study.

In this comparative study, we examine the safety of the sheeppox (SPP) and goatpox (GTP) vaccines and the protective response of these vaccines in cattle against a virulent lumpy skin disease (LSD) field strain. The vaccine safety was tested in rabbits, mice and cattle using ten times recommended dose. In the safety trial, none of the vaccinated animals showed any deviation from physiological norms or fever, inappetence or local/ generalized skin reactions. In the challenge trial, both SPP and GTP vaccine groups developed virus-neutralizing antibodies with an average titre of 2.1 log2 at 21 days post-vaccination. No significant difference in seroconversion was found in cattle vaccinated with SPP and GTP vaccines (P ≥ 0.05). When challenged with a virulent LSD field strain, one animal vaccinated with the SPP Niskhi vaccine strain showed typical LSD skin lesions at the injection sites of different dilutions of the challenge virus. All animals vaccinated with GTP G20-LKV vaccine strain showed full protection. After infection with the challenge virus, unvaccinated fully susceptible control cattle showed characteristic clinical signs of LSD. The average protective index for SPP and GTP vaccine groups was 5.3 ± 1.42 and 5.9 ± 0.00, respectively.

Development and Evaluation of an Inactivated Lumpy Skin Disease Vaccine for Cattle.

Lumpy skin disease (LSD) of cattle is caused by a virus within Capripoxvirus genus. It leads to huge economic losses in addition to trade and animal movement limitation. Vaccination is the only economically feasible way to control this vector-borne disease. Only live attenuated vaccines have been used so far and no inactivated vaccine has been developed nor tested in cattle. In this study, we developed an inactivated oily adjuvanted vaccine based on Neethling strain and tested it on cattle. Selected criteria of appreciation were safety, antibody response by Virus Neutralization and protection through challenge. A field trial was also performed in Bulgaria. The vaccine was safe and did not cause any adverse reaction, high level of specific antibodies was obtained starting from day 7 post-vaccination and protection against virulent challenge strain that caused typical disease in control animals was total. Induced protection was similar to that obtained with live vaccine, without any adverse effect. In addition, the field study confirmed safety and efficacy of the vaccine, which did not show any adverse reaction and induced a high level of antibodies for up to one year. General prophylaxis based on inactivated vaccine could be of great benefit in endemic countries or at risk regions.

Non-vector-borne transmission of lumpy skin disease virus.

The transmission of "lumpy skin disease virus" (LSDV) has prompted intensive research efforts due to the rapid spread and high impact of the disease in recent years, especially in Eastern Europe and Balkan countries. In this study, we experimentally evaluate the vaccine-derived virulent recombinant LSDV strain (Saratov/2017) and provide solid evidence on the capacity of the virus for transmission in a vector-proof environment. In the 60-day long experiment, we used inoculated bulls (IN group) and two groups of in-contact animals (C1 and C2), with the former (C1) being in contact with the inoculated animals at the onset of the trial and the latter (C2) being introduced at day 33 of the experiment. The infection in both groups of contact animals was confirmed clinically, serologically and virologically, and viremia was demonstrated in blood, nasal and ocular excretions, using molecular tools. Further studies into LSDV biology are a priority to gain insights into whether the hypothesized indirect contact mode evidenced in this study is a de novo-created feature, absent from both parental stains of the novel (recombinant) LSDV isolate used, or whether it was dormant, but then unlocked by the process of genetic recombination. Author summary: In global terms, LSD has been termed a "neglected disease" due to its historic natural occurrence of being restricted to Africa and, occasionally, Israel. However, after its slow spread throughout the Middle East, the disease is now experiencing a resurgence of research interest following a recent and rapid spread into more northern latitudes. Given the dearth of solid findings on potential transmission mechanisms, no efficient or reliable control program currently exists, which does not involve the use of live attenuated vaccines or stamping out policies - both of which are controversial for implementation in non-endemic regions or countries. The vector-borne mode is the only working concept currently available, but with scarce evidence to support the aggressive spread northwards - except for human-assisted spread, including legal or illegal animal transportation. The emergence of outbreaks is not consistently linked to weather conditions, with the potential for new outbreaks to occur and spread rapidly. Here, for the first time, we provide evidence for indirect contact-mode transmission for a naturally-occurring recombinant LSDV isolated from the field. In an insect-proof facility, we obtained solid evidence that the novel LSDV strain can pass to in-contact animals. Given the recombinant nature of the virus utilised, its genetic background relating to the observed transmission pattern within the study needs to be delineated.

Influence of the lumpy skin disease virus (LSDV) superoxide dismutase homologue on host transcriptional activity, apoptosis and histopathology.

Lumpy skin disease virus (LSDV), a Capripoxvirus, is of economic importance in the cattle industry and is controlled by vaccination. A comparison was made of the host response to the two LSDV vaccines Neethling and Herbivac LS, with reference to the well-studied Orthopoxvirus, modified vaccinia Ankara (MVA), in a mouse model. Because the vaccines differ at the superoxide dismutase homologue (SOD) gene locus, recombinant SOD knock-out and knock-in nLSDV vaccines were constructed and all four vaccines were tested for the induction and inhibition of apoptosis. The SOD homologue was associated both with induction of apoptosis as well as inhibition of camptothecin-induced apoptosis. Histological analysis of chorioallantoic membranes of fertilized hens' eggs infected with the four different vaccines indicated marked mesodermal proliferation associated with vaccines containing the full-length SOD homologue as well as increased immune cell infiltration. Our findings suggest that the SOD homologue may influence vaccine immunogenicity.

Emergence of a new lumpy skin disease virus variant in Kurgan Oblast, Russia, in 2018.

In this paper, we report the resurgence of lumpy skin disease (LSD) in Kurgan Oblast, Russia, in 2018. The majority of the outbreaks were silent with no mortality and congregated within an area with a radius of about 30 km located 1-50 km away from the national border with Kazakhstan. Following primary molecular diagnosis, LSD virus (LSDV) isolates were analyzed using a panel of PCR assays targeting different genetic loci, namely, LSD008 (vaccine), LSDV126 (field), and GPCR (vaccine and field), for differentiation and genotype assignment. All isolates were positive for the vaccine genotype of GPCR and negative for the other field targets tested. A PCR assay with melt curve analysis utilizing LSD008, developed in this work, indicated that the strains melted with a profile similar to those of field strains. Surprisingly, sequence analysis of the RPO30 and GPCR genes aligned the Kurgan/2018 isolate with KSGP O-240 at the GPCR locus, but with Saratov/2017 at the RPO30 locus. The latter cluster forms an association with a sub-cluster of the field strains comprising the South African KSGP O-240 strain and NI-2490 strain. Due to these incongruent phylogenetic patterns, the sequences of three additional loci ORF19 (Kelch-like protein), ORF52 (putative transcriptional elongation factor), and ORF87 (mutT motif protein) were investigated. Phylogenetic analysis of these additional loci placed the strain Kurgan/2018 in either vaccine or field groups, strongly suggesting a novel recombinant profile. This is another piece of evidence exposing the potential for recombination in capripoxviruses and the ignored danger of using live homologous vaccines against LSD. The necessity to revise the PCR-based strategy differentiating infected from vaccinated animals is discussed. The potential scenarios of incursion and the contribution of the KSGP/NI-2490-like strain to the emergence of the recently identified vaccine-like recombinant are discussed.

Neethling vaccine proved highly effective in controlling lumpy skin disease epidemics in the Balkans.

Despite the wide use of the live attenuated Neethling lumpy skin disease (LSD) vaccine, only limited data existed on its efficacy and effectiveness prior to the large LSD epidemic in the Balkans, which took place during 2016-2017. In addition, analysis of risk factors for the disease was hardly performed with proper control for vaccination effects and potential differences in exposure to the virus. Data from the LSD epidemics in six Balkan countries (Bulgaria, Greece, Serbia, Montenegro, Former Yugoslav Republic of Macedonia (FYROM) and Albania) affected during 2016 were analyzed to determine vaccine effectiveness (VE) and risk factors for LSD infection at the farm level. Vaccination was performed along the occurrence of the epidemics and thus vaccination status of some of the farms changed during the epidemic. To allow for this, left truncated and right censored survival analysis was used in a mixed effects Cox proportional hazard regression model to calculate VE and risk factors for LSD. The results indicated of an average VE of 79.8% (95% CI: 73.2-84.7)) in the six countries, with the lowest VE of 62.5% documented in Albania and up to VE of more than 97% as documented in Bulgaria and Serbia. Analysis of time from vaccination to development of protective immunity showed that VE mostly developed during the first 14 days after vaccination. Data from Greece showed that the vaccination adjusted hazard ratio for LSD was 5.7 higher in grazing farms compared to non-grazing farms. However, due to a difference in geographical location of grazing and non-grazing farms and higher vaccination rate in non-grazing farms, this effect can be at least partly attributed to indirect protection due to herd immunity provided by surrounding vaccinated farms.

An Immunoperoxidase Monolayer Assay (IPMA) for the detection of lumpy skin disease antibodies.

During this study a new Immunoperoxidase Monolayer Assay (IPMA) was developed for the detection of antibodies against lumpy skin disease virus (LSDV) in an easy and low tech setting. Using two dilutions (1:50 and 1:300) in a duplicate format, the test was shown to be highly sensitive, specific and repeatable. In comparison to the VNT and a commercial ELISA, the LSDV-IPMA was able to detect the LSDV antibodies earlier in infected, vaccinated and vaccinated/infected animals. The assay is very flexible as it can be easily adapted for the detection of sheeppox or goatpox antibodies and it can be scaled-up to handle medium size sample sets by preparing the IPMA plates in advance. These plates are safe and can be handled in low biosafety level labs.

Experimental lumpy skin disease virus infection of cattle: comparison of a field strain and a vaccine strain.

Lumpy skin disease virus (LSDV) infections can cause massive clinical signs in cattle and have great economic impact due to severe trade restrictions. For LSDV control, only live attenuated vaccines are commercially available, but they currently are not authorized in the European Union. Moreover, these vaccine virus strains can induce substantial side effects with clinical signs similar to infections with virulent LSDV. In our study, we compared clinical symptoms, viremia, and seroconversion of cattle inoculated either with a virulent field strain from North Macedonia isolated from diseased cattle in 2016 or with the attenuated LSDV vaccine strain "Neethling". Using specimens from the field and from experimental inoculation, different diagnostic tools, including a pan-capripox real-time qPCR, newly developed duplex real-time qPCR assays for differentiation between virulent and attenuated LSDV strains, and several serological methods (ELISA, indirect immunofluorescence test and serum neutralization test [SNT]) were evaluated. Our data show a high analytical sensitivity of both tested duplex real-time qPCR systems for the reliable distinction of LSDV field and vaccine strains. Moreover, the commercially available capripox double-antigen ELISA seems to be as specific as the SNT and therefore provides an excellent tool for rapid and simple serological examination of LSDV-vaccinated or infected cattle.

Importance of the lumpy skin disease virus (LSDV) LSDV126 gene in differential diagnosis and epidemiology and its possible involvement in attenuation.

Examination of lumpy skin disease virus (LSDV) isolates from different geographic regions and times revealed that assays developed in our laboratory for differentiating between virulent Israeli viruses and Neethling vaccine virus (NVV) are generally useful in most, if not all, endemic areas in which NVV-based vaccines are used. Recently it was revealed that the LSDV126 gene of field isolates contains a duplicated region of 27 bp (9 aa), while the vaccine viruses have only one copy. Phylogenetic analysis of a 532-bp segment carrying the LSDV126 gene and whole virus genome sequences revealed that LSDV isolates formed two groups: virulent and vaccine viruses. In this analysis, all of the capripox viruses that lack the ability to efficiently infect cattle were found to carry only one copy of the 27-bp fragment, suggesting that the LSDV126 gene plays an important role in the ability of capripox viruses to infect cattle. In silico analysis of potential antigenic sites in LSDV126 revealed that LSDV126 variants with only one copy of the repeat lack a potentially important antigenic epitope, supporting its possible significance in cattle infection. This study provides new information about the nature of the LSDV126 gene and its possible role in the life cycle of LSDV.

Detection of vaccine-like strains of lumpy skin disease virus in outbreaks in Russia in 2017.

Lumpy skin disease (LSD) has affected many regions of Russia since its first occurrence in 2015. The most devastating year for Russia was 2016, when the virus resurged following a modified stamping-out campaign, causing 313 outbreaks in 16 regions. To avoid unwanted adverse reactions following the use of live attenuated vaccines against LSD virus (LSDV), sheeppox-based vaccines were administered during vaccination campaigns. As a result, LSD was successfully contained in all Russian regions in 2017. In the same year, however, LSD emerged anew in a few regions of the Privolzhsky Federal District of Russia along the northern border of Kazakhstan, which then necessitated vaccinating cattle with a live attenuated LSDV vaccine. Although live attenuated LSDV vaccines are prohibited in Russia, several vaccine-like LSDV strains were identified in the 2017 outbreaks, including commercial farms and backyard animals exhibiting clinical signs consistent with those of field LSDV strains. Sequence alignments of three vaccine-like LSDV strains showed clear similarity to the corresponding RPO30 and GPCR gene sequences of commercial attenuated viruses. How vaccine-like strains spread into Russian cattle remains to be clarified.

Humoral immune response to repeated lumpy skin disease virus vaccination and performance of serological tests.

BACKGROUND: In the presented study we investigated the development of the humoral immune response against LSDV during the process of re-vaccination of cattle over a time span of 5 months. In addition, the performance of different serological techniques for antibody detection against LSDV was compared. For sample collection, an area without previous LSD outbreak reports in Serbia was selected. Seventy-nine cattle from twenty farms vaccinated in 2016 and re-vaccinated in 2017 were included in the study. Two farms from the same area with good calving management were selected for investigation of passive LSDV antibody transfer from vaccinated mothers to new-borne calves. RESULTS: All investigated cattle were healthy on the day of vaccination and during the whole study. Swelling at the injection site or other side effects of vaccination did not occur after re-vaccination in the study. Detection of LSD-specific antibodies was performed with the standard serological methods VNT and IFAT as well as a commercially available Capripox double antigen multi-species-ELISA. Capripoxvirus-specific antibodies were detected 46 to 47 weeks after vaccination in 2016, with VNT in 35.06% and with IFAT and ELISA in 33.77%. A secondary response was observed in all three tests 1 month after re-vaccination with a significant increase in seropositive animals compared to the results before re-vaccination. With all applied serological methods, the number of animals testing positive was significantly higher at 1 and 5 months post re-vaccination than before re-vaccination. No significant statistical difference (p > 0.05) was observed between the results of all three tests used. The sensitivity and specificity of ELISA was estimated to be SeELISA 91% and SpELISA 87% calculated by the results of VNT and SeELISA 88% and SpELISA 76% calculated by the results of IFAT. Passive antibody transfer from vaccinated mothers to new-born calves was investigated at 14 days after birth. Discrepancies for the detection of LSDV specific antibodies between cows and newborn calves at the age of 14 days were observed in VNT and IFAT, but not in ELISA. CONCLUSION: Of all tests used the commercially available ELISA shows to be the most useful for high throughput analysis compared to VNT or IFAT.

A simple method to estimate the number of doses to include in a bank of vaccines. The case of Lumpy Skin Disease in France.

A simple method to estimate the size of the vaccine bank needed to control an epidemic of an exotic infectious disease in case of introduction into a country is presented. The method was applied to the case of a Lumpy Skin disease (LSD) epidemic in France. The size of the stock of vaccines needed was calculated based on a series of simple equations that use some trigonometric functions and take into account the spread of the disease, the time required to obtain good vaccination coverage and the cattle density in the affected region. Assuming a 7-weeks period to vaccinate all the animals and a spread of the disease of 7.3 km/week, the vaccination of 740 716 cattle would be enough to control an epidemic of LSD in France in 90% of the simulations (608 196 cattle would cover 75% of the simulations). The results of this simple method were then validated using a dynamic simulation model, which served as reference for the calculation of the vaccine stock required. The differences between both models in different scenarios, related with the time needed to vaccinate the animals, ranged from 7% to 10.5% more vaccines using the simple method to cover 90% of the simulations, and from 9.0% to 13.8% for 75% of the simulations. The model is easy to use and may be adapted for the control of different diseases in different countries, just by using some simple formulas and few input data.

Colostrum transfer of neutralizing antibodies against lumpy skin disease virus from vaccinated cows to their calves.

The objective of the present study was to access the titres and duration of maternally derived neutralizing antibodies against lumpy skin disease virus (LSDV) in calves born to immunized dairy cows. The study was conducted in a Greek farm of 200 Holstein cows which were immunized with a homologous Neethling strain-based attenuated vaccine. Composite colostrum samples were obtained from 18 selected cows at the day of calving. Blood samples were obtained from each dam-calf pair prior to the first colostrum feeding and from the calves successively on the third day after calving and on monthly intervals thereafter, until day 150. Passive transfer of antibodies in calves was evaluated by determining the levels of total protein in serum samples collected on day 3. Neutralizing antibody (NAb) titres against LSDV in colostrum and serum samples were determined by virus neutralization test. Colostrum NAb titres >1:160 were associated with the presence of NAbs in serum from calves 3 days after birth. Out of the 18 calves, which received colostrum with NAbs, 16 (88.9%) had detectable NAbs in their serum. Thereafter, a declining percentage of calves with detectable serum NAbs was recorded (38.5% on day 90 and 0% on days 120 and 150). Only calves with high NAb titres on day 3 had detectable serum NAbs until day 90 after calving. Thus, a significant number of calves were not protected by maternal antibodies against the disease after the age of 3 months and likely even after the age of 2 months. The findings of the present study substantiate that current recommendation for vaccination can be amended, so as to minimize the susceptible bovine population and enable optimized LSD prevention and eradication.

Characterisation of putative immunomodulatory gene knockouts of lumpy skin disease virus in cattle towards an improved vaccine.

Lumpy skin disease virus (LSDV) is responsible for causing severe economic losses to cattle farmers throughout Africa, the Middle East, and more recently, South-Eastern Europe and Russia. It belongs to the Capripoxvirus genus of the Poxviridae family, with closely related sheeppox and goatpox viruses. Like other poxviruses, the viral genome codes for a number of genes with putative immunomodulatory capabilities. Current vaccines for protecting cattle against lumpy skin disease (LSD) based on live-attenuated strains of field isolates passaged by cell culture, resulting in random mutations. Although generally effective, these vaccines can have drawbacks, including injection site reactions and/or limited immunogenicity. A pilot study was conducted using a more targeted approach where two putative immunomodulatory genes were deleted separately from the genome of a virulent LSDV field isolate. These were open reading frame (ORF) 005 and ORF008, coding for homologues of an interleukin 10-like and interferon-gamma receptor-like gene, respectively. The resulting knockout constructs were evaluated in cattle for safety, immunogenicity and protection. Severe post-vaccinal reactions and febrile responses were observed for both constructs. Two calves inoculated with the ORF008 knockout construct developed multiple lesions and were euthanised. Following challenge, none of the animals inoculated with the knockout constructs showed any external clinical signs of LSD, compared to the negative controls. Improved cellular and humoral immune responses were recorded in both of these groups compared to the positive control. The results indicate that at the high inoculation doses used, the degree of attenuation achieved was insufficient for further use in cattle due to the adverse reactions observed.

Serological and clinical evaluation of the Yugoslavian RM65 sheep pox strain vaccine use in cattle against lumpy skin disease.

Lumpy skin disease (LSD) is an emerging infectious disease of cattle. Since 2012, it has been seen throughout the Middle East region. The aim of this study was to compare the humoral response of three different dosages of the RM65 sheep pox (SPP) vaccine to assess the use of ten times sheep dose of the RM65 vaccine against lumpy skin disease, and to explore the possible causes of, and characterize the side effects caused by the RM65 vaccine. A blinded randomized collected study comprised 57 clinically normal, Holstein Friesian cattle which were randomly assigned into three experimental groups of 17 cattle according to the vaccine dose used (one, five and ten times the dose used for sheep in the field, and a control group of six cattle that did not receive the vaccine. Experimental animals were monitored closely for the development of any abnormality or side effects. Serum samples were collected for 6 weeks and were tested using serum neutralization assay. Decrease in total milk production was observed a week after vaccination and by the fifth week of the experiment, it had returned to prevaccination levels. Clinical side effects were seen in five animals that belong only to the group that received ten times of the SPP vaccine dose. Observed side effects included fever, decreased feed intake and milk production, as well as skin lesions. Skin nodules appeared between 7 and 17 days postvaccination, and remained for 11-17 days. Systemic reactions were likely to be associated with higher dosage and all affected cattle recovered uneventfully. Animals that received the highest dose (ten times the sheep dose) showed the best humoral response. The actual efficacy of the different concentration of the SPP RM65 should be evaluated based on a challenge experiment in a controlled environment.