Persistence of the protective immunity and kinetics of the isotype specific antibody response against the viral nucleocapsid protein after experimental Schmallenberg virus infection of sheep

Schmallenberg virus (SBV) is an Orthobunyavirus that induces abortion, stillbirths and congenital malformations in ruminants. SBV infection induces a long lasting seroconversion under natural conditions. The persistence of the protective immunity and the isotype specific antibody response upon SBV infection of sheep has however not been studied in detail. Five sheep were kept in BSL3 facilities for more than 16 months and subjected to repeated SBV infections. Blood was regularly sampled and organs were collected at euthanasia. The presence of SBV RNA in serum and organs was measured with quantitative real-time PCR. The appearance and persistence of neutralizing and SBV nucleoprotein (N) isotype specific antibodies was determined with virus neutralization tests (VNT) and ELISAs. The primo SBV infection protected ewes against clinical signs, viraemia and virus replication in organs upon challenge infections more than 15 months later. Production of neutralizing SBV specific antibodies was first detected around 6 days post primo-inoculation with VNT and correlated with the appearance of SBV-N specific IgM antibodies. These IgM antibodies remained present for 2 weeks. SBV-N specific IgG antibodies were first detected between 10 and 21 dpi and reached a plateau at 28 dpi. This plateau remained consistently high and no significant decrease in titre was found over a period of more than 1 year. Similar results were found for the neutralising antibody response. In conclusion, the SBV specific IgM response probably eliminates SBV from the blood and the protective immunity induced by SBV infection protects sheep against reinfection for at least 16 months

Strong protection induced by an experimental DIVA subunit vaccine against bluetongue virus serotype 8 in cattle

AbstractBluetongue virus (BTV) infections in ruminants pose a permanent agricultural threat since new serotypes are constantly emerging in new locations. Clinical disease is mainly observed in sheep, but cattle were unusually affected during an outbreak of BTV seroype 8 (BTV-8) in Europe. We previously developed an experimental vaccine based on recombinant viral protein 2 (VP2) of BTV-8 and non-structural proteins 1 (NS1) and NS2 of BTV-2, mixed with an immunostimulating complex (ISCOM)–matrix adjuvant. We demonstrated that bovine immune responses induced by this vaccine were as good or superior to those induced by a classic commercial inactivated vaccine. In this study, we evaluated the protective efficacy of the experimental vaccine in cattle and, based on the detection of VP7 antibodies, assessed its DIVA compliancy following virus challenge. Two groups of BTV-seronegative calves were subcutaneously immunized twice at a 3-week interval with the subunit vaccine (n=6) or with adjuvant alone (n=6). Following BTV-8 challenge 3 weeks after second immunization, controls developed viremia and fever associated with other mild clinical signs of bluetongue disease, whereas vaccinated animals were clinically and virologically protected. The vaccine-induced protection was likely mediated by high virus-neutralizing antibody titers directed against VP2 and perhaps by cellular responses to NS1 and NS2. T lymphocyte responses were cross-reactive between BTV-2 and BTV-8, suggesting that NS1 and NS2 may provide the basis of an adaptable vaccine that can be varied by using VP2 of different serotypes. The detection of different levels of VP7 antibodies in vaccinated animals and controls after challenge suggested a compliancy between the vaccine and the DIVA companion test. This BTV subunit vaccine is a promising candidate that should be further evaluated and developed to protect against different serotypes

Madin-Darby bovine kidney (MDBK) cells are a suitable cell line for the propagation and study of the bovine poxvirus lumpy skin disease virus

Lumpy skin disease virus (LSDV) is a poxvirus that causes systemic disease in cattle, resulting in substantial economic loss to affected communities. LSDV is a rapidly emerging pathogen of growing global concern that recently spread from Africa and the Middle East into Europe and Asia, impacting the cattle population in these regions. An increase in research efforts into LSDV is required to address key knowledge gaps, however this is hampered by lack of suitable cell lines on which to propagate and study the virus. In this work we describe the replication and spread of LSDV on Madin-Darby bovine kidney (MDBK) cells, and the formation of foci-type poxvirus plaques by LSDV on MDBK cells. Methods utilising MDBK cells to quantify neutralising antibodies to LSDV, and to purify LSDV genomic DNA suitable for short read sequencing are described. These research methods broaden the tools available for LSDV researchers and will facilitate the gathering of evidence to underpin the development of LSD control and prevention programmes

Diagnostic evaluation of assays for detection of antibodies against porcine epidemic diarrhea virus (PEDV) in pigs exposed to different PEDV strains

Porcine epidemic diarrhea virus (PEDV) has caused economic losses in the Americas, Asia and Europe in recent years. Reliable serological assays are essential for epidemiological studies and vaccine evaluation. The objective of this study was to compare the ability of five enzymelinked immunosorbent assays (ELISAs) to detect antibodies against different PEDV strains in pig serum. A total of 732 serum samples from North American or European pigs were tested. Samples included experimental samples from pigs infected with classical (G1a PEDV) or variant genogroup 1 PEDV (G1b PEDV), pandemic genogroup 2 PEDV (G2b PEDV) or noninfected controls. Field samples from herds with confirmed or unknown PEDV exposure were also used. Three indirect ELISAs based on G2b antigens (ELISAs 1, 2 and 3), a competitive ELISA based on the G2b antigen (ELISA 4) and a competitive ELISA based on the G1a antigen (ELISA 5) were compared. Overall, the tests had a moderate agreement (κ = 0.61). G1a PEDV infected pigs were earliest detected by ELISA 3, G1b PEDV infected pigs were earliest detected by ELISAs 4 and 5 and the performance of all tests was similar for the G2b PEDV group. ELISA 1 showed the overall lowest detection on experimentally and field derived samples. Diagnostic sensitivity and specificity with a 95% probability interval were estimated to be 68.2% (62.1 – 74.4%) and 97.5% (95.2 – 99.0%) for ELISA 1, 73.7% (71.5 – 79.6%) and 98.4% (96.6 – 99.5%) for ELISA 2, 86.2% (81.1 – 90.6%) and 91.6% (87.7 – 94.8%) for ELISA 3, 78.3% (72.8 – 83.5%) and 99.7% (98.2 – 100%) for ELISA 4, and 93.5% (90.3 – 96.0%) and 91.2% (83.8 – 97.9%) for ELISA 5. Differences in detection among assays seem to be more related to intrinsic factors of an assay than to the PEDV antigen used

Evaluation of the Immunogenicity of an Experimental Subunit Vaccine That Allows Differentiation between Infected and Vaccinated Animals against Bluetongue Virus Serotype 8 in Cattle

International audienceBluetongue virus (BTV), the causative agent of bluetongue in ruminants, is an emerging virus in northern Europe. The 2006 out-break of BTV serotype 8 (BTV-8) in Europe was marked by an unusual teratogenic effect and a high frequency of clinical signs in cattle. Conventional control strategies targeting small ruminants were therefore extended to include cattle. Since cattle were not routinely vaccinated before 2006, the immune responses to BTV have not been studied extensively in this species. With the aims of developing a subunit vaccine against BTV-8 for differentiation between infected and vaccinated animals based on viral protein 7 (VP7) antibody detection and of improving the current understanding of the immunogenicity of BTV proteins in cattle, the immune responses induced by recombinant VP2 (BTV-8) and nonstructural protein 1 (NS1) and NS2 (BTV-2) were studied. Cows were immunized twice (with a 3-week interval) with the experimental vaccine, a commercial inactivated vaccine, or a placebo. The two vaccines induced similar neutralizing antibody responses to BTV-8. Furthermore, the antibody responses detected against VP2, NS1, and NS2 were strongest in the animals immunized with the experimental vaccine, and for the first time, a serotype cross-reactive antibody response to NS2 was shown in cattle vaccinated with the commercial vaccine. The two vaccines evoked measurable T cell responses against NS1, thereby supporting a bovine cross-reactive T cell response. Finally, VP7 sero-conversion was observed after vaccination with the commercial vaccine, as in natural infections, but not after vaccination with the experimental vaccine, indicating that the experimental vaccine may allow the differentiation of vaccinated animals from infected animals regardless of BTV serotype. The experimental vaccine will be further evaluated during a virulent challenge in a high-containment facility

Evaluation of an IGM-specific ELISA for early detection of bluetongue virus infections in domestic ruminants sera

International audienceCompetitive-ELISA (c-ELISA) is the most widely used serological test for the detection of Bluetongue virus (BTV) viral protein 7 (VP7) antibodies (Ab). However, these BTV c-ELISAs cannot to distinguish between IgG and IgM. IgM Ab are generated shortly after the primary immune response against an infectious agent, indicating a recent infection or exposure to antigens, such as after vaccination. Because the BTV genome or anti-VP7 Ab can be detected in ruminant blood months after infection, BTV diagnostic tools cannot discriminate between recent and old infections. In this study, we evaluated an IgM-capture ELISA prototype to detect ruminant anti-BTV VP7 IgM on 1,650 serum samples from cattle, sheep, or goats. Animals were BTV-naive, infected, or/and vaccinated with BTV-1, -2, -4, -8, -9, -16, or -27, and we also included 30 sera from cattle infected with the Epizootic haemorrhagic disease virus (EHDV) serotype 6. Results demonstrated that this ELISA kit is specific and can detect the presence of IgM with satisfactory diagnostic specificity and sensitivity from 1 to 5 weeks after BTV infection in domestic ruminants (for goats and cattle; for sheep, at least up to 24 days). The peak of anti-VP7 IgM was reached when the level of infectious viruses and BTV RNA in blood were the highest. The possibility of detecting BTV-RNA in IgM-positive sera allows the amplification and sequencing of the partial RNA segment 2 (encoding the serotype specific to VP2) to determine the causative BTV serotype/strain. Therefore, BTV IgM ELISA can detect the introduction of BTV (or EHDV) in an area with BTV-seropositive domestic animals regardless of their serological BTV status. This approach may also be of particular interest for retrospective epidemiological studies on frozen serum samples

Cell mediated immune response to L5P in longitudinal study of heifers from naturally<em> Mycobacterium avium</em> subsp <em>paratuberculosis</em> infected herd

National audiencePeptidyl moiety of cell wall lipopentapeptide (L5P) specific of Mycobacterium avium subsp paratuberculosis (Map) is immunogenic and a target for specific humoral response in Map infected animals. A chemically synthesized L5P is able to induce specific cell mediated immune response (CMIR) in IFN-γ release assay (IGRA) in selected cows from Map infected herds comparatively to non-infected or M. bovis infected. Following these observations, the aim of this study was to evaluate if L5P was an antigen of early specific immune response and potentially a predictive tool of Map infection. 113 heifers of 6 herds were included in a two years’ longitudinal study: 71 animals from three Map culture-confirmed herds, 11 animals from a Map infected herd Silirum® vaccinated during the study and 31 animals from two certified Map free herds. The analysis of the CMIR was investigated by IGRA following whole blood stimulation with synthetic L5P or mycobacterium purified protein derivative (PPD) from M. avium (PPDa), M. bovis (PPDb), Map (PPDj) and M. phlei (PPDp). Humoral immune response was quantified by L5P-based ELISA using an internal procedure and two commercial Map kits. Moreover, bacilli excretion was estimated by isolation and culture from faecal sample. PPDs’ CMIR was more or less high depending of infected herds context, became high over 2 S/P ratio for 10/11 animals just after Silirum® vaccination and was low, less than 0.1 S/P ratio, in certified Map free herds. L5P CMIR was observed in 9 of 71 animals from Map culture-confirmed herds. These 9 animals with a L5P CMIR positive between 0.05 and 0.6 S/P ratio were from the same herd, knowing that L5P CMIR was previously detected in all included Map culture-confirmed herds. L5P CMIR was fluctuant as already described for PPD but was significantly correlated with PPDj CMIR. For 2 of the 9 animals, the L5P CMIR was predictive of the Map positive serology, whereas it was concomitant with seropositivity for 2 others and that for 5 animals was several times observed without seroconversion. And no seroconversion was observed in other herds. The continuation of this study would assess the predictive potential of L5P CMIR for paratuberculosis diagnosis

Development and evaluation of a DIVA subunit vaccine against Bluetongue virus serotype 8 in cattle

Session 3 : Antivirals & VaccinesThe incursion and circulation of more than nine different serotypes of Bluetongue virus (BTV) in central and northern Europe over the past fifteen years has resulted in decreased animal welfare and production losses. Vaccination is crucial for controlling BTV since conventional biosecurity measures have limited impact on vector-borne diseases. However, as current vaccines have no accepted characteristic allowing for the differentiation of infected from vaccinated animals (DIVA), the use of these products impairs sero-epidemiological surveillance and thus a quick return to BTV-free status as well as serological monitoring of vaccine efficacy in the field. Small ruminants show the most severe clinical signs following BTV infection, but cattle are the virus’s main amplifying host and when infected with certain BTV strains, such as BTV-8, they can also display clinical signs. However, protective bovine immune responses against BTV are poorly characterized and there is limited information available about the ability of commercial and experimental vaccines to induce clinical and virological protection against BTV infection in cattle. Our objective was to develop and evaluate the efficacy of a novel DIVA subunit vaccine against BTV-8 in cattle, that later may be developed into a multi-serotype vaccine. We produced recombinant VP2 of BTV-8 and NS1 and NS2 of BTV-2 and formulated the vaccine with the purified proteins and an ISCOM-based adjuvant (SubV). The DIVA characteristic of this vaccine is based on the detection of VP7 antibodies in infected animals, thereby enabling the detection of BTV infection of any serotype. In the first study, we evaluated the immunogenicity of SubV in comparison with a commercial inactivated vaccine against BTV-8. Cows were subcutaneously immunized twice at a 3-weeks interval with SubV (n=5), the commercial vaccine (n=5), or with placebo (n=5). Humoral and cell-mediated immune responses were monitored before each vaccination as well as at 3 and 6 weeks after the second immunization. Both vaccines induced similar serum neutralizing antibody titers and the specific IgG1 antibody responses detected against VP2, NS1, and NS2 were strongest in cows immunized with SubV. Furthermore, serotype cross-reactive humoral and cell-mediated immunological responses to BTV-8 were detected to NS2 and NS1 of BTV-2, respectively. In the second study, calves were immunized twice at a 3-weeks interval with either SubV (n=6) or placebo (n=6), then challenged with BTV-8 three weeks later in a Biosecurity level 3 facility. Whereas controls developed strong viremia and clinical signs of Bluetongue disease including fever, mucosal congestion, and stiffness, vaccinated calves did not show any of these signs and were strongly protected against BTV infection. Preliminary data indicate that vaccinated animals developed very strong serum neutralizing antibody responses following vaccination. Humoral and cellular immunological analyses are ongoing and clinical, virological, and immunological results of both studies will be presented during the meeting. Taken together, these data indicate that our novel subunit DIVA vaccine composed of only three BTV proteins is very effective against BTV-8 infection in cattle, and will also enable serological monitoring of any BTV serotype in circulation in vaccinated populations. Furthermore, the serotype cross-reactivity of NS1 and NS2 suggests that this vaccine may be expanded in the future to target multiple BTV serotypes through the addition of purified recombinant VP2 of other serotypes

Development and evaluation of a DIVA subunit vaccine against Bluetongue virus serotype 8 in cattle

Session 3 : Antivirals & VaccinesThe incursion and circulation of more than nine different serotypes of Bluetongue virus (BTV) in central and northern Europe over the past fifteen years has resulted in decreased animal welfare and production losses. Vaccination is crucial for controlling BTV since conventional biosecurity measures have limited impact on vector-borne diseases. However, as current vaccines have no accepted characteristic allowing for the differentiation of infected from vaccinated animals (DIVA), the use of these products impairs sero-epidemiological surveillance and thus a quick return to BTV-free status as well as serological monitoring of vaccine efficacy in the field. Small ruminants show the most severe clinical signs following BTV infection, but cattle are the virus’s main amplifying host and when infected with certain BTV strains, such as BTV-8, they can also display clinical signs. However, protective bovine immune responses against BTV are poorly characterized and there is limited information available about the ability of commercial and experimental vaccines to induce clinical and virological protection against BTV infection in cattle. Our objective was to develop and evaluate the efficacy of a novel DIVA subunit vaccine against BTV-8 in cattle, that later may be developed into a multi-serotype vaccine. We produced recombinant VP2 of BTV-8 and NS1 and NS2 of BTV-2 and formulated the vaccine with the purified proteins and an ISCOM-based adjuvant (SubV). The DIVA characteristic of this vaccine is based on the detection of VP7 antibodies in infected animals, thereby enabling the detection of BTV infection of any serotype. In the first study, we evaluated the immunogenicity of SubV in comparison with a commercial inactivated vaccine against BTV-8. Cows were subcutaneously immunized twice at a 3-weeks interval with SubV (n=5), the commercial vaccine (n=5), or with placebo (n=5). Humoral and cell-mediated immune responses were monitored before each vaccination as well as at 3 and 6 weeks after the second immunization. Both vaccines induced similar serum neutralizing antibody titers and the specific IgG1 antibody responses detected against VP2, NS1, and NS2 were strongest in cows immunized with SubV. Furthermore, serotype cross-reactive humoral and cell-mediated immunological responses to BTV-8 were detected to NS2 and NS1 of BTV-2, respectively. In the second study, calves were immunized twice at a 3-weeks interval with either SubV (n=6) or placebo (n=6), then challenged with BTV-8 three weeks later in a Biosecurity level 3 facility. Whereas controls developed strong viremia and clinical signs of Bluetongue disease including fever, mucosal congestion, and stiffness, vaccinated calves did not show any of these signs and were strongly protected against BTV infection. Preliminary data indicate that vaccinated animals developed very strong serum neutralizing antibody responses following vaccination. Humoral and cellular immunological analyses are ongoing and clinical, virological, and immunological results of both studies will be presented during the meeting. Taken together, these data indicate that our novel subunit DIVA vaccine composed of only three BTV proteins is very effective against BTV-8 infection in cattle, and will also enable serological monitoring of any BTV serotype in circulation in vaccinated populations. Furthermore, the serotype cross-reactivity of NS1 and NS2 suggests that this vaccine may be expanded in the future to target multiple BTV serotypes through the addition of purified recombinant VP2 of other serotypes

Diagnostic evaluation of assays for detection of antibodies against porcine epidemic diarrhea virus (PEDV) in pigs exposed to different PEDV strains

Porcine epidemic diarrhea virus (PEDV) has caused economic losses in the Americas, Asia and Europe in recent years. Reliable serological assays are essential for epidemiological studies and vaccine evaluation. The objective of this study was to compare the ability of five enzymelinked immunosorbent assays (ELISAs) to detect antibodies against different PEDV strains in pig serum. A total of 732 serum samples from North American or European pigs were tested. Samples included experimental samples from pigs infected with classical (G1a PEDV) or variant genogroup 1 PEDV (G1b PEDV), pandemic genogroup 2 PEDV (G2b PEDV) or noninfected controls. Field samples from herds with confirmed or unknown PEDV exposure were also used. Three indirect ELISAs based on G2b antigens (ELISAs 1, 2 and 3), a competitive ELISA based on the G2b antigen (ELISA 4) and a competitive ELISA based on the G1a antigen (ELISA 5) were compared. Overall, the tests had a moderate agreement (κ = 0.61). G1a PEDV infected pigs were earliest detected by ELISA 3, G1b PEDV infected pigs were earliest detected by ELISAs 4 and 5 and the performance of all tests was similar for the G2b PEDV group. ELISA 1 showed the overall lowest detection on experimentally and field derived samples. Diagnostic sensitivity and specificity with a 95% probability interval were estimated to be 68.2% (62.1 – 74.4%) and 97.5% (95.2 – 99.0%) for ELISA 1, 73.7% (71.5 – 79.6%) and 98.4% (96.6 – 99.5%) for ELISA 2, 86.2% (81.1 – 90.6%) and 91.6% (87.7 – 94.8%) for ELISA 3, 78.3% (72.8 – 83.5%) and 99.7% (98.2 – 100%) for ELISA 4, and 93.5% (90.3 – 96.0%) and 91.2% (83.8 – 97.9%) for ELISA 5. Differences in detection among assays seem to be more related to intrinsic factors of an assay than to the PEDV antigen used.This is a manuscript of an article published as Gerber, Priscilla F., Davide Lelli, Jianqiang Zhang, Bertel Strandbygaard, Ana Moreno, Antonio Lavazza, Simona Perulli et al. "Diagnostic evaluation of assays for detection of antibodies against porcine epidemic diarrhea virus (PEDV) in pigs exposed to different PEDV strains." Preventive veterinary medicine 135 (2016): 87-94. doi: 10.1016/j.prevetmed.2016.11.005. Posted with permission.</p