A Serological Protein Microarray for Detection of Multiple Cross‐Reactive Flavivirus Infections in Horses for Veterinary and Public Health Surveillance

The genus Flavivirus in the family Flaviviridae includes some of the most important examples of emerging zoonotic arboviruses that are rapidly spreading across the globe. Japanese encephalitis virus (JEV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV) and Usutu virus (USUV) are mosquito‐borne members of the JEV serological group. Although most infections in humans are asymptomatic or present with mild flu‐like symptoms, clinical manifestations of JEV, WNV, SLEV, USUV and tick‐borne encephalitis virus (TBEV) can include severe neurological disease and death. In horses, infection with WNV and JEV can lead to severe neurological disease and death, while USUV, SLEV and TBEV infections are mainly asymptomatic, however, and induce antibody responses. Horses often serve as sentinels to monitor active virus circulation in serological surveillance programmes specifically for WNV, USUV and JEV. Here, we developed and validated a NS1‐antigen protein microarray for the serological differential diagnosis of flavivirus infections in horses using sera of experimentally and naturally infected symptomatic as well as asymptomatic horses. Using samples from experimentally infected horses, an IgG and IgM specificity of 100% and a sensitivity of 95% for WNV and 100% for JEV was achieved with a cut‐off titre of 1 : 20 based on ROC calculation. In field settings, the microarray identified 93–100% of IgG‐positive horses with recent WNV infections and 87% of TBEV IgG‐positive horses. WNV IgM sensitivity was 80%. Differentiation between closely related flaviviruses by the NS1‐antigen protein microarray is possible, even though we identified some instances of cross‐reactivity among antibodies. However, the assay is not able to differentiate between naturally infected horses and animals vaccinated with an inactivated WNV whole‐virus vaccine. We showed that the NS1‐microarray can potentially be used for diagnosing and distinguishing flavivirus infections in horses and for public health purposes within a surveillance setting. This allows for fast, cheap, syndrome‐based laboratory testing for multiple viruses simultaneously for veterinary and public health purposes.     

Influence of flavivirus co‐circulation in serological diagnostics and surveillance: A model of study using West Nile,Usutu and Bagaza viruses

This study aims at assessing the serological cross‐reactions existing between three mosquito‐borne flaviviruses with avian reservoirs co‐circulating in Europe: West Nile (WNV), Usutu (USUV) and Bagaza (BAGV). The study is useful for a better interpretation of serological results in diagnostics and surveillance. Serum samples obtained from a natural host, the red‐legged partridge (Alectoris rufa), experimentally infected with WNV, USUV or BAGV were analysed using two commercially available WNV competition ELISAs suitable for serological surveillance, and by the confirmatory virus neutralization test (VNT). The ELISAs examined showed different levels of specificity for WNV, as judged by cross‐reaction observed with the other flaviviruses. By VNT, virus‐specific antibodies were confirmed in 80%, 50% or 0% of sera from WNV‐, BAGV‐, or USUV‐inoculated birds, respectively. The results indicate how the co‐circulation of cross‐reacting flaviviruses may affect the outcomes of WNV serological surveillance when applying currently available serological tools. On the one hand, the choice of the ELISA test for antibody screening should consider the differences found in specificity, since one test is more specific for WNV while the other one is more suitable for detection of a broader range of flavivirus antibodies. On the other hand, besides corroborating that cross‐neutralization occurs between flaviviruses from different serocomplexes (WNV/USUV and BAGV), this study points out that cross‐neutralization between WNV and USUV is not symmetric, and reveals the difficulty to identify USUV infections serologically. This finding indicates that actual USUV infections might be underestimated in the current diagnostic schemes.     

Are tree squirrels involved in the circulation of flaviviruses in Italy?

West Nile virus (WNV ), Usutu virus (USUV ) and tick‐borne encephalitis virus (TBEV ) are emerging zoonotic flaviviruses (family Flaviviridae ), which have circulated in Europe in the past decade. A cross‐sectional study was conducted to assess exposure to these antigenically related flaviviruses in eastern grey squirrels (Sciurus carolinensis ) in Italy. Seventeen out of 158 (10.8%; CI _95%: 5.9‐15.6) squirrels’ sera tested through bELISA had antibodies against flaviviruses. Specific neutralizing antibodies to WNV , USUV and TBEV were detected by virus neutralization tests. Our results indicate that tree squirrels are exposed to Culex and tick‐borne zoonotic flaviviruses in Italy. Moreover, this study shows for the first time USUV and TBEV exposure in grey squirrels, broadening the host range reported for these viruses. Even though further studies are needed to define the real role of tree squirrels in the epidemiology of flaviviruses in Europe, this study highlights that serology could be an effective approach for future investigations aimed at broadening our knowledge about the species exposed to these zoonotic infections.     

Exposure to West Nile virus and tick‐borne encephalitis virus in dogs in Spain

In the past decade, the spread of emerging zoonotic flaviviruses (genus Flavivirus , family Flaviviridae ) has been reported in many regions worldwide, representing a threat to both human and animal health. A serosurvey was carried out to assess exposure and risk factors associated with antigenically related flaviviruses, particularly West Nile virus (WNV ), Usutu virus (USUV ) and tick‐borne encephalitis virus (TBEV ), in dogs in Spain. Flavivirus antibodies were detected in 39 of 815 dogs (4.8%; 95% CI : 3.3–6.3) by bELISA . Significantly higher seropositivity was observed in hunting dogs compared to pet dogs. Virus neutralization tests confirmed WNV ‐specific and TBEV ‐specific antibodies in 11 and 14 bELISA ‐positive dogs, respectively. This is the first serosurvey of WNV and TBEV in dogs in Spain and the first report of TBEV circulation in this country. The seropositivity obtained indicates widespread, but not homogeneous, distribution of WNV and TBEV in dogs in Spain. In 2013 and 2015, WNV ‐seropositive dogs were detected in those areas of Andalusia where the highest number of WNV outbreaks were reported in both horses and humans. Antibodies against TBEV have been found in dogs sampled in two different periods and regions in Spain. Serosurveillance in dogs could be a complementary way of monitoring the activity of emerging flaviviruses in Spain.     

Prevalence and molecular epidemiology of West Nile and Usutu virus infections in Croatia in the ‘One health’ context, 2018

In 2018, Croatia reported the largest outbreak of West Nile virus (WNV) infections as well as the re‐occurrence of human Usutu virus (USUV) infections. For the first time, fatal WNV and USUV infections were detected in wild birds. We analysed epidemiological characteristics and molecular epidemiology of WNV and USUV infections detected during 2018 transmission season. From April to November, 178 patients with neuroinvasive disease and 68 patients with febrile disease were tested for WNV and USUV. Viral RNA was detected in cerebrospinal fluid (CSF) and urine samples using a real‐time RT‐PCR. Positive samples were tested by nested RT‐PCR and nucleotide sequencing. IgM/IgG antibodies were detected in serum/CSF samples using ELISA with confirmation of cross‐reactive samples by virus neutralization test (VNT). WNV neuroinvasive disease was confirmed in 54 and WNV fever in seven patients from 10 continental Croatian counties. Areas affected in 2018 were those in which cases occurred in previous seasons, while in three areas human cases were reported for the first time. Phylogenetic analysis of six strains from patients residing in different geographic areas showed circulation of WNV lineage 2. In three patients, neuroinvasive USUV infection was confirmed by RT‐PCR or VNT. Sequence analysis of one detected strain revealed USUV Europe 2 lineage. During the same period, a total of 2,574 horse and 1,069 poultry serum samples were tested for WNV antibodies using ELISA. Acute asymptomatic WNV infection (IgM antibodies) was documented in 20/0.7% horses. WNV IgG antibodies were found in 307/11.9% horses and in 125/12.7% poultry. WNV RNA was detected in two goshawks and USUV RNA was detected in one blackbird from north‐western Croatia. In the Zagreb area, 3,670 female mosquitoes were collected. One Culex pipiens pool collected in July tested positive for USUV RNA. Our results highlight the importance of continuous multidisciplinary ‘One health’ surveillance of these emerging arboviruses.     

Development of a Double‐Antigen Microsphere Immunoassay for Simultaneous Group and Serotype Detection of Bluetongue Virus Antibodies

Bluetongue viruses (BTV) are arboviruses responsible for infections in ruminants. The confirmation of BTV infections is based on rapid serological tests such as enzyme‐linked immunosorbent assays (ELISAs) using the BTV viral protein 7 (VP7) as antigen. The determination of the BTV serotype by serological analyses could be only performed by neutralization tests (VNT) which are time‐consuming and require BSL3 facilities. VP2 protein is considered the major serotype‐defining protein of BTV. To improve the serological characterization of BTV infections, the recombinant VP7 and BTV serotype 8 (BTV‐8) VP2 were synthesized using insect cells expression system. The purified antigens were covalently bound to fluorescent beads and then assayed with 822 characterized ruminant sera from BTV vaccinations or infections in a duplex microsphere immunoassay (MIA). The revelation step of this serological duplex assay was performed with biotinylated antigens instead of antispecies conjugates to use it on different ruminant species. The results demonstrated that MIA detected the anti‐VP7 antibodies with a high specificity as well as a competitive ELISA approved for BTV diagnosis, with a better efficiency for the early detection of the anti‐VP7 antibodies. The VP2 MIA results showed that this technology is also an alternative to VNT for BTV diagnosis. Comparisons between the VP2 MIA and VNT results showed that VNT detects the anti‐VP2 antibodies in an early stage and that the VP2 MIA is as specific as VNT. This novel immunoassay provides a platform for developing multiplex assays, in which the presence of antibodies against multiple BTV serotypes can be detected simultaneously.     

Emergence of Equine West Nile Encephalitis in Central Macedonia,Greece, 2010

During the summer of 2010, an outbreak of West Nile virus (WNV) infections attributed to a lineage 2 WNV strain was reported among humans and horses in Central Macedonia, Northern Greece. Here, the clinical and laboratory investigation of horses that showed severe neurological signs due to WNV infection is being described. Specifically, between August and September 2010, 17 horses with neurological signs were detected. WNV infection was confirmed in all 17 clinical cases by applying laboratory testing. The duration of WNV‐specific IgM antibodies in sera obtained from seven of the clinically affected horses was relatively short (10–60 days; mean 44 days). In the regional unit of Thessaloniki, (i) seroprevalence of WNV and fatality rate in horses were high (33% and 30%, respectively), and (ii) the ratio of neurological manifestations‐to‐infections for this virus strain was high (19%). These observations indicate that the strain responsible for the massive human epidemic of 2010 in Greece was also highly pathogenic for horses. This is the first time that WNV infection has been documented in horses with clinical manifestations in Greece. WNV infection should be included in the differential diagnosis of horses with encephalitis in Greece.     

Mapping the Serological Prevalence Rate of West Nile fever in Equids,Tunisia

West Nile fever (WNF) is a viral disease of wild birds transmitted by mosquitoes. Humans and equids can also be affected and suffer from meningoencephalitis. In Tunisia, two outbreaks of WNF occurred in humans in 1997 and 2003; sporadic cases were reported on several other years. Small‐scale serological surveys revealed the presence of antibodies against WN virus (WNV) in equid sera. However, clinical cases were never reported in equids, although their population is abundant in Tunisia. This study was achieved to characterize the nationwide serological status of WNV in Tunisian equids. In total, 1189 sera were collected in 2009 during a cross‐sectional survey. Sera were tested for IgG antibodies, using ELISA and microneutralization tests. The estimated overall seroprevalence rate was 28%, 95% confidence interval [22; 34]. The highest rates were observed (i) in the north‐eastern governorates (Jendouba, 74%), (ii) on the eastern coast (Monastir, 64%) and (iii) in the lowlands of Chott El Jerid and Chott el Gharsa (Kebili, 58%; Tozeur, 52%). Environmental risk factors were assessed, including various indicators of wetlands, wild avifauna, night temperature and chlorophyllous activity (normalized difference vegetation index: NDVI). Multimodel inference showed that lower distance to ornithological sites and wetlands, lower night‐time temperature, and higher NDVI in late spring and late fall were associated with higher serological prevalence rate. The model‐predicted nationwide map of WNF seroprevalence rate in Tunisian equids highlighted different areas with high seroprevalence probability. These findings are discussed in the perspective of implementing a better WNF surveillance system in Tunisia. This system might rely on (i) a longitudinal survey of sentinel birds in high‐risk areas and time periods for WNV transmission, (ii) investigations of bird die‐offs and (iii) syndromic surveillance of equine meningoencephalitis.     

Development and evaluation of swine vesicular disease isotype‐specific antibody ELISAs based on recombinant virus‐like particles

Swine vesicular disease (SVD) is a contagious viral disease of pigs. The clinical signs of SVD are indistinguishable from other vesicular diseases, such as senecavirus A infection (SVA) and foot‐and‐mouth disease (FMD). Rapid and accurate diagnostic tests of SVD are considered essential in countries free of vesicular diseases. Competitive ELISA (cELISA) is the serological test used routinely. However, although cELISA is the standard test for SVD antibody testing, this test produces a small number of false‐positive results, which caused problems in international trade. The current project developed a SVD isotype antibody ELISA using recombinant SVD virus‐like particles (VLP) and an SVD‐specific monoclonal antibody (mAb) to reduce the percentage of false positives. The diagnostic specificities of SVD‐VLP isotype ELISAs were 98.7% and 99.6% for IgM and IgG. The SVD isotype ELISAs were SVD‐specific, without cross‐reactivity to other vesicular diseases. A panel of 16 SVD‐positive reference sera was evaluated using the SVD‐VLP isotype ELISAs. All sera were correctly identified as positive by the two combined SVD‐VLP isotype ELISAs. Comparison of the test results showed a high level of correlation between the SVDV antigen isotype ELISAs and SVD‐VLP isotype ELISAs. 303 sera from animals lacking clinical signs and history of SVDV exposure were identified positive using SVD cELISA. These samples were examined using SVD‐VLP isotype ELISAs. Of the 303 serum samples, five were positive for IgM, and five of 303 were positive for IgG. Comparable to virus neutralization test results, SVD isotype ELISAs significantly reduced the false‐positive samples. Based on above test results, the combined use of cELISA and isotype ELISAs can reduce the number of false‐positive samples and the use of time‐consuming virus neutralization tests, with benefit for international trade in swine and related products.     

Evaluation of Screening Assays for the Detection of Influenza A Virus Serum Antibodies in Swine

Increased surveillance of influenza A virus (IAV) infections in human and swine populations is mandated by public health and animal health concerns. Antibody assays have proven useful in previous surveillance programmes because antibodies provide a record of prior exposure and the technology is inexpensive. The objective of this research was to compare the performance of influenza serum antibody assays using samples collected from pigs (vaccinated or unvaccinated) inoculated with either A/Swine/OH/511445/2007 γ H1N1 virus or A/Swine/Illinois/02907/2009 Cluster IV H3N2 virus and followed for 42 days. Weekly serum samples were tested for anti‐IAV antibodies using homologous and heterologous haemagglutination‐inhibition (HI) assays, commercial swine influenza H1N1 and H3N2 indirect ELISAs, and a commercial influenza nucleoprotein (NP)‐blocking ELISA. The homologous HIs showed 100% diagnostic sensitivity, but largely failed to detect infection with the heterologous virus. With diagnostic sensitivities of 1.4% and 4.9%, respectively, the H1N1 and H3N2 indirect ELISAs were ineffective at detecting IAV antibodies in swine infected with the contemporary influenza viruses used in the study. At a cut‐off of S/N ≤ 0.60, the sensitivity and specificity of the NP‐blocking ELISA were estimated at 95.5% and 99.6%, respectively. Statistically significant factors which affected S/N results include vaccination status, inoculum (virus subtype), day post‐inoculation and the interactions between those factors (P < 0.0001). Serum antibodies against NP provide an ideal universal diagnostic screening target and could provide a cost‐effective approach for the detection and surveillance of IAV infections in swine populations.     

Retrospective serological analysis reveals presence of the emerging lagovirus RHDV2 in Australia in wild rabbits at least five months prior to its first detection

The lagovirus rabbit haemorrhagic disease virus (RHDV) has been circulating in Australia since the mid‐1990s when it was released to control overabundant rabbit populations. In recent years, the viral diversity of different RHDVs in Australia has increased, and currently four different types of RHDV are known to be circulating. To allow for ongoing epidemiological studies and impact assessments of these viruses on Australian wild rabbit populations, it is essential that serological tools are updated. To this end, reference sera were produced against all four virulent RHDVs (RHDV, RHDV2 and two different strains of RHDVa) known to be present in Australia and tested in a series of available immunological assays originally developed for the prototype RHDV, to assess patterns of cross‐reactivity and the usefulness of these assays to detect lagovirus antibodies, either in a generic or specific manner. Enzyme‐linked immunosorbent assays (ELISAs) developed to detect antibody isotypes IgM, IgA and IgG were sufficiently cross‐reactive to detect antibodies raised against all four virulent lagoviruses. For the more specific detection of antibodies to the antigenically more different RHDV2, a competition ELISA was adapted using RHDV2‐specific monoclonal antibodies in combination with Australian viral antigen. Archival serum banks from a long‐term rabbit monitoring site where rabbits were sampled quarterly over a period of 6 years were re‐screened using this assay and revealed serological evidence for the arrival of RHDV2 in this population at least 5 months prior to its initial detection in Australia in a dead rabbit in May 2015. The serological methods and reference reagents described here will provide valuable tools to study presence, prevalence and impact of RHDV2 on Australian rabbit populations; however, the discrimination of different antigenic variants of RHDVs as well as mixed infections at the serological level remains challenging.     

Analysis of the Complete Genome of Tembusu Virus,a Flavivirus Isolated from Ducks in China

During investigations into the outbreak of duck viral infection in 2010 in China, with a severe drop in egg production, a flavivirus was isolated from the affected ducks. It was characterized as a Tembusu virus (TMUV). In this study, we obtained a complete genome sequence of Tembusu virus using RT‐PCR and RACE techniques. TMUV genome is a singled‐stranded RNA, with 10 990 nucleotides in length, and contains a single open reading frame (3410 amino acids) encoding 11 viral proteins with 5′and 3′non‐translated regions (NTRs) of 142 and 618 nt, respectively. We characterized the open reading frame (ORF) with respect to gene sizes, cleavage sites and potential glycosylation sites. The different genomic regions of the virus were also compared with those of six other flaviviruses including Japanese encephalitis virus, West Nile virus (WNV), dengue‐2 virus, yellow fever virus, tick‐borne encephalitis virus (TBEV) and Bagaza virus. TMUV demonstrated the highest similarity to Bagaza virus. The result of entire ORF scanning shows that TMUV was close to Bagaza viruses in genetic relatedness. These data demonstrate that TMUV is a unique virus among the mosquito‐borne flaviviruses and also provide a useful reference for a critically important study to determine why TMUV is a serious pathogen for ducks.     

Serological Evidence Indicates that Foot‐and‐Mouth Disease Virus Serotype O,C and SAT1 are most Dominant in Eritrea

Foot‐and‐mouth disease (FMD) is endemic in Eritrea and in most parts of Africa. To be able to control FMD using vaccination, information on the occurrence of various foot‐and‐mouth disease serotypes in Eritrea is needed. In this cross‐sectional study, 212 sera samples were collected from FMD infected and recovered animals in Eritrea. These samples were tested for the presence of antibodies against FMD non‐structural proteins (NSP) and neutralizing antibodies against six of the seven (all but SAT 3) serotypes of FMD virus (FMDV). Of these, 67.0% tested positive to non‐structural protein antibodies in the FMD NS ELISA. By virus neutralization, FMDV serotype O antibodies were shown to be the most dominant (approximately 50%). Virus neutralization test results indicate that infection with serotype C and SAT 1 might have occurred, although there are no reports of isolation of these two serotypes. Because the samples were not randomly selected, further random serological surveillance in all age group animals is necessary both to estimate the prevalence of FMD in the country and to confirm the serological results with serotype C and SAT 1.     

The serologic investigation and viral isolation of bluetongue virus in Shangri‐La in Southwest China

Bluetongue is an arthropod‐borne viral disease of ruminants caused by bluetongue virus (BTV). In China, BTV is relatively common in Yunnan Province with the exception of northern regions around Shangri‐La, where the average altitude is approximately 3,450 metres. Recently, the seroprevalence of BTV has been measured in yaks in Shangri‐La; therefore, this study investigated BTV infections in this area. The serological investigation in five villages in Shangri‐La showed that there were sporadic BTV infections in yaks (20 of 507 positive) during 2014 to 2017, while the seroprevalence of BTV at three goat farms in a nearby river valley was 35%–65% in 2017. Subsequently, 20 sentinel goats were kept on two separate farms in the river valley and monitored for seroconversion between May and September of 2017. Five of the sentinel animals were tested positive for antibodies to BTV by C‐ELISA during the study period, and 13 BTV isolates were isolated from ten sentinel animals. All isolates were identified as the same serotype, and the complete nucleotide sequence of one was determined. The genomic sequences showed that the isolated BTV strain belonged to serotype 21 and had approximately 99.8%–100% homology with three Indonesian BTV‐21 strains (D151, RIVS‐66 and RIVS‐60) between their coding sequences (CDSs) except for Seg4 (99.5%). Besides, our data suggested that this BTV‐21 strain might have also infected some local yaks and sheep.     

The impact of serum incubation time on IgM/IgG binding to porcine aortic endothelial cells

The results of the assay for measuring anti‐non‐Gal antibodies (which affect pig xenograft survival) in recipients are important. Serum incubation time and concentration may be important factors in the extent of antibody binding to the graft. The aim of this in vitro study was to determine the optimal incubation time and serum concentration for measuring anti‐non‐Gal antibody binding to porcine aortic endothelial cells (pAECs). Pooled human , naive, and sensitized baboon sera were incubated with wild‐type, α1,3‐galactosyltransferase gene‐knockout (GTKO), and GTKO/human CD55 pAECs. IgM/IgG binding to pAECs after varying serum incubation times (0.5, 1, 2, and 3 hour) and concentrations (5, 10, 20, and 40 μL) was determined by flow cytometry. An increase in incubation time from 30 minutes to 2 hour was associated with increases in anti‐non‐Gal IgM/IgG binding to GTKO and GTKO/hCD55 pAECs of pooled human , naive and sensitized baboon sera (P<.05). Pooled human serum showed a significant increase in anti‐non‐Gal IgM (1.5 times) and a minimal increase in anti‐non‐Gal IgG antibody binding. IgM/IgG binding of sensitized baboon serum to GTKO pAECs after 2‐hour incubation was 1.5 times and 2 times greater than after 30‐minutes incubation, respectively, whereas naïve baboon sera showed minimal (non‐significant) increase in anti‐non‐Gal IgM/IgG antibody binding. With 2‐hour incubation, increasing the serum concentration from 5 μL to 20 μL significantly increased antibody binding to non‐Gal antigens in pooled human and sensitized baboon serum. With naïve baboon serum, only IgG was significantly increased. Increasing the serum incubation time contributed to improve the sensitivity of detecting anti‐non‐Gal antibodies, without affecting cell viability in vitro.     

Incidence and cytotoxicity of antibodies in cynomolgus monkeys directed to nonGal antigens, and their relevance for experimental models

The recent availability of pigs homozygous for alpha1,3-galactosyltransferase gene-knockout (GT-KO) has enabled the study of incidence and cytotoxicity of antibodies of cynomolgus monkeys directed to antigens other than Galalpha1,3Gal (Gal), termed nonGal antigens. To this aim, sera from 21 cynomolgus monkeys were tested by flow cytometry for binding of IgM and IgG to peripheral blood mononuclear cells (PBMC) from wild-type (WT) and GT-KO pigs. The sera were also tested for complement-dependent cytotoxicity to WT and GT-KO PBMC. Anti-WT IgM and IgG were found in 100% and 95%, respectively, and anti-GT-KO IgM and IgG in 76% and 66%, respectively, in the sera of the monkeys tested (P < 0.01). Whereas 100% of sera were cytotoxic to WT PBMC, only 76% were cytotoxic to GT-KO PBMC, and the level of cytotoxicity was significantly less (P < 0.01). Although the incidence and cytotoxicity of antibodies in monkey sera to GT-KO pig PBMC are significantly less than to WT PBMC, approximately three-quarters of the monkeys tested had cytotoxic antibodies to GT-KO PBMC. This incidence of cytotoxicity is significantly higher than that found in baboons and humans, suggesting the baboon may be an easier and possibly more suitable model to study antibody-mediated rejection of transplanted GT-KO pig organs and cells.     

Allosensitized humans are at no greater risk of humoral rejection of GT-KO pig organs than other humans

BACKGROUND: The availability of pigs homozygous for alpha1,3-galactosyltransferase gene-knockout (GT-KO) has enabled study of the incidence and cytotoxicity of primate antibodies directed to antigens other than Galalpha1,3Gal (Gal), termed non-Gal antigens. METHODS: Sera from 27 healthy humans and 31 patients awaiting renal allotransplantation, who were either unsensitized [panel reactive antibodies (PRA) < 10%] or allosensitized (PRA > 70%), were tested by flow cytometry for binding of immunoglobulin M (IgM) and IgG to peripheral blood mononuclear cells (PBMC) from both wild-type (WT) and GT-KO pigs. Complement-dependent cytotoxicity to WT and GT-KO PBMC was also measured. RESULTS: IgM and IgG from all 27 (100%) healthy human sera bound to WT PBMC, while 78% and 63% of these sera had IgM and IgG that bound to GT-KO PBMC, respectively. Mean binding to WT PBMC was significantly greater than GT-KO PBMC. Whereas 100% of sera were cytotoxic to WT PBMC, only 61% were cytotoxic to GT-KO PBMC, and the extent of lysis was significantly less. Neither mean binding of IgM and IgG nor cytotoxicity of unsensitized and allosensitized sera to WT and GT-KO PBMC was significantly different to that of healthy sera. CONCLUSIONS: More than half of the healthy humans tested had cytotoxic antibodies to GT-KO PBMC, but allosensitized patients will be at no greater risk of rejecting a pig xenograft by a humoral mechanism.     

Human leukocyte antigen class II DQ alpha and beta epitopes identified from sera of kidney allograft recipients

BACKGROUND: Epitopes are the sites to which antibodies bind. Both alpha and beta peptide chains of the human leukocyte antigen-DQ heterodimers (DQA1 and DQB1, respectively) contain polymorphic regions. We can identify DQA1 and DQB1 epitopes by DQ single antigen beads assay of the antibodies, correlating the beads' reaction patterns with either DQA1 or DQB1 alleles. METHODS: Sera from 74 transplant patients and 35 mouse DQB1 monoclonal antibodies were tested with DQ single antigen beads for their DQ allelic and serological specificities. Epitopes were defined by amino acids shared by the positive antigens of the antibodies. Unique amino acids were identified as potential epitope sites by comparing the peptide sequences of all human leukocyte antigen class II alleles. For the absorption or elution, patient's serum sample was absorbed by a homozygous B-lymphoblast cell line of specific DQ typing, the eluted antibody then tested with single antigen beads to demonstrate that the antibody reacted to a single epitope shared by multiple DQ antigens. RESULTS: Three DQA1 and 15 DQB1 epitopes were identified. We found that 21 patients produced antibodies against one of the DQA1 epitopes; 27 patients produced antibodies against one of the DQB1 epitopes. CONCLUSION: The DQA1 and DQB1 epitopes identified here seem to be immunogenic and to elicit DQ antibodies. For the DQB1 epitopes, multiple DQ serological specificities that were detected in the serum of a transplant patient could be explained as a single donor-specific DQ antibody reacting to a mismatched DQ epitope of the donor. Ten examples are shown here.     

Pretransplant Donor‐Specific HLA Class‐I and ‐II Antibodies Are Associated With an Increased Risk for Kidney Graft Failure

Pretransplant risk assessment of graft failure is important for donor selection and choice of immunosuppressive treatment. We examined the relation between kidney graft failure and presence of IgG donor specific HLA antibodies (DSA) or C1q‐fixing DSA, detected by single antigen bead array (SAB) in pretransplant sera from 837 transplantations. IgG‐DSA were found in 290 (35%) sera, whereas only 30 (4%) sera had C1q‐fixing DSA. Patients with both class‐I plus ‐II DSA had a 10 yr graft survival of 30% versus 72% in patients without HLA antibodies (p < 0.001). No significant difference was observed in graft survival between patients with or without C1q‐fixing DSA. Direct comparison of both assays showed that high mean fluorescence intensity values on the pan‐IgG SAB assay are generally related to C1q‐fixation. We conclude that the presence of class‐I plus ‐II IgG DSA as detected by SAB in pretransplant sera of crossmatch negative kidney recipients is indicative for an increased risk for graft failure, whereas the clinical significance of C1q‐fixing IgG‐DSA could not be assessed due to their low prevalence.     

Proteomic identification of sperm antigens using serum samples from individuals with and without antisperm antibodies

The aim of the study was to identify human sperm antigens reacting with polyclonal antisperm antibodies. Protein sperm extracts were subjected to electrofocusing, and next immune reactions (immunoblotting) were carried out with positive for antisperm antibodies and control (not containing antisperm antibodies) serum samples. Proteomic analysis of human sperm proteins resulted in identification of 80 sperm antigens that could be divided into three groups: antigens specific for patients with antisperm antibodies (32), antigens recognised by both infertile patients and control sera (35) and antigens detected by control serum samples only (13). Among antigens specific for infertile patients, there were 12 sperm entities known to be involved in fertilisation process. We have also characterised three protein entities identified only by sera of infertile women. Altogether, the proteomic analysis resulted in identification of 27 sperm entities not reported previously in human sperm proteome. Identified proteins are sperm antigens that could be potentially responsible for immunological infertility. The study also sheds new light on the sperm antigens in aspect of gender specificity. The investigation of human sperm proteome by the use of antisperm antibodies‐containing sera of infertile individuals not only may indicate new proteins but also can draft their immunological nature.