Search

The emerging coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the causative agent of coronavirus disease 2019 (COVID-19), which has become a severe threat to global public health and local economies. In this study, several single-chain antibody fragments that bind to the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein were identified and used to construct human-mouse chimeric antibodies and humanized antibodies. These antibodies exhibited strong binding to RBD and neutralization activity towards a SARS-CoV-2 pseudovirus. Moreover, these antibodies recognize different RBD epitopes and exhibit synergistic neutralizing activity. These provide candidate to combination use or bispecific antibody to potential clinical therapy for COVID-19.

Novel SARS-CoV-2 variants of concern (VOC) Delta and Omicron are able to escape some monoclonal antibody therapies, making again COVID-19 convalescent plasma (CCP) a potential frontline treatment.In this study, we investigated the kinetics of anti-SARS-CoV-2 neutralizing antibodies (nAbs) against VOCs Delta and Omicron in vaccine breakthrough infected plasma donors. Serum samples from 19 donors were collected at the time of plasma donation and tested for anti-SARS-CoV-2 nAbs (using live authentic VOC viral neutralization test) and IgG (Liaison® SARS-CoV-2 S1/S2 and Liaison® SARS-CoV-2 TrimericS IgG assays, DiaSorin). Measures were correlated with different variables, including the time between last vaccine dose and CCP donation, and time between SARS-COV-2 infection and CCP donation.nAb titers against VOC Delta and Omicron were directly related to the time interval since last vaccine dose to CCP donation, but inversely related to time since COVID19 breakthrough infection.SARS-CoV-2 breakthrough infection in vaccinated in donors boosts nAb titers against VOCs Delta and Omicron, but such titers decay shortly after infection. Therefore, CCP must be collected early after vaccine breakthrough infection.

Several studies have described associations between ABO blood group and SARS-CoV-2 infection severity in hospitalized patients where group A individuals are over-represented and group O individuals may have a lower infection rate. In convalescent individuals, group B blood donors have higher neutralizing SARS-CoV-2 antibody titers. We analyzed whether there was any correlation of ABO Rh(D) blood group with SARS-CoV-2 infection and with neutralizing antibodies in Australian convalescent plasma (CP) donors.ABO Rh(D) distribution and demographics of CP donors (n = 765) were compared with the total blood donor panel (n = 488,028), plasmapheresis donors (n = 203,176) and whole blood donors (n = 282,437) from 2020. The presence of neutralizing antibodies in CP donors was measured using the Vero E6 cell microneutralization assay.The distribution of ABO group in CP donors compared to the total donor panel was not significantly different (p = .177). There were significantly more group AB donors in the plasmapheresis subset (p = .005) and group O individuals were over-represented in the whole blood donor subset (p .0001). There was no significant difference in neutralizing antibody levels among CP donors with differing ABO blood groups (p = .872).ABO Rh(D) blood group distribution was not found to be significantly different between convalescent plasma donors and general blood donors in our large sample group. Inherent blood donor selection biases associated with clinical demand accounted for some differences within CP donors. The levels of SARS-CoV-2 neutralizing antibodies were also not significantly associated with ABO Rh(D) group.

Coronavirus disease 2019 (COVID‐19) manifests with high clinical variability and warrants sensitive and specific assays to analyze immune responses in infected and vaccinated individuals. Using Single Molecule Arrays (Simoa), we developed an assay to assess antibody neutralization with high sensitivity and multiplexing capabilities based on antibody‐mediated blockage of the ACE2‐spike interaction. The assay does not require live viruses or cells and can be performed in a biosafety level 2 laboratory within two hours. We used this assay to assess neutralization and antibody levels in patients who died of COVID‐19 and patients hospitalized for a short period of time and show that neutralization and antibody levels increase over time. We also adapted the assay for SARS‐CoV‐2 variants and measured neutralization capacity in pre‐pandemic healthy, COVID‐19 infected, and vaccinated individuals. This assay is highly adaptable for clinical applications, such as vaccine development and epidemiological studies., Using Single Molecule Arrays (Simoa), we developed a simple neutralization assay based on antibody‐mediated blockage of the ACE2‐spike interaction. This assay can assess antibody neutralization with high sensitivity and multiplexing capabilities. We used this assay to profile neutralization capacity in patients infected with SARS‐CoV‐2 and vaccinated individuals and show that this assay is useful for various clinical applications.

Background Convalescent plasma (CP) therapy for coronavirus disease (COVID‐19) provides virus‐neutralizing antibodies that may ameliorate the outcome of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infections. The effectiveness of CP likely depends on its antiviral neutralizing potency and is determined using in vitro neutralizing antibody assays. Study design and methods We evaluated abilities of three immunoassays for anti‐spike antibodies (EUROimmun, Ortho, Roche), a pseudotype‐based neutralization assay, and two assays that quantify ACE2 binding of spike protein (GenScript and hemagglutination test [HAT]‐based assay) to predict neutralizing antibody titers in 113 CP donations. Assay outputs were analyzed through linear regression and calculation of sensitivities and specificities by receiver operator characteristic (ROC) analysis. Results Median values of plasma samples containing neutralizing antibodies produced conversion factors for assay unitage of ×6.5 (pseudotype), ×19 (GenScript), ×3.4 (HAT assay), ×0.08 (EUROimmun), ×1.64 (Roche), and ×0.10 (Ortho). All selected assays were sufficient in identifying the high titer donations based on ROC analysis; area over curve ranged from 91.7% for HAT and GenScript assay to 95.6% for pseudotype assay. However, their ability to predict the actual neutralizing antibody levels varied substantially as shown by linear regression correlation values (from 0.27 for Ortho to 0.61 for pseudotype assay). Discussion Overall, the study data demonstrate that all selected assays were effective in identifying donations with high neutralizing antibody levels and are potentially suitable as surrogate assays for donation selection for CP therapy.

The pandemic of COVID‐19 has caused enormous fatalities worldwide. Serological assays are important for detection of asymptomatic or mild cases of COVID‐19, and sero‐prevalence and vaccine efficacy studies. Here, we evaluated and compared the performance of seven commercially available enzyme‐linked immunosorbent assay (ELISA)s for detection of anti‐severe acute respiratory syndrome corona virus 2 (SARS‐CoV‐2) immunoglobulin G (IgG). The ELISAs were evaluated with a characterized panel of 100 serum samples from qRT‐PCR confirmed COVID‐19 patients, collected 14 days post onset disease, 100 SARS‐CoV‐2 negative samples and compared the results with that of neutralization assay. Results were analysed by creating the receiver operating characteristic curve of all the assays in reference to the neutralization assay. All kits, were found to be suitable for detection of IgG against SARS‐CoV‐2 with high accuracy. The DiaPro COVID‐19 IgG ELISA showed the highest sensitivity (98%) among the kits. The assays demonstrated high sensitivity and specificity in detecting the IgG antibodies against SARS‐CoV‐2. However, the presence of IgG antibodies does not always correspond to neutralizing antibodies. Due to their good accuracy indices, these assays can also aid in tracing mild infections, in cohort studies and in pre‐vaccine evaluations.

Coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic even after vaccination. We aimed to identify immunological heterogeneity over time in vaccinated healthcare workers using neutralization antibodies and neutralizing activity tests.Serum samples were collected from 214 healthcare workers before vaccination (pre) and on days 22, 90, and 180 after receiving the first dose of BNT162b2 vaccine (day 0). Neutralization antibody (NAb, SARS-CoV-2 S-RBD IgM/IgG) titers and two kinds of surrogate virus neutralization tests (sVNTs) were analyzed (UMIN000043851).The NAb (SARS-CoV-2 S-RBD IgG) titer peaked on day 90 after vaccination (30,808.0 μg/ml ± 35,211; p 0.0001) and declined on day 180 (11,678.0 μg/ml ± 33,770.0; p 0.0001). The neutralizing activity also peaked on day 90 and declined with larger individual differences than those of IgG titer on day 180 (88.9% ± 15.0%, 64.8% ± 23.7%, p 0.0001). We also found that the results of POCT-sVNT (immunochromatography) were highly correlated with those of conventional sVNT (ELISA).Neutralizing activity is the gold standard for vaccine efficacy evaluation. Our results using conventional sVNT showed large individual differences in neutralizing activity reduction on day 180 (64.8% ± 23.7%), suggesting an association with the difference in vaccine efficacy. POCT-sVNT is rapid and user-friendly; it might be used for triage in homes, isolation facilities, and event venues without restrictions on the medical testing environment.

While COVID‐19 convalescent plasma (CCP) efficacy is still under investigation in randomized controlled trials (RCT), CCP collections continue worldwide with largely variable criteria. Since it is well known that only a minority of patients develop high‐titer neutralizing antibodies (nAb), as assessed by the viral neutralization tests (VNT), strategies to maximize cost‐effectiveness of CCP collection are urgently needed. A growing amount of the population is having exposure to the virus and is hence becoming a candidate CCP donor. Laboratory screening with high‐throughput serology has good correlations with the VNT titer, but upstream screening using clinical surrogates would be advisable. We review here the existing literature on clinical predictors of high‐titer nAb. Older age, male sex, and hospitalization are the main proxies of high VNT and should drive CCP donor recruitment.

Data regarding antibody responses to severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) in patients infected with COVID‐19 are not yet available. In this study, we aimed to evaluate serum antibody responses in patients regardless of the outcome. We measured the circulating immunoglobulin G (IgG) antibody levels in 60 subjects with a certified history of SARS‐CoV‐2 infection by using immunoenzymatic, chemiluminescent, and Neutralization assays. Half patients had a severe infection, the other half were pauci‐symptomatic. We analyzed their antibody response to see the trend of the humoral response. Our results showed a significant difference in circulating IgG level among the two groups. The neutralizing antibody response against SARS‐CoV‐2 was significantly higher among those who had severe disease. Furthermore, ten subjects from each group were screened twice, and a declining antibody trend was observed in pauci‐symptomatic individuals. These findings provide evidence that humoral immunity against SARS‐CoV‐2 in pauci‐symptomatic people is weak and may not be long‐lasting. This may have implications for immunity strategy and prevention, since it is still not clear whether a time‐dependent decrease of both circulating and neutralizing antibodies to nonprotective levels could occur in a longer time span and whether potential vaccines are able to induce a herd immunity and a durable response.

The micro‐neutralization assay is a fundamental test in virology, immunology, vaccine assessment, and epidemiology studies. Since the SARS‐CoV‐2 outbreak at the end of December 2019 in China, it has become extremely important to have well‐established and validated diagnostic and serological assays for this new emerging virus. Here, we present a micro‐neutralization assay with the use of SARS‐CoV‐2 wild type virus with two different methods of read‐out. We evaluated the performance of this assay using human serum samples taken from an Italian seroepidemiological study being performed at the University of Siena, along with the human monoclonal antibody CR3022 and some iper‐immune animal serum samples against Influenza and Adenovirus strains. The same panel of human samples have been previously tested in enzyme‐linked immunosorbent assay (ELISA) as a pre‐screening. Positive, borderline, and negative ELISA samples were evaluated in neutralization assay using two different methods of read‐out: subjective (by means of an inverted optical microscope) and objective (by means of a spectrophotometer). Our findings suggest that at least 50% of positive ELISA samples are positive in neutralization as well, and that method is able to quantify different antibody concentrations in a specific manner. Taken together, our results confirm that the colorimetric cytopathic effect‐based microneutralization assay could be used as a valid clinical test method for epidemiological and vaccine studies., Highlights High sensitivity of the Micro‐Neutralization assay for anti‐SARS‐CoV‐2 neutralizing antibodies detection in serum samples.CPE‐based (Subjective) and Colorimetric‐based (Objective) Micro‐Neutralization read out are comparable in terms of results provided.50% of S1 ELISA positive serum samples present neutralizing antibodies against wild type SARS‐CoV‐2 virus.

BACKGROUND: Several studies have described associations between ABO blood group and SARS-CoV-2 infection severity in hospitalized patients where group A individuals are over-represented and group O individuals may have a lower infection rate. In convalescent individuals, group B blood donors have higher neutralizing SARS-CoV-2 antibody titers. We analyzed whether there was any correlation of ABO Rh(D) blood group with SARS-CoV-2 infection and with neutralizing antibodies in Australian convalescent plasma (CP) donors. STUDY DESIGN AND METHODS: ABO Rh(D) distribution and demographics of CP donors (n = 765) were compared with the total blood donor panel (n = 488,028), plasmapheresis donors (n = 203,176) and whole blood donors (n = 282,437) from 2020. The presence of neutralizing antibodies in CP donors was measured using the Vero E6 cell microneutralization assay. RESULTS: The distribution of ABO group in CP donors compared to the total donor panel was not significantly different (p = .177). There were significantly more group AB donors in the plasmapheresis subset (p = .005) and group O individuals were over-represented in the whole blood donor subset (p

The emergence of Zika virus in the Americas has caused an increase of babies born with microcephaly or other neurological malformations. The differential diagnosis of Zika infection, particularly serological diagnosis, is an important but complex issue. In this study, we describe clinical manifestations of 94 suspected cases of congenital Zika from Bahia state, Brazil, and the results of serological tests performed on children and/or their mothers at an average of 71 days after birth. Anti‐Zika immunoglobulin M (IgM) antibodies were detected in 44.4% and in 7.1% of samples from mothers and children, respectively. Nearly all the IgM, and 92% of immunoglobulin G positive results were confirmed by neutralization test. Zika specific neutralizing antibodies were detected in as much as 90.4% of the cases. Moreover, dengue specific neutralizing antibodies were detected in 79.0% of Zika seropositive mothers. In conclusion, Zika IgM negative results should be considered with caution, due to a possible rapid loss of sensitivity after birth, while the NS1‐based Zika IgM enzyme‐linked immunosorbent assay test we have used has demonstrated to be highly specific. In a high percentage of cases, Zika specific neutralizing antibodies were detected, which are indicative of a past Zika infection, probably occurred during pregnancy in this population., Highlight In this study we have analyzed serum samples collected a mean of 71 days after birth from 94 suspected congenital Zika cases.Our results have shown a high percentage of positive neutralization test results for ZIKV, and also DENV, but low prevalence of anti‐ZIKV IgM antibodies.Our data confirm the difficulty of an accurate retrospective diagnosis of ZIKV congenital infection.

The identification of a novel class of shark-derived single domain antibodies, named vnarbodies that show picomolar affinities binding to the receptor binding domain (RBD) of Wuhan and Alpha, Beta, Kappa, Delta, Delta-plus, and Lambda variants, is reported. Vnarbody 20G6 and 17F6 have broad neutralizing activities against all these SARS-CoV-2 viruses as well as other sarbecoviruses, including Pangolin coronavirus and Bat coronavirus. Intranasal administration of 20G6 effectively protects mice from the challenges of SARS-CoV-2 Wuhan and Beta variants. 20G6 and 17F6 contain a unique "WXGY" motif in the complementary determining region 3 that binds to a hidden epitope on RBD, which is highly conserved in sarbecoviruses through a novel β-sheet interaction. It is found that the S375F mutation on Omicron RBD disrupts the structure of β-strand, thus impair the binding with 20G6. The study demonstrates that shark-derived vnarbodies offer a prophylactic and therapeutic option against most SARS-CoV-2 variants and provide insights into antibody evasion by the Omicron variant.

SARS‐CoV‐2 serological assays are urgently needed for rapid diagnosis, contact tracing and for epidemiological studies. So far, there is limited data on how commercially available tests perform with real patient samples and if positive tested samples show neutralizing abilities. Focusing on IgG antibodies, we demonstrate the performance of two ELISA assays (Euroimmun SARS‐CoV‐2 IgG and Vircell COVID‐19 ELISA IgG) in comparison to one lateral flow assay ((LFA) FaStep COVID‐19 IgG/IgM Rapid Test Device) and two in‐house developed assays (immunofluorescence assay (IFA) and plaque reduction neutralization test (PRNT)). We tested follow up serum/plasma samples of individuals PCR‐diagnosed with COVID‐19. Most of the SARS‐CoV‐2 samples were from individuals with moderate to severe clinical course, who required an in‐patient hospital stay. For all examined assays, the sensitivity ranged from 58.8 to 76.5% for the early phase of infection (days 5‐9) and from 93.8 to 100% for the later period (days 10‐18). With exception of one sample, all positive tested COVID‐19 follow up‐samples, using the commercially available assays examined (including the in‐house developed IFA), demonstrated neutralizing properties in the PRNT. Regarding specificity, some samples of endemic coronavirus (HCoV‐OC43, HCoV‐229E) and Epstein Barr virus (EBV) infected individuals cross‐reacted in the ELISA assays and IFA, in one case generating a false positive result. This article is protected by copyright. All rights reserved.

West Nile virus (WNV) is an important emerging zoonotic arbovirus giving rise to clinical syndromes of varying severity in humans and horses. Culex mosquitoes are the main vector. Although WNV has been reported in many countries in the Middle East and Asia, little is known about its prevalence in equine populations in the Arabian Peninsula. We have carried out a serological study on 200 horses to assess WNV infection in the Eastern and Central regions of Saudi Arabia in 2013-2015. Sera were tested for the presence of WNV antibodies in parallel using a commercial enzyme-linked immunosorbent assay (ELISA) kit and microneutralization (MN) tests. In comparison with the MN assay used as "gold standard," we find the ELISA had a sensitivity of 94.7% and specificity of 80.1%. The prevalence of WNV neutralizing antibody ranged from 5 (17.3%) of 29 sera collected in Riyadh up to 15 (55.6%) of 27 sera collected from Al-Qateef. These findings highlight the need to be aware of the possibility of WNV disease in humans and horses presenting with central nervous system disease in the Kingdom of Saudi Arabia.

A serosurvey for Tahyna virus (TAHV), a mosquito-borne California encephalitis orthobunyavirus (Peribunyaviridae) endemic to Europe, was performed to estimate the activity of TAHV on a broad geographic scale. Sera from wild boar (Sus scrofa), roe deer (Capreolus capreolus) and red deer (Cervus elaphus) were collected from Austria, Hungary and Romania. Samples were tested for neutralizing antibodies against TAHV using a virus microneutralization assay. The results demonstrate that TAHV transmission to mammals is widespread in Europe, particularly in the wild boar population where the mean rate of seroconversion is 15.2%.

Summary In order to enhance vaccine uptake by the immune cells in vivo, molecular engineering approach was employed to construct a polymeric immunoglobulin G scaffold (PIGS) that incorporates multiple copies of an antigen and targets the Fc gamma receptors on antigen‐presenting cells. These self‐adjuvanting immunogens were tested in the context of dengue infection, for which there is currently no globally licensed vaccine yet. Thus, the consensus domain III sequence (cEDIII) of dengue glycoprotein E was incorporated into PIGS and expressed in both tobacco plants and Chinese Ovary Hamster cells. Purified mouse and human cEDIII‐PIGS were fractionated by HPLC into low and high molecular weight forms, corresponding to monomers, dimers and polymers. cEDIII‐PIGS were shown to retain important Fc receptor functions associated with immunoglobulins, including binding to C1q component of the complement and the low affinity Fcγ receptor II, as well as to macrophage cells in vitro. These molecules were shown to be immunogenic in mice, with or without an adjuvant, inducing a high level IgG antibody response which showed a neutralizing potential against the dengue virus serotype 2. The cEDIII‐PIGS also induced a significant cellular immune response, IFN‐γ production and polyfunctional T cells in both the CD4+ and CD8+ compartments. This proof‐of‐principle study shows that the potent antibody Fc‐mediated cellular functions can be harnessed to improve vaccine design, underscoring the potential of this technology to induce and modulate a broad‐ranging immune response.

The mechanism for the co-existence of hepatitis B surface antigen (HBsAg) and antibodies to HBsAg (anti-HBs) in chronic HBV infected patients remains controversial. This study aimed to explore the role of HBV S gene mutation and anti-HBs subtype-nonspecificity in patients with simultaneous HBsAg/anti-HBs positivity. Chronic HBV infections with (n = 145, group I) and without (n = 141, group II) anti-HBs were included. The S gene was amplified and sequenced. The neutralization experiment was used in group I patients' sera to determine the specificity of anti-HBs. Additionally, the HBV vaccinated persons' sera were used to estimate the neutralize capacity of anti-HBs against HBsAg in group I patients. Results showed that 2.63% (145/5513) chronic HBV infected patients had positive results for anti-HBs. HBsAg amino acid (aa) substitution rate in 35 patients of group I was significantly higher than that in 58 patients of group II (1.89% vs 0.95%, P

The role of bone morphogenic protein 9 (BMP9) signaling in angiogenesis has been controversial, with a number of studies showing that it acts either as a pro-angiogenic or, conversely, as an anti-angiogenic factor in a context-dependent manner. Notably, BMP9 was also reported to function in both pro- or anti-tumorigenic roles during tumor progression. It has therefore remained unclear, whether selective BMP9 inhibition is a useful target for antibody therapy of cancer. To shed light on these questions, we characterized BMP9 expression in plasma of patients with different cancer indications and found elevated levels of pro-domains and precursor BMP9 with a strong response in renal cell carcinoma (RCC). These studies prompted us to evaluate the potential of selective anti-BMP9 cancer therapy in RCC. We generated a novel monoclonal therapeutic antibody candidate, mAb BMP9-0093, that selectively targets all different BMP9 variants but does not bind to the closest homolog BMP10. In vitro , mAb BMP9-0093 treatment inhibited signaling, endothelin-1 (ET-1) production and spreading of endothelial cells and restored BMP9-induced decrease in pericyte migration and attachment. Furthermore, BMP9-mediated epithelial–mesenchymal transition of renal cell carcinoma cells was reversed by mAb BMP9-0093 treatment in vitro . In vivo , mAb BMP9-0093 showed significant anti-tumor activity that was associated with an increase in apoptosis as well as a decrease in tumor cell proliferation and ET-1 release. Furthermore, mAb BMP9-0093 induced mural cell coverage of endothelial cells, which was corroborated by a reduction in vascular permeability, demonstrated by a diminished penetration of omalizumab-Alexa 647 into tumor tissue. Our findings provide new evidence for a better understanding of BMP9 contribution in tumor progression and angiogenesis that may result in the development of effective targeted therapeutic interventions.

Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of a serious infective, notifiable disease affecting common carp and varieties. In survivors, infection is generally characterized by a subclinical latency phase with restricted viral replication. The CyHV-3 genome is difficult to detect in such carrier fish that represent a potential source of dissemination if viral reactivation occurs. In this study, the analytical and diagnostic performance of an alternative serum neutralization (SN) method based on the detection of CyHV-3-specific antibodies was assessed using 151 serum or plasma samples from healthy and naturally or experimentally CyHV-3-infected carp. French CyHV-3 isolate 07/108b was neutralized efficiently by sera from carp infected with European, American and Taiwanese CyHV-3 isolates, but no neutralization was observed using sera specific to other aquatic herpesviruses. Diagnostic sensitivity, diagnostic specificity and repeatability of 95.9%, 99.0% and 99.3%, respectively, were obtained, as well as a compliance rate of 89.9% in reproducibility testing. Neutralizing antibodies were steadily detected in infected carp subjected to restrictive or permissive temperature variations over more than 25 months post-infection. The results suggest that this non-lethal diagnostic test could be used in the future to improve the epidemiological surveillance and control of CyHV-3 disease.

BACKGROUND: Serum antibody to influenza can be used to identify past exposure and measure current immune status. The two most common methods for measuring this are the hemagglutination inhibition assay (HI) and the viral neutralization assay (NT), which have not been systematically compared for a large number of influenza viruses. METHODS: 151 study participants from near Guangzhou, China were enrolled in 2009 and provided serum. HI and NT assays were performed for 12 historic and recently circulating strains of seasonal influenza A. We compared titers using Spearman correlation and fit models to predict NT using HI results. RESULTS: We observed high positive mean correlation between HI and NT assays (Spearman's rank correlation, rho=0.86) across all strains. Correlation was highest within subtypes and within close proximity in time. Overall, an HI=20 corresponded to NT=10, and HI=40 corresponded to NT=20. Linear regression of log(NT) on log(HI) was statistically significant, with age modifying this relationship. Strain-specific area under a curve (AUC) indicated good accuracy (>80%) for predicting NT with HI. CONCLUSIONS: While we found high overall correspondence of titers between NT and HI assays for seasonal influenza A, no exact equivalence between assays could be determined. This was further complicated by correspondence between titers changing with age. These findings support generalized comparison of results between assays and give further support for use of the hemagglutination inhibition assay over the more resource intensive viral neutralization assay for seasonal influenza A, though attention should be given to the effect of age on these assays. This article is protected by copyright. All rights reserved.

Background Overexpression or administration of interleukin 31 (IL-31) has been shown to induce a profound itch response in mice and dogs. The chronic pruritus observed in mouse IL-31 transgenic mice results in the development of skin lesions and alopecia through excoriation from excessive scratching, a condition similar to that observed in patients with atopic dermatitis (AD). Objective To test whether IL-31 induces pruritus in non-human primates and, if so, whether treatment with an anti-IL-31 neutralizing monoclonal antibody (mAb) can block the response. Methods A series of studies was conducted in cynomolgus monkeys to evaluate the itch response to recombinant cynomolgus IL-31 (cIL-31) administration. Three routes of cIL-31 administration (intravenous, intradermal, and subcutaneous) were evaluated. Subcutaneous treatment with a humanized anti-human IL-31 mAb cross-reactive to cIL-31 was subsequently tested for its ability to block the response to intradermal cIL-31 administration. Results Each route of cIL-31 delivery elicited a scratching response immediately after cIL-31 administration and lasted at least 3 h. Treatment with the IL-31 mAb inhibited the cIL-31-mediated scratching response in a dose-dependent manner. Conclusion These results demonstrate that an IL-31 mAb can inhibit IL-31-mediated pruritus in vivo, and could be an effective therapy for pruritic skin conditions like AD where IL-31 upregulation may play a role.

Background & Aim Hepatitis B virus-e-antigen (HBeAg) is an affordable viral marker to assess viral replication kinetics and response to antiviral therapy. In the absence of confirmatory assays, discrepant or false-positive HBeAg results are resolved by screening for other HBV markers. We standardized an in-house HBeAg neutralization assay (HBeAg-NT) to confirm HBeAg in clinical samples. Methods The performance and reliability of this assay were evaluated by first WHO International Standard for HBeAg (first WHO-IS HBeAg) from Paul Ehrlich Institute and clinical samples (n = 150) from chronic HBV carriers. Of these, 71 HBeAg-positive sera were used for HBeAg-NT. Results Concentrations spanning 0.25–10 U of first WHO-IS HBeAg and clinical samples (S/Co ranges from 1.00 to 10.00) were neutralized completely in the HBeAg-NT. Conclusions HBeAg-NT is a simple, cost-effective, and reliable direct approach to confirm HBeAg in clinical samples which precludes the need for screening additional HBV markers in low resource settings.

Previously, we reported the ability of the chimeric protein DIIIC-2 (domain III of the dengue envelope protein fused to the capsid protein of dengue-2 virus), to induce immunity and protection in mice, when it is highly aggregated with a non-defined oligodeoxynucleotide (ODN) and adjuvanted in alum. In this work, three different defined ODNs were studied as aggregating agents. Our results suggest that the nature of the ODN influences the capacity of protein DIIIC-2 to activate cell-mediated immunity in mice. Consequently, the ODN 39M was selected to perform further experiments in mice and nonhuman primates. Mice receiving the preparation 39M-DIIIC-2 were solidly protected against dengue virus (DENV) challenge. Moreover, monkeys immunized with the same preparation developed neutralizing antibodies, as measured by four different neutralization tests varying the virus strains and the cell lines used. Two of the immunized monkeys were completely protected against challenge, whereas the third animal had a single day of low-titer viremia. This is the first work describing the induction of short-term protection in monkeys by a formulation that is suitable for human use combining a recombinant protein from DENV with alum.

Previously, our group engineered a plant-derived monoclonal antibody (MAb pE16) that efficiently treated West Nile virus (WNV) infection in mice. In this study, we developed a pE16 variant consisting of a single-chain variable fragment (scFv) fused to the heavy chain constant domains (CH) of human IgG (pE16scFv-CH). pE16 and pE16scFv-CH were expressed and assembled efficiently in Nicotiana benthamiana ∆XF plants, a glycosylation mutant lacking plant-specific N-glycan residues. Glycan analysis revealed that ∆XF plant-derived pE16scFv-CH (∆XFpE16scFv-CH) and pE16 (∆XFpE16) both displayed a mammalian glycosylation profile. ∆XFpE16 and ∆XFpE16scFv-CH demonstrated equivalent antigen-binding affinity and kinetics, and slightly enhanced neutralization of WNV in vitro compared with the parent mammalian cell-produced E16 (mE16). A single dose of ∆XFpE16 or ∆XFpE16scFv-CH protected mice against WNV-induced mortality even 4 days after infection at equivalent rates as mE16. This study provides a detailed tandem comparison of the expression, structure and function of a therapeutic MAb and its single-chain variant produced in glycoengineered plants. Moreover, it demonstrates the development of anti-WNV MAb therapeutic variants that are equivalent in efficacy to pE16, simpler to produce, and likely safer to use as therapeutics due to their mammalian N-glycosylation. This platform may lead to a more robust and cost-effective production of antibody-based therapeutics against WNV infection and other infectious, inflammatory or neoplastic diseases.

We tested, using a low starting dilution, sequential serum samples from dromedary camels, sheep and horses collected in Dubai from February/April to October of 2005 and from dromedary camels for export/import testing between Canada and USA in 2000-2001. Using a standard Middle East respiratory syndrome coronavirus (MERS-CoV) neutralization test, serial sera from three sheep and three horses were all negative while sera from 9 of 11 dromedary camels from Dubai were positive for antibodies supported by similar results in a MERS-CoV recombinant partial spike protein antibody ELISA. The two negative Dubai camels were both dromedary calves and remained negative over the 5 months studied. The six dromedary samples from USA and Canada were negative in both tests. These results support the recent findings that infection with MERS-CoV or a closely related virus is not a new occurrence in camels in the Middle East. Therefore, interactions of MERS-CoV at the human-animal interface may have been ongoing for several, perhaps many, years and by inference, a widespread pandemic may be less likely unless significant evolution of the virus allow accelerated infection and spread potential in the human population.

Induction of broadly neutralizing antibody is considered important for an effective HIV-1 vaccine. Identification and characterization of broadly neutralizing antibodies in HIV-1-infected patients will facilitate our understanding of the immune correlates to protection and the design of an effective prophylactic vaccine. A number of studies to probe the specificity of antibodies in broadly neutralizing sera in the United States and Europe have been reported. However, little is known about and would be interesting to investigate the immunological characteristics of HIV-1-positive sera in China where non-clade B viruses are prevalent and the circulating viral subtypes are distinct and more complex than both the United States and Europe. Here, we screened 80 Chinese HIV-1-positive sera against a minipanel of pseudoviruses representing various circulating HIV-1 subtypes in China and identified 8 cross-clade neutralizing sera (CNsera). Immunological characterization of the sera showed that gp120-targeting antibodies with multiple epitope specificities contributed to the cross-clade neutralizing activity of these CNsera. V3-directed antibodies were prevalent in these CNsera, but did not mainly contribute to their neutralization breath and potency while CD4bs-specific, 2F5- and 4E10-like antibodies were rarely detected. 2G12-like neutralizing antibodies were more frequently detected in HIV-1 patients from China where recombinant subtype viruses are prevalent than in United States and Europe. One broadly neutralizing serum (Serum 45) was identified to contain antibodies with unknown epitope specificities that were sensitive to terminal glycan modifications on virus Env and insensitive to N160K mutagenesis, and correlated with the cross-clade neutralization activity of Serum 45.

It has previously been shown that patients with haemophilia A may develop non-neutralizing anti-factor VIII (FVIII) antibodies (NNA) that escape detection by the Bethesda assay, but are detected using immune-based assays. We and others found NNAs to be directed not only towards non-functional parts of the protein, but towards all regions of the FVIII protein. We also showed a heterogeneous antibody response towards different FVIII products. However, the clinical relevance and the natural history of NNA remain unclear. Therefore, we followed a cohort of unrelated subjects with haemophilia A for 4 years with the goal of exploring the long-term development of NNA using an enzyme-linked immunosorbent assay (ELISA). Ten of 78 subjects (12·8%) exhibited an immune response that was transient and heterogeneous, and none of the subjects developed an FVIII inhibitor. The result of the ELISA was examined in relation to clinical variables and no significant associations between a positive ELISA and age, F8 mutation, port-à-cath implantation and HCV infection were shown. Interestingly, patients with NNA had significantly fewer bleeding episodes (P = 0·048) compared with NNA-negative subjects. The results indicate that the immune response to FVIII products within an individual may vary over time. However, the clinical impact of NNA remains unclear.

The serological reactivity between strains of each of the six currently genetically defined subtypes of salmonid alphavirus (SAV) was examined by comparison of homologous and heterologous virus neutralization titres on sera from experimentally infected fish. With the exception of the level of SAV subtype 6 neutralization by heterologous sera, good cross-neutralization was detected between all subtypes, albeit with variation in geometric mean titres when each subtype-specific serum set was tested against the panel of virus subtypes. A similar pattern was evident with field sera, except that heterologous neutralization of the SAV6 strain was more evident. In only 23% of available pairwise comparisons was the homologous titre recorded with an experimentally derived serum fourfold or greater than the heterologous titre, and in only two instances was this difference demonstrated in both directions. No virus strains consistently met the old serology-based criteria (Sub-committee on Inter-relationships Among Catalogued Alphaviruses) to be considered separate subtypes within an alphavirus species. Only when testing with an SAV subtype-2-specific monoclonal antibody was a major difference between homologous and heterologous neutralization capacity evident. These results provide new direct or indirect information in terms of SAV classification, vaccine efficacy and the selection and validation of reagents for serological and immunological diagnostic purposes.

The replication fidelity of reassortant A/Fujian/411/02(H3N2)-like influenza viruses was assessed by in vitro detection of escape mutants developing under selective pressure from monoclonal antibodies. The results showed A/Wyoming/3/03(H3N2) possessed lower fidelity relative to the A/California/7/04(H3N2) and A/Wisconsin/57/05(H3N2) viruses through the emergence of mutant viruses carrying H156Q hemagglutinin mutation which allows antibody escape. Using the neutralization assay to compare the fidelity of reassortant pandemic A/California/7/09(H1N1) viruses, the pandemic virus was shown to possess relative higher fidelity compared to A/Wyoming/3/03. This higher fidelity may contribute to the lack of major antigenic changes in the pandemic virus since the emergence in 2009. J. Med. Virol. 85: 1090–1094, 2013. © 2013 Wiley Periodicals, Inc.

Rabies kills many people throughout the developing world every year. The murine monoclonal antibody (mAb) 62-71-3 was recently identified for its potential application in rabies postexposure prophylaxis (PEP). The purpose here was to establish a plant-based production system for a chimeric mouse-human version of mAb 62-71-3, to characterize the recombinant antibody and investigate at a molecular level its interaction with rabies virus glycoprotein. Chimeric 62-71-3 was successfully expressed in Nicotiana benthamiana. Glycosylation was analyzed by mass spectroscopy; functionality was confirmed by antigen ELISA, as well as rabies and pseudotype virus neutralization. Epitope characterization was performed using pseudotype virus expressing mutagenized rabies glycoproteins. Purified mAb demonstrated potent viral neutralization at 500 IU/mg. A critical role for antigenic site I of the glycoprotein, as well as for two specific amino acid residues (K226 and G229) within site I, was identified with regard to mAb 62-71-3 neutralization. Pseudotype viruses expressing glycoprotein from lyssaviruses known not to be neutralized by this antibody were the controls. The results provide the molecular rationale for developing 62-71-3 mAb for rabies PEP; they also establish the basis for developing an inexpensive plant-based antibody product to benefit low-income families in developing countries.—Both, L., van Dolleweerd, C., Wright, E., Banyard, A. C., Bulmer-Thomas, B., Selden, D., Altmann, F., Fooks, A. R., Ma, J. K.-C. Production, characterization, and antigen specificity of recombinant 62-71-3, a candidate monoclonal antibody for rabies prophylaxis in humans.

T cell tolerance to tumor antigens represents a major hurdle in generating tumor immunity. Combined administration of agonistic monoclonal antibodies to the costimulatory receptors CD134 plus CD137 can program T cells responding to tolerogenic antigen to undergo expansion and effector T cell differentiation, and also elicits tumor immunity. Nevertheless, CD134 and CD137 agonists can also engage inhibitory immune components. To understand how immune stimulatory versus inhibitory components are regulated during CD134 plus CD137 dual costimulation, the current study utilized a model where dual costimulation programs T cells encountering a highly tolerogenic self-antigen to undergo effector differentiation. IFN-γ was found to play a pivotal role in maximizing the function of effector T cells while simultaneously limiting the expansion of CD4+CD25+Foxp3+ Tregs. In antigen-responding effector T cells, IFN-γ operates via a direct cell-intrinsic mechanism to cooperate with IL-2 to program maximal expression of granzyme B. Simultaneously, IFN-γ limits expression of the IL-2 receptor alpha chain (CD25) and IL-2 signaling through a mechanism that does not involve T-bet-mediated repression of IL-2. IFN-γ also limited CD25 and Foxp3 expression on bystanding CD4+Foxp3+ Tregs, and limited the potential of these Tregs to expand. These effects could not be explained by the ability of IFN-γ to limit IL-2 availability. Taken together, during dual costimulation IFN-γ interacts with IL-2 through distinct mechanisms to program maximal expression of effector molecules in antigen-responding T cells while simultaneously limiting Treg expansion.

Objective To compare the performance of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies specific to bovine viral diarrhoea virus (BVDV) with a virus neutralisation test (VNT) and agarose gel immunodiffusion (AGID) test. Design A total of 125 cattle serum samples were tested by a commercially available ELISA for antibodies specific to BVDV and by a VNT as the reference standard. A comparison between AGID and ELISA for detection of BVDV antibodies was also carried out, using 1182 serum samples from unvaccinated South Australian cattle. Methods Two-graph receiver operating characteristics (TG-ROC) analysis was used to confirm that the manufacturer's recommended cut-off value for the ELISA was appropriate. Two-by-two tables were constructed to analyse the concordance of serological results among the three assays. McNemar tests were used to assess the agreement among serological tests. Results and conclusions Using the manufacturer's cut-off threshold, supported by TG-ROC analysis, the ELISA's sensitivity and specificity were calculated to be 96.7% and 97.1%, respectively, compared with the VNT. Compared with AGID, ELISA with specific BVDV antibodies may be more sensitive and detect 5.8% more samples than AGID. McNemar test also showed a significant difference (P < 0.001) between AGID and ELISA.

The milestone in polio eradication program is to protect effectively children aged 0–5 years against the three serotypes of poliovirus. It became necessary to measure the level of neutralizing antibodies to the three poliovirus types in an endemic State in Nigeria. Neutralizing antibodies to the poliovirus types among children aged 0–5 years was estimated using micro neutralization assay. Of 129 children, 99 (76.8%), 95 (73.6%), and 95 (73.6%) had neutralizing antibodies with the geometric mean titer of 42.7, 31.3, and 33.2 for the poliovirus type 1, 2, and 3, respectively. Fifty-three percent of the children were protected against the three types of poliovirus. Combination of poliovirus types 1 and 2, 1 and 3, and 2 and 3 were neutralized by 62.8, 58.9, and 61.2% of the children studied, respectively. Only poliovirus type 1 induced antibody titres ≥1:1,024. The number of children with neutralizing antibodies after receiving three doses was significantly higher than those who received one or two doses of oral polio vaccine (P ≤ 0.05). However, those who received more than three doses of oral polio vaccine showed no significant difference in their antibody response. The existence of immunity gap poses a risk of re-emergence of the paralytic poliovirus. The existence of unimmunized and unprotected children along with high birth rate could impede the success of polio vaccination in Nigeria. Elimination of non-compliance to polio vaccine, promotion of health education and documented evidence of vaccination of each child with the parents may facilitate the success of polio eradication program in Nigeria. J. Med. Virol. 84:691–696, 2012. © 2011 Wiley Periodicals, Inc.

BACKGROUND: Xenotropic murine leukemia virus (MLV)-related virus (XMRV) and other related MLVs have been described with chronic fatigue syndrome and certain types of prostate cancer. In addition, prevalence rates as high as 7% have been reported in blood donors, raising the risk of transfusion-related transmission. Several laboratories have utilized microneutralization assays as a surrogate marker for detection of anti-MLV serologic responses—with up to 25% of prostate cancer patients reported to harbor neutralizing antibody responses. STUDY DESIGN AND METHODS: We developed a high-throughput microneutralization assay for research studies on blood donors using retroviral vectors pseudotyped with XMRV-specific envelopes. Infection with these pseudotypes was neutralized by sera from both macaques and mice challenged with XMRV, but not preimmune serum. A total of 354 plasma samples from blood donors in the Reno/Tahoe area were screened for neutralization. RESULTS: A total of 6.5% of donor samples gave moderate neutralization of XMRV, but not control pseudotypes. However, further testing by Western blot revealed no evidence of antibodies against MLVs in any of these samples. Furthermore, no evidence of infectious virus or viral nucleic acid was observed. CONCLUSION: A microneutralization assay was developed for detection of XMRV and can be applied in a high-throughput format for large-scale studies. Although a proportion of blood donors demonstrated the ability to block XMRV envelope-mediated infection, we found no evidence that this inhibition was mediated by specific antibodies elicited by exposure to XMRV or MLV. It is likely that this moderate neutralization is mediated through another, nonspecific mechanism.

Human adenoviruses species D (HAdV-D) are known to cause severe epidemic keratoconjunctivitis. However, the isolation rate of HAdV-D is not high, because HAdV-D is usually slow to propagate. Although new types of HAdV-D have been reported, accurate surveillance has not been performed because of difficulties in culturing the viruses and lack of a practical identification method. In this study, HAdV-Ds were detected and identified from patients with epidemic keratoconjunctivitis in the Fukui Prefecture during 1995–2010 by PCR, loop-mediated isothermal amplification (LAMP) of DNA, and conventional virus isolation and neutralization tests. All samples were subjected to culture and PCR and LAMP. A total of 124 strains of HAdV-D were detected from 157 patients with epidemic keratoconjunctivitis. The strains consisted of the following types: D8 (n = 8), D19 (n = 4), D37 (n = 40), D53 (n = 5), D54 (n = 66), and D56 (n = 1). Among these, D53, D54, and D56 are new types that have been reported recently. The results of this study demonstrated that new types of HAdV-D caused epidemic keratoconjunctivitis during 1995–2010, and included an outbreak of keratoconjunctivitis caused by HAdV-D54. The LAMP method was able to detect and identify HAdV-D53 and HAdV–D54 in 1 hr, and may therefore be applicable for use at the bedside. J. Med. Virol. 84:81–86, 2011. © 2011 Wiley Periodicals, Inc.

This study aimed to examine the application of hepatitis B surface antigen (HBsAg) confirmatory testing when diagnosing hepatitis B infection among young persons in Taiwan with a low prevalence rate of hepatitis B infection. HBsAg status, the presence of antibodies against HBsAg (anti-HBs), and the presence of antibodies against hepatitis B core antigen (anti-HBc) were compared among 403 graduate students (mean age 22.8 ± 0.7 years) and 1,745 undergraduate students (18.6 ± 1.0 years) from one university, and 367 adult subjects (41.1 ± 15.8 years) in 2008. Any HBsAg-positive subjects were tested with an HBsAg confirmatory test. Chi-square tests for trend and predictive values of positivity (PVP) when using HBsAg-positive only for determining confirmed cases of hepatitis B infection were compared across the three cohorts. The prevalence of HBsAg positivity among subjects decreased from 16.3% in the adults to 5.2% in the graduate students and then to 2.8% for the undergraduate students (P = 0.0007). The PVP of HBsAg testing when determining cases of hepatitis B decreased from 0.97 for the adults to 0.81 for the graduate students and then to 0.56 for the undergraduate students (P < 0.0001). Thus, a significant decrease in the true-positive rate of HBsAg among the students born after the introduction of hepatitis B vaccination was observed only when HBsAg testing was applied. Additional neutralization tests may therefore become mandatory for persons with a positive HBsAg test result who were born after the commencement of the universal neonatal hepatitis B vaccination program in Taiwan.

Enterovirus (EV) infection is a common disease of childhood and associated not uncommonly with aseptic meningitis. In the summer of 2008, laboratory surveillance has detected increased number of coxsackievirus B3 (CVB3) associated aseptic meningitis in Hong Kong, constituting 11.6% of those infected. This study analyzed the epidemiology, circulating pattern, and clinical presentations of CVB3 in Hong Kong over the last 10 years with reference to the circulation of EV in the locality. Enteroviruses (EV) were isolated from respiratory, cerebrospinal fluid (CSF), stool, and vesicular samples using human rhabdomyosarcoma, human laryngeal carcinoma (HEp2-C), human lung fibroblast (MRC-5), and African green monkey kidney (Vero) cell lines. Virus isolates were identified and characterized by indirect immunofluorescence (IF) using monoclonal antibodies (mAB), neutralization test as well as partial VP1 sequencing. Different from previous years, IF test result showed that majority of the isolates from 2008 were untypeable by the mAB suggesting antigenic change. Sequence analysis revealed that these isolates were clustered with recent isolate from Fuyang, China. Review of data from 1999 to 2008 showed increased activity of CVB3 in the years 2005 and 2008, and isolates in these 2 years displayed an amino acid change from threonine to alanine at codon 277 of the VP1 gene, which may be associated with central nervous system (CNS) disease.

The purpose of this study was to evaluate the frequency of human metapneumovirus (hMPV) infection in adults and to determine the association between the levels of serum antibody titers and the susceptibility to reinfection. Serum samples collected at the periodic occupational medical checkup for employees of a hospital were subjected to an ELISA test. Of the 289 subjects, 288 (99.7%) had hMPV antibody titers that were more than 1:100 in May 2006. The percentage of subjects with a titer of ≥ 3,200 was significantly higher in adults aged 40-65 years old (30.2-31.5%) compared to young adults 20-39 years old (13.6%) (P < 0.05). To investigate the longitudinal course of the hMPV antibody titer, a total of 649 serum samples collected from 59 subjects who had participated in all biannual medical checkups between 2001 and 2006 were tested. We found that ten serum pairs showed a greater than fourfold increase in hMPV antibody titers. Additionally, the 5-year reinfection rate was estimated at 16.9% (10 of 59 subjects). The baseline titer before the fourfold increase ranged from 1:100 to 1:3,200, and the titer returned to baseline levels 2 or 3 years after the fourfold increase. The antibody titer of the person with the baseline titer of 1:100 showed a greater than fourfold increase twice within a year. Sixty to 80% of adults had an ELISA titer of 1:800 to 1:1,600, suggesting that such an antibody titer is not enough to protect from hMPV infection and that reinfection could occur among adults.

Summary Cross-neutralization tests with rabbit antisera I-1 (I serotype), P-1 (II serotype) and T80 (II serotype) were carried out with 14 porcine enteroviruses isolated in Bulgaria and 10 viruses received from other countries. On the basis of these investigations 10 of the 14 viruses isolated in Bulgaria were shown to be related to the Teschen-Talfan group. One virus (P-1) isolated in Bulgaria gave reciprocal cross-reaction with T80, T52A, S-180/4 (II serotype). Three viruses (K-1, P-2, Si-1) gave unilateral neutralization test with virus P-1 (II serotype). Out of 10 viruses received from abroad one (L-1) was neutralized with antisera I-1 (I serotype) and P-1 (II serotype); 5 strains (S-180/4, S-159, T-1, U-6, L-1) supplied from abroad were neutralized with 3 antisera (I-1, P-1, L-1). On the basis of these experiments, the author suggests that the classification should include intermediate serological types of the swine enteroviruses giving cross-reactions with more than one serotype. Zusammenfassung Serologische Klassifizierung von Schweine-Enterovirusstammen aus Bulgarien und anderen Landern Zwischen 14 Schweine-Enterovirusstammen, die in Bulgarien isoliert worden waren, sowie 10 anderen aus dem Ausland herruhrenden Stammen und den Kaninchenantiseren I-1 (Serotyp I), P-1 (Serotyp II) und T80 (Serotyp II) wurden kreuzweise Neutralisationsteste ausgefuhrt. Auf der Grundlage dieser Untersuchungen zeigte sich bei 10 der in Bulgarien isolierten 14 Virusstamme eine Verwandtschaft zu der Teschen-Talfan-Gruppe. Ein Virusstamm, der gleichfalls in Bulgarien isoliert worden war, lieferte reziproke Kreuzreaktionen mit T80, T52A, S-180/4 (Serotyp II). Drei Stamme (K-1, P-2, Si-1) ergaben eine einseitige Neutralisation mit Virus P-1 (Serotyp II). Von den 10 von auserhalb stammenden Virusstammen wurde einer (L-1) durch die Antiseren I-1 (Serotyp I) und P-1 (Serotyp II) und 5 andere (S-180/4, S-159, T-1, U-6, L-1) durch 3 Antiseren (I-1, P-1, L-1) neutralisiert. Auf Grund der Versuche schlagt der Autor vor, das eine Klassifizierung auch diejenigen serologischen Intermediartypen der Schweine-Enteroviren einbeziehen sollte, die mit mehr als einem Serotyp Kreuzreaktionen aufweisen. Resume La classification serologique des souches porcines d'enterovirus en Bulgarie et dans d'autres pays On effectua des tests de neutralisation croisee sur 14 souches porcines d'enterovirus isolees en Bulgarie ainsi que sur 10 autres souches de provenance etrangere avec les antiserums de lapin I-1 (serotype I), P-1 (serotype II) et T80 (serotype II). Ces recherches permirent de constater chez 10 des 14 souches isolees en Bulgarie une parente avec le groupe Teschen-Talfan. Une souche de virus egalement isolee en Bulgarie montra des reactions reciproques croisees avec T80, T52A, S-180/4 (serotype II). Trois souches (K-1, P-2, Si-1) donnerent une neutralisation unilaterale avec le virus P-1 (serotype II). Sur les 10 souches de virus de provenance exterieure, l'une (L-1) fut neutralisee par les antiserums I-1 (serotype I) et P-1 (serotype II) et 5 autres (S-180/4, S-159, T-1, U-6, L-1) par 3 anteserums (I-1, P-1, L-1). Sur la base de ces essais, l'auteur propose qu'une classification prenne egalement en consideration les types serologiques intermediaires des enterovirus porcins qui donnent des reactions croisees avec plus d'un serotype. Resumen Clasificacion serologica de las cepas de enterovirus porcinos de Bulgaria y otros paises Entre 14 cepas de enterovirus porcinos, que fueron aislados en Bulgaria, asi como otras 10, procedentes del extranjero, y los antisueros I-1 de conejo (serotipo I), P-1 (serotipo II) y T-80 (serotipo II) se efectuaron pruebas de neutralizacion cruzada. Sobre la base de estos estudios, en 10 de las 14 cepas de virus aisladas en Bulgaria se hallo cierto parentesco con el grupo Teschen-Talfan. Una cepa de virus, aislada tambien en Bulgaria, daba reacciones cruzadas reciprocas con T-80, T-52A, S-180/4 (serotipo II). Tres cepas (K-1, P-2, Si-1) produjeron una neutralizacion unilateral con el virus P-1 (serotipo II). De las 10 cepas virosicas de origen extranjero, una (L-1) se neutralizo mediante los antisueros I-1 (serotipo I) y P-1 (serotipo II) y otras 5 (S-180/4, S-159, T-1, U-6, L-1) mediante 3 antisueros (I-1, P-1, L-1). Con arreglo a los ensayos, el autor propone que la clasificacion tambien deberia englobar aquellos tipos serologicos intermediarios de los enterovirus porcinos que muestran reacciones cruzadas con mas de un serotipo.

Summary A partially purified preparation of E. coli heat-stable enterotoxin of porcine origin, active at the nanogram level, was polymerised by glutaraldehyde treatment. Inoculation of the modified ST toxin into rabbits and pigs induced a significant level of antitoxic antibody response. Zusammenfassung Untersuchungen zur Immunitatsbildung gegen das E. coli ST-Enterotoxin Ein teilweise gereinigtes, hitzestabiles Enterotoxin (ST) eines vom Schwein isolierten E. coli-Stammes (aktiv im Nanogramm-Bereich) wurde mit Glutaraldehyd polymerisiert. Das modifizierte ST-Toxin induzierte nach parenteraler Applikation bei Kaninchen und Schweinen einen signifikanten Anstieg der antitoxischen Antikorper. Resume Induction d'une immunite contre la toxine ST de E. coli Une enterotoxine thermostable (ST) provenant d'une souche E. coli de porc a ete purifiee partiellement (active secifique » 5 nanogrammes) et polymerisee a l'aide de l'aldehyde glutarique. La toxine modifiee induit une quantite significative d'anticorps anti-toxine. Resumen Estudios sobre la inmunogenia frente a la enterotoxina ST de E. coli Se polimerizo con glutaraldehido una enterotoxina (ST) termoestable, purificada parcialmente, de una estirpe E. coli aislada del cerdo (activa en el ambito del nanograma). La toxina ST modificada estimulo, tras aplicacion parenteral en conejo y cerdos, un aumento significante de los anticuerpos antitoxicos.

Summary Sera from young cattle hyperimmunized with inactivated oil-adjuvant Aujeszky's (AVa) or IBR vaccine and developing high titres of homologous antibodies (1: 256–1: 4,096) neutralized also the heterologous virus. Mutual cross-neutralization activity was recorded in nasal secretions from calves at the time of the highest titres of serum antibodies. Susceptible calves with no antibodies against IBR virus produced, after vaccination and re-vaccination with AVa or IBR vaccine, antibodies at medium or high titres against only the homologous virus; they responded anamnestically however to the application of heterologous vaccine as if they were immunologically stimulated. Sera from animals naturally infected with IBR virus neutralized AVi even at low titres of homologous antibodies. In natural infection IBR seropositive calves responded to the application of AVa by an increase in IBR antibodies, and to IBR vaccine by developing, in addition to an increase of homologous antibodies, neutralizing activity against AVi. Conjugates prepared from the sera of cattle hyperimmunized with AVa or IBR vaccine visualized in the infected cell cultures both homologous and heterologous viruses. Sera of pigs with high titres of antibodies against AVi did not neutralize IBR virus and rabbits immunized with IBR vaccine remained fully sensitive to AVi infection. The possibility of cross-protection of cattle immune to Aujeszky's disease, or IBR immune cattle against infection with heterologous virus is discussed. Zusammenfassung Untersuchungen uber immunologische Beziehungen zwischen Infektiosem Bovinem Rhinotracheitis-Virus (IBR) und dem Virus der Aujeszkyschen Krankheit mit Kalbern, Schweinen und Kaninchen Seren von Jungrindern, die mit inaktivierter Aujeszky- oder IBR-Oladjuvansvakzine vakziniert worden waren, neutralisierten im Falle hoher homologer Antikorpertiter (1: 256–1: 4096) auch das heterologe Virus. Kreuzneutralisierende Aktivitaten konnten auch in Nasensekreten von Kalbern nachgewiesen werden, die zum Zeitpunkt des Auftretens von Hochsttitern der Serumantikorper entnommen wurden. Empfangliche Kalber ohne IBR-Virusantikorper produzierten nach Vakzinierung und Revakzinierung mit Aujeszky- oder IBR-Vakzinen nur Antikorper gegen das homologe Virus. Sie zeigten jedoch eine anamnestische Reaktion nach Applikation der jeweils heterologen Vakzine. Seren von naturlich mit IBR-Virus infizierten Tieren neutralisierten Aujeszkyvirus sogar dann wenn nur niedrige homologe Antikorpertiter vorlagen. Naturlich mit IBR-Virus infizierte seropositive Kalber reagierten nach Impfung mit Aujeszky-Vakzinen mit einem Anstieg der IBR-Virusantikorpertiter. Nach Applikation der IBR-Vakzine wurde neben dem Anstieg homologer Antikorpertiter auch die Entwicklung von Antikorpern gegen Aujeszkyvirus beobachtet. Mit Immunfluoreszenz-Konjugaten, die von Rinderseren hergestellt wurden, deren Spender zuvor mit Aujeszky- oder IBR-Vakzine hyperimmunisiert worden waren, liesen sich in infizierten Zellkulturen sowohl homologe als auch heterologe Viren nachweisen. Seren von Schweinen mit hohen Antikorpertitern gegen Aujeszkyvirus neutralisierten IBR-Virus nicht. Mit IBR-Vakzinen immunisierte Kaninchen blieben vollempfanglich fur eine Infektion mit Aujeszkyvirus. Die Moglichkeiten eines Schutzes von Rindern die gegen die Aujeszkysche Krankheit immun sind, oder Rindern die gegen IBR immun sind gegen Infektionen mit dem jeweils heterologen Virus werden diskutiert. Resume Recherches sur les rapports immunologiques entre le virus de la rhinotracheite infectieuse du bovin (IBR) et le virus de la maladie d'Aujeszky avec des veaux, des porcs et des lapins Des serums de jeunes bovins qui avaient ete vaccines avec des vaccins inactives a adjuvant huileux d'Aujeszky ou d'IBR ont egalement neutralise le virus heterologue dans les cas de titre eleve d'anticorps homologues (1: 256–1: 4096). Des activites neutralisantes croisees ont egalement pu etre mises en evidence dans les secretions nasales des veaux qui avaient ete preleves au moment de l'apparition des titres les plus eleves des anticorps seriques. Des veaux receptifs sans anticorps viraux IBR ont produit seulement des anticorps contre le virus homologue apres une vaccination et une revaccination avec des vaccins Aujeszky ou IBR. Ils ont montre toutefois une reaction anamnestique selon le cas apres l'application de vaccins heterologues. Des serums d'animaux infectes naturellement avec le virus IBR ont neutralise le virus d'Aujeszky meme lorsqu'il n'y avait qu'un titre bas d'anticorps homologue. Des veaux seropositifs infectes naturellement avec le virus IBR ont reagi apres vaccination avec des vaccins d'Aujeszky par une montee du titre des anticorps viraux IBR. On a observe a cote de la montee du titre des anticorps homologues egalement le developpement d'anticorps contre le virus d'Aujeszky apres l'application de vaccins IBR. Des conjuges immunofluorescents obtenus a partir de serums bovins dont les donneurs avaient ete auparavant hyperimmunises avec des vaccins Aujeszky ou IBR ont permis de mettre en evidence aussi bien du virus homologue qu'heterologue dans des cultures cellulaires infectees. Des serums de porcs n'ont pas neutralise le virus IBR. Des lapins immunises avec des vaccins IBR sont restes totalement receptifs a une infection avec le virus d'Aujeszky. Les possibilites d'une protection des bovins qui sont immunises contre la maladie d'Aujeszky ou contre l'IBR vis-a-vis d'infections avec le virus heterologue sont discutees. Resumen Estudios en terneros, cerdos y conejos sobre las relaciones inmunologicas que hay entre el virus de la rinotraqueitis bovina infecciosa (IBR) y el virus de la enfermedad de Aujeszky Los sueros sanguineos de vacunos jovenes, los cuales habian sido vacunados con una vacuna Aujeszky inactivada o una IBR con adyuvante aceitoso, neutralizaban tambien el virus heterologo en el caso de titulos de anticuerpos homologos elevados (1: 256–1: 4.096). Asimismo se pudieron poner en evidencia actividades neutralizantes cruzadas en secreciones nasales de terneros que habian sido recogidas en el momento de aparecer los titulos maximos de seroanticuerpos. Los terneros receptibles sin anticuerpos virosicos IBR producian solo, tras vacunacion y revacunacion con vacunas Aujeszky o IBR, anticuerpos contra el virus homologo. Sin embargo, mostraban una reaccion anamnesica tras la aplicacion de la vacuna cada vez heterologa. Los sueros de animales infectados naturalmente con virus IBR neutralizaban el virus de Aujeszky incluso cuando solo estaban presentes titulos bajos de anticuerpos homologos. Los terneros seropositivos infectados naturalmente con virus IBR reaccionaban tras inoculacion con vacunas Aujeszky con el aumento de los titulos de anticuerpos virosicos IBR. Tras la aplicacion de la vacuna IBR se observo tambien al lado del aumento de titulos de anticuerpos homologos el desarrollo de anticuerpos contra el virus de Aujeszky. Con conjugados de inmunofluorescencia, obtenidos a partir de sueros vacunos, cuyos donadores habian sido hiperinmunizados previamente con la vacuna Aujeszky o la IBR, se pudieron identificar en los cultivos celulares infectados tanto virus homologos como heterologos. Los sueros de cerdos con titulos elevados de anticuerpos contra el virus de Aujeszky no neutralizaban el virus IBR. Los conejos inmunizados con vacunas IBR quedaron completamente receptibles para una infeccion con el virus de Aujeszky. Se discuten las posibilidades de proteger los vacunos que eran inmunes frente a la enfermedad de Aujeszky, o de los bovinos que son inmunes contra la IBR, con cada vez el virus heterologo.

Zusammenfassung Die Testinfektion geimpfter Schweine mit Maul- und Klauenseuchevirus Empfangliche Schweine schieden nach oraler Infektion (Einreibung mittels Tuch) und nach parenteraler Infektion bis zu 107 plaquebildende Einheiten (PBE) Maul- und Klauenseuchevirus pro ml Maulhohlenflussigkeit aus. Nach oraler Testinfektion 6 Wochen zuvor vakzinierter Schweine war die Virusausscheidung vergleichsweise viel geringer. Bei oraler Testinfektion 20 Wochen nach der Impfung betrug die Virusausscheidung bis zu 105,5 PBE pro ml Maulhohlenflussigkeit, die Tiere blieben jedoch gesund. Die Art der Auf-stallung (Einzel- oder Gruppenhaltung) hatte auf die Virusausscheidung keinen Einflus. Nach parenteraler Testinfektion einzeln aufgestallter vakzinierter Schweine war die Virusausscheidung uber die Maulhohle abhangig von der Immunitatslage des jeweiligen Tieres. Wurden solche Schweine zusammen aufgestallt, so schieden selbst voll immune Tiere Virus aus. Bei parenteral testinfizierten Schweinen mit nachfolgender generalisierter Maul- und Klauenseuche fielen die Titer neutralisierender Serumantikorper zunachst geringgradig, stiegen dann aber am 4. Tag wieder deutlich an. Die Abhangigkeit der Ergebnisse von Impfstoff-Wirksamkeitsprufungen vom Modus der Testinfektion wird diskutiert. Resume L'infection d'epreuve de porcs vaccines avec un virus de la fievre aphteuse Des porcs receptifs ont excrete jusqu'a 107 unites formant des plaques (UFP) de virus de la fievre aphteuse par ml de salive apres une infection orale (frottement avec un linge) et une infection parenterale. Une infection d'epreuve orale chez des porcs vaccines 6 semaines auparavant n'a provoque comparativement qu'une excretion viral beaucoup plus faible. Une infection d'epreuve 20 semaines apres la vaccination a donne une elimination virale jusqu'a 105,5 UFP par ml de salive, les animaux restant sains. Le genre d'installation (animal isole ou porcs en groupes) n'a eu aucune influence sur l'excretion de virus. L'excretion virale salivaire chez le porc isole vaccine apres une infection parenterale fut dependante de la situation immunitaire de chaque animal. Si les porcs etaient tenus en groupe, meme les animaux totalement immunises, excreterent du virus. Chez les porcs infectes par voie parenterale presentant une fievre aphteuse generalisee, les titres d'anticorps seriques neutralisants deminuerent faiblement au debut pour nettement augmenter le quatrieme jour. On discute l'influence du mode d'infection-test sur les resultats d'examens d'efficacite d'un vaccin. Resumen La infeccion de prueba con el virus aftoso en cerdos vacunados Cerdos receptibles secretaron tras infeccion oral (frotamiento con un pano) y tras infeccion parenteral hasta 107 unidades formadoras de plaques (UFP) de virus aftoso por ml. de liquido de la cavidad bucal. Tras la infeccion oral de prueba en cerdos vacunados seis semanas antes era mucho menor comparativamente la eliminacion de virus. En la infeccion oral de prueba 20 semanas despues de la vacunacion importo la eliminacion de virus hasta 105,5 UFP por ml. de liquido de la cavidad bucal, aunque los animales permanecieron sanos. El tipo de estabulacion (alojamiento individual o colectivo) no ejercia ningun influjo sobre la excrecion de virus. Tras la infeccion parenteral de prueba en cerdos vacunados en porquerizas individuales dependia la eliminacion de virus a traves de la cavidad bucal de la situacion inmunitaria del animal correspondiente. Cuando se estabularon juntos cerdos semejantes, excretaron virus incluso animales con inmunidad plena. En cerdos de prueba infectados por via parenteral con fiebre aftosa generalizada subsiguiente descendieron por lo pronto ligeramente los titulos de los seroanticuerpos neutralizantes, pero volvieron a aumentar con claridad el dia 4°. Se discute la dependencia de los resultados de las pruebas de efectividad de la vacuna del modo de efectuar la infeccion de prueba.