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Detection of Serum Antibody Responses in Nipah Virus-Infected Pigs

Fischer K, Pickering B, Diederich S

Methods Mol Biol, 2023;2610:17-29.
PMID: 36534278 DOI: 10.1007/978-1-0716-2895-9_2

Nipah virus (NiV) is an emerging, zoonotic paramyxovirus that is among the most pathogenic of viruses in humans. During the first reported outbreak of NiV in Malaysia and Singapore in the late 1990s, pigs served as an intermediate host, which enabled the transmission to humans. Although subsequent outbreaks in Asia only reported direct bat-to-human and human-to-human transmission, pigs are still considered a potential source for viral dissemination in the epidemiology of the disease. Thus, serological assays such as Enzyme-linked immunosorbent assay (ELISA) or virus neutralization test (VNT) represent powerful tools to characterize the serum antibody responses in NiV-infected pigs as well as to perform seroepidemiological surveillance studies on the potential circulation of NiV or NiV-related viruses among pig populations worldwide. This chapter describes both methods in detail. Furthermore, we discuss some of the major pitfalls and indicate how to avoid them.
Cytoplasmic and periplasmic expression of recombinant shark VNAR antibody in Escherichia coli

Leow HC, Fischer K, Leow YC, Braet K, Cheng Q, McCarthy J

Prep Biochem Biotechnol, 2019;49(4):315-327.
PMID: 30767708 DOI: 10.1080/10826068.2019.1566145

Shark variable new antigen receptors (VNARs) are known to possess excellent heat-stability, and the long complementarity determining region 3 (CDR3) has permitted it to penetrate into the cleft region of antigens. The number of cysteine (Cys) residues contained within VNAR is greater than in conventional antibodies, entailing disulfide bond formation in both the inter- or intra-loop regions is required for interactions with the target protein antigens. Therefore, the selection of a suitable expression system is important to ensure the solubility and correct folding of functional VNAR protein production. Unlike higher organisms, the machinery for effecting posttranslational modifications of proteins in Escherichia coli (E. coli) are less sophisticated. To overcome this circumstance, a pDSB-28Y vector fusion with DsbA signal peptide was engineered for periplasmic H8VNAR production. Despite the periplasmic proteins showing a lower yield (62 µg/mL) than cytosolic proteins (468 µg/mL) that is obtained from pET-28a vector, it has demonstrated better performance than that of a cytosolic protein in terms of absorbance. However, these readings were still inferior to that of positive control mouse monoclonal antibody (mAb) C1-13 in this experiment. Therefore, further investigation is required to improve the binding affinity of selected recombinant VNAR towards malaria biomarkers.
Fulltext Safety, efficacy and pharmacokinetics of rVIII-SingleChain in children with severe hemophilia A: results of a multicenter clinical trial

Stasyshyn O, Djambas Khayat C, Iosava G, Ong J, Abdul Karim F, Fischer K, et al.

J Thromb Haemost, 2017 Apr;15(4):636-644.
PMID: 28166608 DOI: 10.1111/jth.13647

Essentials rVIII-SingleChain is a novel recombinant factor VIII with covalently bonded heavy and light chains. Efficacy, safety and pharmacokinetics were studied in pediatric patients with severe hemophilia A. Across all prophylaxis regimens, the median annualized spontaneous bleeding rate was 0.00. rVIII-SingleChain showed excellent hemostatic efficacy and a favorable safety profile.
SUMMARY: Background rVIII-SingleChain is a novel B-domain truncated recombinant factor VIII (rFVIII) comprised of covalently bonded FVIII heavy and light chains, demonstrating a high binding affinity to von Willebrand factor. Objectives This phase III study investigated the safety, efficacy and pharmacokinetics of rVIII-SingleChain in previously treated pediatric patients < 12 years of age with severe hemophilia A. Patients/Methods Patients could be assigned to prophylaxis or on-demand therapy by the investigator. For patients assigned to prophylaxis, the treatment regimen and dose were based on the bleeding phenotype. For patients receiving on-demand therapy, dosing was guided by World Federation of Hemophilia recommendations. The primary endpoint was treatment success, defined as a rating of 'excellent' or 'good' on the investigator's clinical assessment of hemostatic efficacy for all treated bleeding events. Results The study enrolled 84 patients (0 to < 6 years, n = 35; ≥ 6 to < 12 years, n = 49); 81 were assigned to prophylaxis and three to an on-demand regimen. Patients accumulated a total of 5239 exposure days (EDs), with 65 participants reaching > 50 EDs. In the 347 bleeds treated and evaluated by the investigator, hemostatic efficacy was rated as excellent or good in 96.3%. The median annualized spontaneous bleeding rate was 0.00 (Q1, Q3: 0.00, 2.20), and the median annualized bleeding rate was 3.69 (Q1, Q3: 0.00, 7.20) across all prophylaxis regimens. No participant developed an inhibitor. Conclusions rVIII-SingleChain is a novel rFVIII molecule showing excellent hemostatic efficacy and a favorable safety profile in a clinical study in children < 12 years of age with severe hemophilia A.
Fulltext Isolation and characterization of malaria PfHRP2 specific VNAR antibody fragments from immunized shark phage display library

Leow CH, Fischer K, Leow CY, Braet K, Cheng Q, McCarthy J

Malar J, 2018 Oct 24;17(1):383.
PMID: 30355309 DOI: 10.1186/s12936-018-2531-y

BACKGROUND: Malaria rapid diagnostic tests (RDTs) represent an important antibody based immunoassay platform. Unfortunately, conventional monoclonal antibodies are subject to degradation shortening shelf lives of RDTs. The variable region of the receptor (VNAR) from shark has a potential as alternative to monoclonal antibodies in RDTs due to high thermal stability.
METHODS: In this study, new binders derived from shark VNAR domains library were investigated. Following immunization of a wobbegong shark (Orectolobus ornatus) with three recombinant malaria biomarker proteins (PfHRP2, PfpLDH and Pvaldolase), a single domain antibody (sdAb) library was constructed from splenocytes. Target-specific VNAR phage were isolated by panning. One specific clone was selected for expression in Escherichia coli expression system, and study of binding reactivity undertaken.
RESULTS: The primary VNAR domain library possessed a titre of 1.16 × 106 pfu/mL. DNA sequence analysis showed 82.5% of isolated fragments appearing to contain an in-frame sequence. After multiple rounds of biopanning, a highly dominant clone specific to PfHRP2 was identified and selected for protein production in an E. coli expression system. Biological characterization showed the recombinant protein expressed in periplasmic has better detection sensitivity than that of cytoplasmic proteins. Assays of binding activity indicated that its reactivity was inferior to the positive control mAb C1-13.
CONCLUSIONS: Target-specific bacteriophage VNARs were successfully isolated after a series of immunization, demonstrating that phage display technology is a useful tool for selection of antigen binders. Generation of new binding reagents such as VNAR antibodies that specifically recognize the malaria biomarkers represents an appealing approach to improve the performance of RDTs.
Fulltext Evidence of exposure to henipaviruses in domestic pigs in Uganda

Atherstone C, Diederich S, Weingartl HM, Fischer K, Balkema-Buschmann A, Grace D, et al.

Transbound Emerg Dis, 2019 Mar;66(2):921-928.
PMID: 30576076 DOI: 10.1111/tbed.13105

Hendra virus (HeV) and Nipah virus (NiV), belonging to the genus Henipavirus, are among the most pathogenic of viruses in humans. Old World fruit bats (family Pteropodidae) are the natural reservoir hosts. Molecular and serological studies found evidence of henipavirus infection in fruit bats from several African countries. However, little is known about the potential for spillover into domestic animals in East Africa, particularly pigs, which served as amplifying hosts during the first outbreak of NiV in Malaysia and Singapore. We collected sera from 661 pigs presented for slaughter in Uganda between December 2015 and October 2016. Using HeV G and NiV G indirect ELISAs, 14 pigs (2%) were seroreactive in at least one ELISA. Seroprevalence increased to 5.4% in October 2016, when pigs were 9.5 times more likely to be seroreactive than pigs sampled in December 2015 (p = 0.04). Eight of the 14 ELISA-positive samples reacted with HeV N antigen in Western blot. None of the sera neutralized HeV or NiV in plaque reduction neutralization tests. Although we did not detect neutralizing antibodies, our results suggest that pigs in Uganda are exposed to henipaviruses or henipa-like viruses. Pigs in this study were sourced from many farms throughout Uganda, suggesting multiple (albeit rare) introductions of henipaviruses into the pig population. We postulate that given the widespread distribution of Old World fruit bats in Africa, spillover of henipaviruses from fruit bats to pigs in Uganda could result in exposure of pigs at multiple locations. A higher risk of a spillover event at the end of the dry season might be explained by higher densities of bats and contact with pigs at this time of the year, exacerbated by nutritional stress in bat populations and their reproductive cycle. Future studies should prioritize determining the risk of spillover of henipaviruses from pigs to people, so that potential risks can be mitigated.
Henipaviruses at the Interface Between Bats, Livestock and Human Population in Africa

Mbu'u CM, Mbacham WF, Gontao P, Sado Kamdem SL, Nlôga AMN, Groschup MH, et al.

Vector Borne Zoonotic Dis, 2019 07;19(7):455-465.
PMID: 30985268 DOI: 10.1089/vbz.2018.2365

Nipah virus (NiV) and Hendra virus (HeV) are closely related members within the genus Henipavirus, family Paramyxoviridae, for which fruit bats serve as the reservoir. The initial emergence of NiV infections in pigs and humans in Malaysia, and HeV infections in horses and humans in Australia, posed severe impacts on human and animal health, and continues threatening lives of humans and livestock within Southeast Asia and Australia. Recently, henipavirus-specific antibodies have also been detected in fruit bats in a number of sub-Saharan African countries and in Brazil, thereby considerably increasing the known geographic distribution of henipaviruses. Africa is progressively being recognized as a new high prevalence zone for henipaviruses, as deduced from serological and molecular evidence of past infections in Madagascar, Ghana, Republic of Congo, Gulf of Guinea, Zambia, Tanzania, Cameroon, and Nigeria lately. Serological data suggest henipavirus spillover from bats to livestock and human populations in Africa without reported clinical disease in any of these species. All virus isolation attempts have been abortive, highlighting the need for further investigations. The genome of the Ghanaian bat henipavirus designated Ghana virus (GhV), which was detected in a pteropid Eidolon helvum bat, is the only African henipavirus that has been completely sequenced limiting our current knowledge on the genetic diversity and pathogenesis of African henipaviruses. In this review, we summarize the available data on the circulation of henipaviruses in Africa, discuss potential sources for virus spillover, and highlight existing research gaps.
Fulltext Indirect ELISA based on Hendra and Nipah virus proteins for the detection of henipavirus specific antibodies in pigs

Fischer K, Diederich S, Smith G, Reiche S, Pinho Dos Reis V, Stroh E, et al.

PLoS One, 2018;13(4):e0194385.
PMID: 29708971 DOI: 10.1371/journal.pone.0194385

Hendra virus (HeV) and Nipah virus (NiV) belong to the genus Henipavirus in the family Paramyxoviridae. Henipavirus infections were first reported in the 1990's causing severe and often fatal outbreaks in domestic animals and humans in Southeast Asia and Australia. NiV infections were observed in humans in Bangladesh, India and in the first outbreak in Malaysia, where pigs were also infected. HeV infections occurred in horses in the North-Eastern regions of Australia, with singular transmission events to humans. Bats of the genus Pteropus have been identified as the reservoir hosts for henipaviruses. Molecular and serological indications for the presence of henipa-like viruses in African fruit bats, pigs and humans have been published recently. In our study, truncated forms of HeV and NiV attachment (G) proteins as well as the full-length NiV nucleocapsid (N) protein were expressed using different expression systems. Based on these recombinant proteins, Enzyme-linked Immunosorbent Assays (ELISA) were developed for the detection of HeV or NiV specific antibodies in porcine serum samples. We used the NiV N ELISA for initial serum screening considering the general reactivity against henipaviruses. The G protein based ELISAs enabled the differentiation between HeV and NiV infections, since as expected, the sera displayed higher reactivity with the respective homologous antigens. In the future, these assays will present valuable tools for serosurveillance of swine and possibly other livestock or wildlife species in affected areas. Such studies will help assessing the potential risk for human and animal health worldwide by elucidating the distribution of henipaviruses.
Health-related quality of life in patients with generalized pustular psoriasis: A systematic literature review

Choon SE, De La Cruz C, Wolf P, Jha RK, Fischer KI, Goncalves-Bradley DC, et al.

J Eur Acad Dermatol Venereol, 2024 Feb;38(2):265-280.
PMID: 37750484 DOI: 10.1111/jdv.19530

Generalized pustular psoriasis (GPP) is a rare, chronic, neutrophilic inflammatory skin disease characterized by episodes of widespread eruption of sterile, macroscopic pustules that can be accompanied by systemic inflammation and symptoms. A systematic literature review and narrative synthesis were conducted to determine the impact of GPP on patients' health-related quality of life (HRQoL) and patient-reported severity of symptoms and to compare its impact to patients with plaque psoriasis (plaque PsO). Searches were undertaken in Embase, MEDLINE and the Cochrane Library from 1 January 2002 to 15 September 2022. Screening was carried out by two reviewers independently. Outcome measures included generic (e.g. EQ-5D, SF-36) and dermatology-specific (e.g. DLQI) clinical outcome assessments, and other relevant patient-reported outcome measures (PROMs) (e.g. severity of pain measured by a numerical rating scale). Overall, 20 studies were found to be eligible for inclusion, of which seven also had data for plaque PsO. The DLQI was the most frequently reported outcome measure (16 out of 20 studies). When reported, mean DLQI (SD) scores varied from 5.7 (1.2) to 15.8 (9.6) across the studies, indicating a moderate to very large effect on HRQoL; the wide range of scores and large SDs were explained by the small population sizes (n ≤ 12 for all studies except two). Similar ranges and large SDs were also observed for other measures within individual studies. However, in general, people with GPP reported a greater impact of their skin condition on HRQoL, when compared to people with plaque PsO (i.e. higher DLQI scores) and higher severity for itch, pain and fatigue. This systematic review highlighted the need for studies with a larger population size, a better understanding of the impact of cutaneous and extracutaneous symptoms and comorbidities on HRQoL during and between GPP flares, and outcome measures specifically tailored to the unique symptoms and the natural course/history of GPP.
Fulltext First international external quality assessment scheme of nucleic acid amplification tests for the detection of Schistosoma and soil-transmitted helminths, including Strongyloides: A pilot study

Cools P, van Lieshout L, Koelewijn R, Addiss D, Ajjampur SSR, Ayana M, et al.

PLoS Negl Trop Dis, 2020 06;14(6):e0008231.
PMID: 32544158 DOI: 10.1371/journal.pntd.0008231

BACKGROUND: Nucleic acid amplification tests (NAATs) are increasingly being used as diagnostic tools for soil-transmitted helminths (STHs; Ascaris lumbricoides, Trichuris trichiura, Necator americanus, Ancylostoma duodenale and A. ceylanicum), Strongyloides stercoralis and Schistosoma in human stool. Currently, there is a large diversity of NAATs being applied, but an external quality assessment scheme (EQAS) for these diagnostics is lacking. An EQAS involves a blinded process where test results reported by a laboratory are compared to those reported by reference or expert laboratories, allowing for an objective assessment of the diagnostic performance of a laboratory. In the current study, we piloted an international EQAS for these helminths (i) to investigate the feasibility of designing and delivering an EQAS; (ii) to assess the diagnostic performance of laboratories; and (iii) to gain insights into the different NAAT protocols used.
METHODS AND PRINCIPAL FINDINGS: A panel of twelve stool samples and eight DNA samples was validated by six expert laboratories for the presence of six helminths (Ascaris, Trichuris, N. americanus, Ancylostoma, Strongyloides and Schistosoma). Subsequently this panel was sent to 15 globally dispersed laboratories. We found a high degree of diversity among the different DNA extraction and NAAT protocols. Although most laboratories performed well, we could clearly identify the laboratories that were poorly performing.
CONCLUSIONS/SIGNIFICANCE: We showed the technical feasibility of an international EQAS for the NAAT of STHs, Strongyloides and Schistosoma. In addition, we documented that there are clear benefits for participating laboratories, as they can confirm and/or improve the diagnostic performance of their NAATs. Further research should aim to identify factors that explain poor performance of NAATs.
Fulltext Efficacy and safety of rVIII-SingleChain: results of a phase 1/3 multicenter clinical trial in severe hemophilia A

Mahlangu J, Kuliczkowski K, Karim FA, Stasyshyn O, Kosinova MV, Lepatan LM, et al.

Blood, 2016 Aug 04;128(5):630-7.
PMID: 27330001 DOI: 10.1182/blood-2016-01-687434

Recombinant VIII (rVIII)-SingleChain is a novel B-domain-truncated recombinant factor VIII (rFVIII), comprised of covalently bonded factor VIII (FVIII) heavy and light chains. It was designed to have a higher binding affinity for von Willebrand factor (VWF). This phase 1/3 study investigated the efficacy and safety of rVIII-SingleChain in the treatment of bleeding episodes, routine prophylaxis, and surgical prophylaxis. Participants were ≥12 years of age, with severe hemophilia A (endogenous FVIII <1%). The participants were allocated by the investigator to receive rVIII-SingleChain in either an on-demand or prophylaxis regimen. Of the 175 patients meeting study eligibility criteria, 173 were treated with rVIII-SingleChain, prophylactically (N = 146) or on-demand (N = 27). The total cumulative exposure was 14 306 exposure days (EDs), with 120 participants reaching ≥50 EDs and 52 participants having ≥100 EDs. Hemostatic efficacy was rated by the investigator as excellent or good in 93.8% of the 835 bleeds treated and assessed. Across all prophylaxis regimens, the median annualized spontaneous bleeding rate was 0.00 (Q1, Q3: 0.0, 2.4) and the median overall annualized bleeding rate (ABR) was 1.14 (Q1, Q3: 0.0, 4.2). Surgical hemostasis was rated as excellent/good in 100% of major surgeries by the investigator. No participant developed FVIII inhibitors. In conclusion, rVIII-SingleChain is a novel rFVIII molecule showing excellent hemostatic efficacy in surgery and in the control of bleeding events, low ABR in patients on prophylaxis, and a favorable safety profile in this large clinical study. This trial was registered at www.clinicaltrials.gov as #NCT01486927.

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