Development of a Genus-Specific Antigen Capture ELISA for Orthopoxviruses – Target Selection and Optimized Screening

Orthopoxvirus species like cowpox, vaccinia and monkeypox virus cause zoonotic infections in humans worldwide. Infections often occur in rural areas lacking proper diagnostic infrastructure as exemplified by monkeypox, which is endemic in Western and Central Africa. While PCR detection requires demanding equipment and is restricted to genome detection, the evidence of virus particles can complement or replace PCR. Therefore, an easily distributable and manageable antigen capture enzyme-linked immunosorbent assay (ELISA) for the detection of orthopoxviruses was developed to facilitate particle detection. By comparing the virus particle binding properties of polyclonal antibodies developed against surface-exposed attachment or fusion proteins, the surface protein A27 was found to be a well-bound, highly immunogenic and exposed target for antibodies aiming at virus particle detection. Subsequently, eight monoclonal anti-A27 antibodies were generated and characterized by peptide epitope mapping and surface plasmon resonance measurements. All antibodies were found to bind with high affinity to two epitopes at the heparin binding site of A27, toward either the N- or C-terminal of the crucial KKEP-segment of A27. Two antibodies recognizing different epitopes were implemented in an antigen capture ELISA. Validation showed robust detection of virus particles from 11 different orthopoxvirus isolates pathogenic to humans, with the exception of MVA, which is apathogenic to humans. Most orthopoxviruses could be detected reliably for viral loads above 1 × 103 PFU/mL. To our knowledge, this is the first solely monoclonal and therefore reproducible antibody-based antigen capture ELISA able to detect all human pathogenic orthopoxviruses including monkeypox virus, except variola virus which was not included. Therefore, the newly developed antibody-based assay represents important progress towards feasible particle detection of this important genus of viruses

Genomic Expression Libraries for the Identification of Cross-Reactive Orthopoxvirus Antigens

Increasing numbers of human cowpox virus infections that are being observed and that particularly affect young non-vaccinated persons have renewed interest in this zoonotic disease. Usually causing a self-limiting local infection, human cowpox can in fact be fatal for immunocompromised individuals. Conventional smallpox vaccination presumably protects an individual from infections with other Orthopoxviruses, including cowpox virus. However, available live vaccines are causing severe adverse reactions especially in individuals with impaired immunity. Because of a decrease in protective immunity against Orthopoxviruses and a coincident increase in the proportion of immunodeficient individuals in today's population, safer vaccines need to be developed. Recombinant subunit vaccines containing cross-reactive antigens are promising candidates, which avoid the application of infectious virus. However, subunit vaccines should contain carefully selected antigens to confer a solid cross-protection against different Orthopoxvirus species. Little is known about the cross-reactivity of antibodies elicited to cowpox virus proteins. Here, we first identified 21 immunogenic proteins of cowpox and vaccinia virus by serological screenings of genomic Orthopoxvirus expression libraries. Screenings were performed using sera from vaccinated humans and animals as well as clinical sera from patients and animals with a naturally acquired cowpox virus infection. We further analyzed the cross-reactivity of the identified immunogenic proteins. Out of 21 identified proteins 16 were found to be cross-reactive between cowpox and vaccinia virus. The presented findings provide important indications for the design of new-generation recombinant subunit vaccines

Expressionsbibliotheken als Werkzeuge für die Entwicklung von Subunit-Impfstoffen und neuartigen Detektionsmolekülen für Orthopockenviren

Nach der erfolgreichen globalen Ausrottung der Pockenkrankheit wurde die Pockenimpfung aufgrund seltener aber schwerer Komplikationen eingestellt. Heute, nach mehr als 30 Jahren, weist die Mehrheit der Welt-Bevölkerung keinen Immunschutz mehr auf. Gleichzeitig steigt neben der Häufigkeit zoonotischer Pockenvirusinfektionen mit Affen- oder Kuhpockenviren auch die Angst vor Bioterror-Anschlägen mit Variolaviren. Diese Entwicklungen unterstreichen die Notwendigkeit von Projekten, die vorbereitende Maßnahmen für den Fall eines Bioterroranschlags treffen, um die Ausbreitung von Krankheitserregern zu verhindern. Hierzu zählt neben der Entwicklung von schnellen und einfach zu bedienenden Diagnostikplattformen auch die Verfügbarkeit von Impfstoffen und Therapeutika, die der Mehrheit der Bevölkerung verabreicht werden können. Obwohl bereits zahlreiche, gut etablierte Methoden für den Nachweis von Pockenviren existieren, sind diese für eine schnelle Vor-Ort-Diagnostik häufig nicht geeignet. Vorhandene konventionelle Pockenimpfstoffe besitzen gute immunogene Eigenschaften, können jedoch einer steigenden Anzahl immunsupprimierter Menschen nicht verabreicht werden. Somit ist eine Entwicklung sicherer Pockenimpfstoffe erforderlich. Hierbei, stellen Subunit-Impfstoffe eine potentiell sicherere Alternative zu konventionellen Impfstoffen dar. Um die Entwicklung von Subunit-Impfstoffen voranzubringen und die Anwendbarkeit synthetischer Peptid-Aptamere für Pockenvirusdetektion zu beurteilen, wurden in der vorliegenden Arbeit Bakteriophagen-basierte Bibliotheken gescreent. Zuerst wurde eine kostengünstige Methode zur Antigen-Identifizierung etabliert und evaluiert. Bei dieser Methode werden Bakteriophagen-basierte Expressionsbibliotheken mit Pocken-Antiseren gescreent. Dank dieser serologischen Screenings konnten 21 immunogene Pockenproteine identifiziert werden. Sechzehn dieser 21 Proteine wurden auch als kreuzreaktiv zwischen Vaccinia-Virus und Kuhpockenvirus identifiziert. Nach einer Auswertung, werden sieben der identifizierten kreuzreaktiven Proteine A3, A4, D13, E2, E3, E9 und H6 aufgrund ihrer Antigenität und Konserviertheit für die Verwendung in Subunit-Impfstoffen vorgeschlagen. Außerdem wird die kombinatorische Phagen-Display-Methode und ihre Verwendung zur Identifizierung pockenspezifischer Peptidliganden als neuartige Detektionsmoleküle vorgestellt. Affinitätsselektionen von Phagen-Display-Bibliotheken gegen infektiöse Vaccinia-Virus-Partikel führten zur erfolgreichen Anreicherung von 17 wiederkehrenden Peptidsequenzen, was auf eine Anreicherung von Pockenvirus-bindenden Phagenklonen hindeutet. Nach einer Charakterisierung dieser 17 bindenden Klone, wurden drei Peptidsequenzen ausgewählt, synthetisiert und weiter charakterisiert. Dabei konnte für eins dieser drei synthetischen Peptide eine spezifische Bindung an Vaccinia-Virus-Partikel gezeigt werden. Dies demonstriert die Eignung synthetischer Peptide für die Detektion von Bioterror-Agenzien.The global eradication of smallpox led to the cessation of routine smallpox vaccination due to rare but severe adverse reactions. Today, more than 30 years later, the majority of the world’s population has no protective immunity against poxviruses. Concurrently, the frequency of zoonotic poxvirus infections with monkeypox and cowpox virus is increasing, accompanied by the fear of bioterrorist attacks with smallpox. These developments emphasize the need for bio-preparedness programs to enable rapid and effective prevention and control of poxvirus-associated disease spread. Bio-preparedness includes the availability of rapid detection methods as well as the existence of safe vaccines and therapeutics that can be administered to the majority of the population. Various existing poxvirus detection methods are well-established and highly sensitive. However, they are usually not suitable for rapid on-site detection of biothreat agents. Conventional smallpox vaccines, on the other hand, show excellent immunogenic properties. Yet today, they can not be administered to a growing proportion of individuals with impaired immunity, urging the development of safer vaccines. Thereby, recombinant subunit vaccines are considered to be a safer alternative to conventional smallpox vaccines. To speed up the development of subunit vaccines and to evaluate the applicability of synthetic peptide aptamers for poxvirus detection, screenings of bacteriophage-based libraries were utilized in the present study. First, a low-cost approach for antigen discovery was established and evaluated. This approach is based on serological screenings of bacteriophage-based genomic expression libraries. These screenings resulted in the identification of 21 antigenic proteins. Sixteen of these 21 antigens were also found to be cross-reactive among cowpox and vaccinia virus. In addition, seven of identified antigenic cross-reactive proteins A3, A4, D13, E2, E3, E9, and H6 are proposed to be included in subunit vaccines due to their antigenicity and conservation among orthopoxviruses. Additionally, combinatorial phage display methodology was utilized to identify poxvirus-specific peptide ligands as novel detection molecules. Affinity selections of random peptide phage display libraries against infectious virus particles yielded 17 recurring peptide sequences indicating an enrichment of poxvirus-binding phage clones. After characterization of these 17 binding clones, three peptide sequences were synthesized and characterized further. Thereby, one phage-derived synthetic peptide was shown to bind selectively and specifically to vaccinia virus particles. This provides important insights into applicability of synthetic molecules for detection of biothreat agents

Rapid and sensitive point-of-care detection of Orthopoxviruses by ABICAP immunofiltration

Background: The rapid and reliable detection of infectious agents is one of the most challenging tasks in scenarios lacking well-equipped laboratory infrastructure, like diagnostics in rural areas of developing countries. Commercially available point-of-care diagnostic tests for emerging and rare diseases are particularly scarce. Results: In this work we present a point-of-care test for the detection of Orthopoxviruses (OPV). The OPV ABICAP assay detects down to 1 × 104 plaque forming units/mL of OPV particles within 45 min. It can be applied to clinical material like skin crusts and detects all zoonotic OPV infecting humans, including Vaccinia, Cowpox, Monkeypox, and most importantly Variola virus. Conclusions: Given the high sensitivity and the ease of handling, the novel assay could be highly useful for on-site diagnostics of suspected Monkeypox virus infections in areas lacking proper laboratory infrastructure as well as rapid on-site testing of suspected bioterrorism samples

Additional file 1: Figure S1. of Rapid and sensitive point-of-care detection of Orthopoxviruses by ABICAP immunofiltration

Titration of SA-pHRP and biotinylated detection antibody A3/710. Working concentrations for both SA-pHRP (A) and A3/710 (B) were determined by testing four concentrations (0.1, 0.5, 1, 5 μg/mL), using the ABICAP protocol to minimize background binding caused by too high antibody or enzyme concentrations (TIF 164 kb

Binding to viral particles by anti-A27, -D8, -H3 and -L1 pAbs.

<p><b>A</b>. Immobilized recombinant proteins or purified UV-inactivated VACV_NYCBOH particles were incubated with a dilution series of purified biotinylated anti-A27, -D8, -H3 and -L1 antibodies in an indirect ELISA. Detection was done with SA-pHRP (1:5000). <b>B</b>. Representative electron microscopic pictures of anti-A27, -D8, or -H3 antibodies binding to purified VACV_NYCBOH. Biotinylated antibodies were detected using 5 nm immunogold labelled streptavidin. Scale bars = 100 nm. <b>C</b>. Box-and-whisker plot for quantification of the number of gold particles per viral particle (whiskers: min to max; A27 n = 111; D8 n = 109 viral particles analyzed).</p

Antigen titration curves for the newly developed antigen capture ELISA with mAb A1/40 as capture and mAb A3/710 as detection antibody.

<p><b>A</b>. Titration curve of rA27 with the antigen capture ELISA. <b>B</b>. To check for cross-reactivity, PPXV, HSV-1 and tanapox virus were tested. <b>C</b>. Detection of different VACV strains by the newly developed assay. <b>D</b>. Detection of different OPV.</p