Convalescent human plasma candidate reference materials protect against Crimean-Congo haemorrhagic fever virus (CCHFV) challenge in an A129 mouse model.

Crimean-Congo Haemorrhagic Fever Virus (CCHFV) is spread by infected ticks or direct contact with blood, tissues and fluids from infected patients or livestock. Infection with CCHFV causes severe haemorrhagic fever in humans which is fatal in up to 83 % of cases. CCHFV is listed as a priority pathogen by the World Health Organization (WHO) and there are currently no widely-approved vaccines. Defining a serological correlate of protection against CCHFV infection would support the development of vaccines by providing a 'target threshold' for pre-clinical and clinical immunogenicity studies to achieve in subjects and potentially obviate the need for in vivo protection studies. We therefore sought to establish titratable protection against CCHFV using pooled human convalescent plasma, in a mouse model. Convalescent plasma collected from seven individuals with a known previous CCHFV virus infection were characterised using binding antibody and neutralisation assays. All plasma recognised nucleoprotein and the Gc glycoprotein, but some had a lower Gn glycoprotein response by ELISA. Pooled plasma and two individual donations from convalescent donors were administered intraperitoneally to A129 mice 24 h prior to intradermal challenge with CCHFV (strain IbAr10200). A partial protective effect was observed with all three convalescent plasmas characterised by longer survival post-challenge and reduced clinical score. These protective responses were titratable. Further characterisation of the serological reactivities within these samples will establish their value as reference materials to support assay harmonisation and accelerate vaccine development for CCHFV.

Seroprevalence and Risk Factors Associated with Phleboviruses and Crimean-Congo Hemorrhagic Fever Virus among Blood Donors in Central Tunisia.

The aim of this study was to determine the prevalence of six viruses, from two families of the order Bunyavirales, in the general population of central Tunisia. Sera collected from 377 asymptomatic blood donors were serologically assayed for Rift Valley fever virus (RVFV), Crimean-Congo hemorrhagic fever virus (CCHFV), and four sandfly-borne phleboviruses: Toscana virus (TOSV), sandfly fever Naples virus (SFNV), sandfly fever Sicilian virus (SFSV), and sandfly fever Cyprus virus (SFCV). Of the 377 subjects enrolled in this study, 17.3% were IgG positive for at least one of the viruses tested. The most frequently detected antibodies were against TOSV (13.3%), followed by SFCV (2.9%), RVFV (1.9%), SFSV (1.3%), and SFNV (1.1%). Only one sample was IgG positive for CCHFV. Dual reactivity was observed in nine cases: SFSV + SFCV in three cases (0.8%) and TOSV + SFNV, TOSV + SFCV, and TOSV + RVFV in two cases (0.5%) each. 15.9% of donors were IgG positive against sandfly-borne phleboviruses. Among the 65 donors IgG positive for phleboviruses, 50.8% were from rural areas compared to 12.3% from urban areas (p < 0.001); 92.3% had animals in their living quarters (p = 0.009); and 70.8% lived in the vicinity of stagnant water (p = 0.062). Seroprevalence was significantly higher among donors living with chronic diseases (p = 0.039). Furthermore, the seroprevalence of phleboviruses was higher in Kairouan, the central governorate, than in the two coastal governorates: Monastir and Sousse, with 33.4%, 24.2%, and 14.9%, respectively. The presence of antibodies in the general population needs further investigation to better assess the extent of these viruses. Only TOSV was known to have an extensive circulation in Tunisia and in North Africa. Continued surveillance and interventions are necessary to detect the emergence of all arboviruses and to prevent further transmission.

Pathogenesis of Rift Valley Fever Virus in a BALB/c Mouse Model Is Affected by Virus Culture Conditions and Sex of the Animals.

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen causing disease in livestock and humans. Whilst initially restricted to the African continent, recent spread to the Arabian Peninsula has highlighted the likelihood of entry into new regions. Due to the absence of a regulatory-approved human vaccine, work is ongoing to develop and assess countermeasures. As such, small animal models play a pivotal role in providing information on disease pathogenesis and elucidating which intervention strategies confer protection. To develop and establish the BALB/c mouse model, we challenged mice with RVFV grown from two separate cell lines: one derived from mosquitoes (C6/36) and the other mammalian derived (Vero E6). Following infection, we assessed the clinical course of disease progression at days 1 and 3 post-challenge and evaluated viral tropism and immune analytes. The results demonstrated that RVFV infection was affected by the cell line used to propagate the challenge virus, with those grown in insect cells resulting in a more rapid disease progression. The lowest dose that caused uniform severe disease remained the same across both virus preparations. In addition, to demonstrate reproducibility, the lowest dose was used for a subsequent infection study using male and female animals. The results further demonstrated that male mice succumbed to infection more rapidly than their female counterparts. Our results establish an RVFV mouse model and key parameters that affect the course of disease progression in BALB/c mice.

Establishment of a Nipah Virus Disease Model in Hamsters, including a Comparison of Intranasal and Intraperitoneal Routes of Challenge.

Nipah virus (NiV) is an emerging pathogen that can cause severe respiratory illness and encephalitis in humans. The main reservoir is fruit bats, distributed across a large geographical area that includes Australia, Southeast Asia, and Africa. Incursion into humans is widely reported through exposure of infected pigs, ingestion of contaminated food, or through contact with an infected person. With no approved treatments or vaccines, NiV poses a threat to human public health and has epidemic potential. To aid with the assessment of emerging interventions being developed, an expansion of preclinical testing capability is required. Given variations in the model parameters observed in different sites during establishment, optimisation of challenge routes and doses is required. Upon evaluating the hamster model, an intranasal route of challenge was compared with intraperitoneal delivery, demonstrating a more rapid dissemination to wider tissues in the latter. A dose effect was observed between those causing respiratory illness and those resulting in neurological disease. The data demonstrate the successful establishment of the hamster model of NiV disease for subsequent use in the evaluation of vaccines and antivirals.

Refinement of an ovine-based immunoglobulin therapy against SARS-CoV-2, with comparison of whole IgG versus F(ab')2 fragments.

The development of new therapies against SARS-CoV-2 is required to extend the toolkit of intervention strategies to combat the global pandemic. In this study, hyperimmune plasma from sheep immunised with whole spike SARS-CoV-2 recombinant protein has been used to generate candidate products. In addition to purified IgG, we have refined candidate therapies by removing non-specific IgG via affinity binding along with fragmentation to eliminate the Fc region to create F(ab')2 fragments. These preparations were evaluated for in vitro activity and demonstrated to be strongly neutralising against a range of SARS-CoV-2 strains, including Omicron B2.2. In addition, their protection against disease manifestations and viral loads were assessed using a hamster SARS-CoV-2 infection model. Results demonstrated protective effects of both IgG and F(ab')2, with the latter requiring sequential dosing to maintain in vivo activity due to rapid clearance from the circulation.

Development of a cost-effective ovine antibody-based therapy against SARS-CoV-2 infection and contribution of antibodies specific to the spike subunit proteins.

Antibodies against SARS-CoV-2 are important to generate protective immunity, with convalescent plasma one of the first therapies approved. An alternative source of polyclonal antibodies suitable for upscaling would be more amendable to regulatory approval and widespread use. In this study, sheep were immunised with SARS-CoV-2 whole spike protein or one of the subunit proteins: S1 and S2. Once substantial antibody titres were generated, plasma was collected and samples pooled for each antigen. Non-specific antibodies were removed via affinity-purification to yield candidate products for testing in a hamster model of SARS-CoV-2 infection. Affinity-purified polyclonal antibodies to whole spike, S1 and S2 proteins were evaluated for in vitro for neutralising activity against SARS-CoV-2 Wuhan-like virus (Australia/VIC01/2020) and a recent variant of concern, B.1.1.529 BA.1 (Omicron), antibody-binding, complement fixation and phagocytosis assays were also performed. All antibody preparations demonstrated an effect against SARS-CoV-2 disease in the hamster model of challenge, with those raised against the S2 subunit providing the most promise. A rapid, cost-effective therapy for COVID-19 was developed which provides a source of highly active immunoglobulin specific to SARS-CoV-2 with multi-functional activity.

Screening of wild deer populations for exposure to SARS-CoV-2 in the United Kingdom, 2020-2021.

Following findings in Northern America of SARS-CoV-2 infections in white-tailed deer, there is concern of similar infections in European deer and their potential as reservoirs of SARS-CoV-2 including opportunities for the emergence of new variants. UK deer sera were collected in 2020-2021 from 6 species and a hybrid with 1748 tested using anti-spike and anti-nucleocapsid serology assays. No samples were positive on both assays nor by surrogate neutralization testing. There is no evidence that spill-over infections of SARS-CoV-2 occurred from the human population to UK deer or that SARS-CoV-2 has been circulating in UK deer (over the study period). Although it cannot be ruled out, study results indicate that spill-over infections followed by circulation of SARS-CoV-2 to the most common European deer species is small.

X-ray inactivation of RNA viruses without loss of biological characteristics.

In the event of an unpredictable viral outbreak requiring high/maximum biosafety containment facilities (i.e. BSL3 and BSL4), X-ray irradiation has the potential to relieve pressures on conventional diagnostic bottlenecks and expediate work at lower containment. Guided by Monte Carlo modelling and in vitro 1-log10 decimal-reduction value (D-value) predictions, the X-ray photon energies required for the effective inactivation of zoonotic viruses belonging to the medically important families of Flaviviridae, Nairoviridae, Phenuiviridae and Togaviridae are demonstrated. Specifically, it is shown that an optimized irradiation approach is attractive for use in a multitude of downstream detection and functional assays, as it preserves key biochemical and immunological properties. This study provides evidence that X-ray irradiation can support emergency preparedness, outbreak response and front-line diagnostics in a safe, reproducible and scalable manner pertinent to operations that are otherwise restricted to higher containment BSL3 or BSL4 laboratories.

Hantavirus infection in type I interferon receptor-deficient (A129) mice.

Type I interferon receptor knockout mice (strain A129) were assessed as a disease model of hantavirus infection. A range of infection routes (intramuscular, intraperitoneal and intranasal) were assessed using minimally passaged Seoul virus (strain Humber). Dissemination of virus to the spleen, kidney and lung was observed at 5 days after intramuscular and intraperitoneal challenge, which was resolved by day 14. In contrast, intranasal challenge of A129 mice demonstrated virus tropism to the lung, which was maintained to day 14 post-challenge. These data support the use of the A129 mouse model for future infection studies and the in vivo evaluation of interventions.

Immunogenicity and Efficacy of Zika Virus Envelope Domain III in DNA, Protein, and ChAdOx1 Adenoviral-Vectored Vaccines.

The flavivirus envelope protein domain III (EDIII) was an effective immunogen against dengue virus (DENV) and other related flaviviruses. Whether this can be applied to the Zika virus (ZIKV) vaccinology remains an open question. Here, we tested the efficacy of ZIKV-EDIII against ZIKV infection, using several vaccine platforms that present the antigen in various ways. We provide data demonstrating that mice vaccinated with a ZIKV-EDIII as DNA or protein-based vaccines failed to raise fully neutralizing antibodies and did not control viremia, following a ZIKV challenge, despite eliciting robust antibody responses. Furthermore, we showed that ZIKV-EDIII encoded in replication-deficient Chimpanzee adenovirus (ChAdOx1-EDIII) elicited anti-ZIKV envelope antibodies in vaccinated mice but also provided limited protection against ZIKV in two physiologically different mouse challenge models. Taken together, our data indicate that contrary to what was shown for other flaviviruses like the dengue virus, which has close similarities with ZIKV-EDIII, this antigen might not be a suitable vaccine candidate for the correct induction of protective immune responses against ZIKV.

Towards quantification of protective antibody responses by passive transfer of the 1st WHO International Standard for Ebola virus antibody in a guinea pig model.

Ebola virus (EBOV) represents a major concern to global health due to the unpredictable nature of outbreaks. Infection with EBOV can cause a severe viral haemorrhagic fever with no licensed vaccine or treatment, restricting work with live EBOV to Containment/Biosafety Level 4 facilities. Whilst the magnitude of recent outbreaks has provided an impetus for vaccine and antiviral development, establishing the efficacy of candidate vaccine materials relies on EBOV challenge models and advanced human trials should outbreaks occur and where logistics and funding allow. To address these hurdles in vaccine development, we investigated whether a recently established serological reference standard, the 1st WHO International Standard for Ebola virus antibody, could be used to provide a quantifiable correlate of immune protection in vivo. Dilutions of the International Standard were inoculated into naïve guinea pigs 24 h before challenge with a lethal dose of Ebola virus. Only subjects receiving the highest dose of the International Standard exhibited evidence of delayed progression. Due to it being a WHO established reagent and available globally upon request, this standard allows for effective comparisons of data between laboratories and may prove valuable to select the candidate vaccines that are most likely to confer humoral immune protection ensuring the most promising candidates progress into efficacy studies.

Absence of Crimean-Congo haemorrhagic fever virus in the tick Hyalomma aegyptium parasitizing the spur-thighed tortoise (Testudo graeca) in Tunisia.

Free-ranging spur-thighed tortoises Testudo graeca, captured in different habitat types of Northern Tunisia from March to April 2017, were examined for tick infestation: 134/147 (91%) were infested. The overall infestation intensity and abundance was 8.5 and 7.8, respectively. From these tortoises, 1174 ticks were collected, of which 10% (n = 120) taken from 18 randomly-selected tortoises were identified at the species level; the remaining ticks were examined for the presence of Crimean-Congo haemorrhagic fever virus (CCHFv) by real time RT-PCR. Only adult Hyalomma aegyptium were found, suggesting a high degree of host specificity to tortoises. No CCHFv was detected in ticks. Considering the absence of CCHFv in Hyalomma aegyptium infesting its main host, the spur-thighed tortoise, this tick species is unlikely to play a major role in the epidemiology of CCHF. Therefore, more studies are needed to investigate the circulation of this arbovirus between livestock and other tick species from North Africa.

Therapeutic Monoclonal Antibodies for Ebola Virus Infection Derived from Vaccinated Humans.

We describe therapeutic monoclonal antibodies isolated from human volunteers vaccinated with recombinant adenovirus expressing Ebola virus glycoprotein (EBOV GP) and boosted with modified vaccinia virus Ankara. Among 82 antibodies isolated from peripheral blood B cells, almost half neutralized GP pseudotyped influenza virus. The antibody response was diverse in gene usage and epitope recognition. Although close to germline in sequence, neutralizing antibodies with binding affinities in the nano- to pico-molar range, similar to "affinity matured" antibodies from convalescent donors, were found. They recognized the mucin-like domain, glycan cap, receptor binding region, and the base of the glycoprotein. A cross-reactive cocktail of four antibodies, targeting the latter three non-overlapping epitopes, given on day 3 of EBOV infection, completely protected guinea pigs. This study highlights the value of experimental vaccine trials as a rich source of therapeutic human monoclonal antibodies.

A novel vaccine against Crimean-Congo Haemorrhagic Fever protects 100% of animals against lethal challenge in a mouse model.

Crimean-Congo Haemorrhagic Fever (CCHF) is a severe tick-borne disease, endemic in many countries in Africa, the Middle East, Eastern Europe and Asia. Between 15-70% of reported cases are fatal. There is no approved vaccine available, and preclinical protection in vivo by an experimental vaccine has not been demonstrated previously. In the present study, the attenuated poxvirus vector, Modified Vaccinia virus Ankara, was used to develop a recombinant candidate vaccine expressing the CCHF virus glycoproteins. Cellular and humoral immunogenicity was confirmed in two mouse strains, including type I interferon receptor knockout mice, which are susceptible to CCHF disease. This vaccine protected all recipient animals from lethal disease in a challenge model adapted to represent infection via a tick bite. Histopathology and viral load analysis of protected animals confirmed that they had been exposed to challenge virus, even though they did not exhibit clinical signs. This is the first demonstration of efficacy of a CCHF vaccine.

Hazara virus infection is lethal for adult type I interferon receptor-knockout mice and may act as a surrogate for infection with the human-pathogenic Crimean-Congo hemorrhagic fever virus.

Hazara virus (HAZV) is closely related to the Crimean-Congo hemorrhagic fever virus (CCHFV). HAZV has not been reported to cause human disease; work with infectious material can be carried out at containment level (CL)-2. By contrast, CCHFV causes a haemorrhagic fever in humans and requires CL-4 facilities. A disease model of HAZV infection in mice deficient in the type I interferon receptor is reported in this study. Dose-response effects were seen with higher doses, resulting in a shorter time to death and earlier detection of viral loads in organs. The lowest dose of 10 p.f.u. was still lethal in over 50 % of the mice. Histopathological findings were identified in the liver, spleen and lymph nodes, with changes similar to a recent mouse model of CCHFV infection. The findings demonstrate that inoculation of mice with HAZV may act as a useful surrogate model for the testing of antiviral agents against CCHFV.