Crimean-Congo haemorrhagic fever virus uses LDLR to bind and enter host cells.

Climate change and population densities accelerated transmission of highly pathogenic viruses to humans, including the Crimean-Congo haemorrhagic fever virus (CCHFV). Here we report that the Low Density Lipoprotein Receptor (LDLR) is a critical receptor for CCHFV cell entry, playing a vital role in CCHFV infection in cell culture and blood vessel organoids. The interaction between CCHFV and LDLR is highly specific, with other members of the LDLR protein family failing to bind to or neutralize the virus. Biosensor experiments demonstrate that LDLR specifically binds the surface glycoproteins of CCHFV. Importantly, mice lacking LDLR exhibit a delay in CCHFV-induced disease. Furthermore, we identified the presence of Apolipoprotein E (ApoE) on CCHFV particles. Our findings highlight the essential role of LDLR in CCHFV infection, irrespective of ApoE presence, when the virus is produced in tick cells. This discovery holds profound implications for the development of future therapies against CCHFV.

Nucleoside-Modified mRNA Vaccines Protect IFNAR-/- Mice against Crimean-Congo Hemorrhagic Fever Virus Infection.

Crimean-Congo hemorrhagic fever (CCHF), caused by Crimean-Congo hemorrhagic fever virus (CCHFV), is on the World Health Organizations' list of prioritized diseases and pathogens. With global distribution, high fatality rate, and no approved vaccine or effective treatment, CCHF constitutes a threat against global health. In the current study, we demonstrate that vaccination with nucleoside-modified mRNA-lipid nanoparticles (mRNA-LNP), encoding for the CCHFV nucleoprotein (N) or glycoproteins (GcGn) protect IFNAR-/- mice against lethal CCHFV infection. In addition, we found that both mRNA-LNP induced strong humoral and cellular immune responses in IFNAR-/- and immunocompetent mice and that neutralizing antibodies are not necessary for protection. When evaluating immune responses induced by immunization including CCHFV Gc and Gn antigens, we found the Gc protein to be more immunogenic compared with the Gn protein. Hepatic injury is prevalent in CCHF and contributes to the severity and mortality of the disease in humans. Thus, to understand the immune response in the liver after infection and the potential effect of the vaccine, we performed a proteomic analysis on liver samples from vaccinated and control mice after CCHFV infection. Similar to observations in humans, vaccination affected the metabolic pathways. In conclusion, this study shows that a CCHFV mRNA-LNP vaccine, based on viral nucleo- or glycoproteins, mediate protection against CCHFV induced disease. Consequently, genetic immunization is an attractive approach to prevent disease caused by CCHFV and we believe we have necessary evidence to bring this vaccine platform to the next step in the development of a vaccine against CCHFV infection. IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is a zoonotic pathogen causing Crimean-Congo hemorrhagic fever (CCHF), a severe fever disease. CCHFV has a wide distribution and is endemic in several areas around the world. Cases of CCHF are also being reported in new areas, indicating an expansion of the disease, which is of high concern. Dispersion of the disease, high fatality rate, and no approved vaccine makes CCHF a threat to global health. The development of a vaccine is thus of great importance. Here we show 100% protection against lethal CCHFV infection in mice immunized with mRNA-LNP encoding for different CCHFV proteins. The vaccination showed both robust humoral and cellular immunity. mRNA-LNP vaccines combine the ability to induce an effective immune response, the safety of a transient carrier, and the flexibility of genetic vaccines. This and our results from the current study support the development of a mRNA-LNP based vaccine against CCHFV.

Immunization with DNA Plasmids Coding for Crimean-Congo Hemorrhagic Fever Virus Capsid and Envelope Proteins and/or Virus-Like Particles Induces Protection and Survival in Challenged Mice.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a bunyavirus causing severe hemorrhagic fever disease in humans, with high mortality rates. The requirement of a high-containment laboratory and the lack of an animal model hampered the study of the immune response and protection of vaccine candidates. Using the recently developed interferon alpha receptor knockout (IFNAR-/-) mouse model, which replicates human disease, we investigated the immunogenicity and protection of two novel CCHFV vaccine candidates: a DNA vaccine encoding a ubiquitin-linked version of CCHFV Gc, Gn, and N and one using transcriptionally competent virus-like particles (tc-VLPs). In contrast to most studies that focus on neutralizing antibodies, we measured both humoral and cellular immune responses. We demonstrated a clear and 100% efficient preventive immunity against lethal CCHFV challenge with the DNA vaccine. Interestingly, there was no correlation with the neutralizing antibody titers alone, which were higher in the tc-VLP-vaccinated mice. However, the animals with a lower neutralizing titer, but a dominant cell-mediated Th1 response and a balanced Th2 response, resisted the CCHFV challenge. Moreover, we found that in challenged mice with a Th1 response (immunized by DNA/DNA and boosted by tc-VLPs), the immune response changed to Th2 at day 9 postchallenge. In addition, we were able to identify new linear B-cell epitope regions that are highly conserved between CCHFV strains. Altogether, our results suggest that a predominantly Th1-type immune response provides the most efficient protective immunity against CCHFV challenge. However, we cannot exclude the importance of the neutralizing antibodies as the surviving immunized mice exhibited substantial amounts of them.IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is responsible for hemorrhagic diseases in humans, with a high mortality rate. There is no FDA-approved vaccine, and there are still gaps in our knowledge of the immune responses to infection. The recently developed mouse models mimic human CCHF disease and are useful to study the immunogenicity and the protection by vaccine candidates. Our study shows that mice vaccinated with a specific DNA vaccine were fully protected. Importantly, we show that neutralizing antibodies are not sufficient for protection against CCHFV challenge but that an extra Th1-specific cellular response is required. Moreover, we describe the identification of five conserved B-cell epitopes, of which only one was previously known, that could be of great importance for the development of diagnostics tools and the improvement of vaccine candidates.

Understanding the relationship between Plasmodium falciparum growth rate and multiplicity of infection.

Natural infections with Plasmodium falciparum are often composed of multiple concurrent genetically distinct parasite clones. Such multiclonal infections are more common in areas of high transmission, and the frequency of multiclonal infection also varies with age. A number of studies have suggested that multiclonal infection predicts the risk of subsequent clinical malaria. The multiplicity of infection is determined by the rate of new infections, the number of clones inoculated at each mosquito bite, and the duration of infections. Here, we used a mathematical modeling approach to understand how variation in the growth rate of blood-stage parasites affects the observed multiplicity of infection (MOI), as well as the relationship between the MOI and the risk of subsequent malaria. We then analyzed data from a study of multiclonal infection and malaria in an malaria-endemic area in Tanzania and show that the proportion of multiclonal infections varies with age and that the observed relationship between multiclonal infection and subsequent clinical events can be explained by a reduction in blood-stage parasite growth with age in this population.

Harmonization of European laboratory response networks by implementing CWA 15793: use of a gap analysis and an "insider" exercise as tools.

Laboratory response networks (LRNs) have been established for security reasons in several countries including the Netherlands, France, and Sweden. LRNs function in these countries as a preparedness measure for a coordinated diagnostic response capability in case of a bioterrorism incident or other biocrimes. Generally, these LRNs are organized on a national level. The EU project AniBioThreat has identified the need for an integrated European LRN to strengthen preparedness against animal bioterrorism. One task of the AniBioThreat project is to suggest a plan to implement laboratory biorisk management CWA 15793:2011 (CWA 15793), a management system built on the principle of continual improvement through the Plan-Do-Check-Act (PDCA) cycle. The implementation of CWA 15793 can facilitate trust and credibility in a future European LRN and is an assurance that the work done at the laboratories is performed in a structured way with continuous improvements. As a first step, a gap analysis was performed to establish the current compliance status of biosafety and laboratory biosecurity management with CWA 15793 in 5 AniBioThreat partner institutes in France (ANSES), the Netherlands (CVI and RIVM), and Sweden (SMI and SVA). All 5 partners are national and/or international laboratory reference institutes in the field of public or animal health and possess high-containment laboratories and animal facilities. The gap analysis showed that the participating institutes already have robust biorisk management programs in place, but several gaps were identified that need to be addressed. Despite differences between the participating institutes in their compliance status, these variations are not significant. Biorisk management exercises also have been identified as a useful tool to control compliance status and thereby implementation of CWA 15793. An exercise concerning an insider threat and loss of a biological agent was performed at SVA in the AniBioThreat project to evaluate implementation of the contingency plans and as an activity in the implementation process of CWA 15793. The outcome of the exercise was perceived as very useful, and improvements to enhance biorisk preparedness were identified. Gap analyses and exercises are important, useful activities to facilitate implementation of CWA 15793. The PDCA cycle will enforce a structured way to work, with continual improvements concerning biorisk management activities. Based on the activities in the AniBioThreat project, the following requirements are suggested to promote implementation: support from the top management of the organizations, knowledge about CWA 15793, a compliance audit checklist and gap analysis, training and exercises, networking in LRNs and other networks, and interinstitutional audits. Implementation of CWA 15793 at each institute would strengthen the European animal bioterrorism response capabilities by establishing a well-prepared LRN.

An antibody against a novel and conserved epitope in the hemagglutinin 1 subunit neutralizes numerous H5N1 influenza viruses.

The spread of the recently emerged, highly pathogenic H5N1 avian influenza virus has raised concern. Preclinical studies suggest that passive immunotherapy could be a new form of treatment for H5N1 virus infection. Here, a neutralizing monoclonal antibody (MAb) against the hemagglutinin (HA) of the influenza A/chicken/Hatay/2004 H5N1 virus, MAb 9F4, was generated and characterized. MAb 9F4 binds both the denatured and native forms of HA. It was shown to recognize the HA proteins of three heterologous strains of H5N1 viruses belonging to clades 1, 2.1, and 2.2, respectively. By use of lentiviral pseudotyped particles carrying HA on the surface, MAb 9F4 was shown to effectively neutralize the homologous strain, Hatay04, and another clade 1 strain, VN04, at a neutralization titer of 8 ng/ml. Furthermore, MAb 9F4 also neutralized two clade 2 viruses at a neutralizing titer of 40 ng/ml. The broad cross-neutralizing activity of MAb 9F4 was confirmed by its ability to neutralize live H5N1 viruses of clade 2.2.2. Epitope-mapping analysis revealed that MAb 9F4 binds a previously uncharacterized epitope below the globular head of the HA1 subunit. Consistently, this epitope is well conserved among the different clades of H5N1 viruses. MAb 9F4 does not block the interaction between HA and its receptor but prevents the pH-mediated conformational change of HA. MAb 9F4 was also found to be protective, both prophylactically and therapeutically, against a lethal viral challenge of mice. Taken together, our results showed that MAb 9F4 is a neutralizing MAb that binds a novel and well-conserved epitope in the HA1 subunit of H5N1 viruses.

Crimean-Congo hemorrhagic fever virus infection is lethal for adult type I interferon receptor-knockout mice.

Crimean-Congo hemorrhagic fever virus (CCHFV) poses a great threat to public health due to its high mortality, transmission and geographical distribution. To date, there is no vaccine or specific treatment available and the knowledge regarding its pathogenesis is highly limited. Using a small-animal model system, this study showed that adult mice missing the type I interferon (IFN) receptor (IFNAR(-/-)) were susceptible to CCHFV and developed an acute disease with fatal outcome. In contrast, infection of wild-type mice (129 Sv/Ew) was asymptomatic. Viral RNA was found in all analysed organs of the infected mice, but the amount of CCHFV RNA was significantly higher in the IFNAR(-/-) mice than in the wild-type mice. Furthermore, the liver of IFNAR(-/-) mice was enlarged significantly, showing that IFN is important for limiting virus spread and protecting against liver damage in mice.

Genetic diversity among Plasmodium falciparum field isolates in Pakistan measured with PCR genotyping of the merozoite surface protein 1 and 2.

BACKGROUND: The genetic diversity of Plasmodium falciparum has been extensively studied in various parts of the world. However, limited data are available from Pakistan. This study aimed to establish molecular characterization of P. falciparum field isolates in Pakistan measured with two highly polymorphic genetic markers, i.e. the merozoite surface protein 1 (msp-1)and 2 (msp-2). METHODS: Between October 2005 and October 2007, 244 blood samples from patients with symptomatic blood-slide confirmed P. falciparum mono-infections attending the Aga Khan University Hospital, Karachi, or its collection units located in Sindh and Baluchistan provinces, Pakistan were collected. The genetic diversity of P. falciparum was analysed by length polymorphism following gel electrophoresis of DNA products from nested polymerase chain reactions (PCR) targeting block 2 of msp-1 and block 3 of msp-2, including their respective allelic families KI, MAD 20, RO33, and FC27, 3D7/IC. RESULTS: A total of 238/244 (98%) patients had a positive PCR outcome in at least one genetic marker; the remaining six were excluded from analysis. A majority of patients had monoclonal infections. Only 56/231 (24%) and 51/236 (22%) carried multiple P. falciparum genotypes in msp-1 and msp-2, respectively. The estimated total number of genotypes was 25 msp-1 (12 KI; 8 MAD20; 5 RO33) and 33 msp-2 (14 FC27; 19 3D7/IC). CONCLUSIONS: This is the first report on molecular characterization of P. falciparum field isolates in Pakistan with regards to multiplicity of infection. The genetic diversity and allelic distribution found in this study is similar to previous reports from India and Southeast Asian countries with low malaria endemicity.

Associations between the IL-4 -590 T allele and Plasmodium falciparum infection prevalence in asymptomatic Fulani of Mali.

In this study, we compared the genotype and allele frequencies of the IL-10 -1087 A/G and IL-4 -590 C/T single nucleotide polymorphisms in asymptomatic subjects of two sympatric ethnic tribes differing in susceptibility to malaria, the Fulani and the Dogon in Mali. The genotype data was correlated with ethnicity and malariometric indexes. A statistically significant inter-ethnic difference in allele and genotype frequency for both loci was noted (P<0.0001). Within the Fulani, the prevalence of Plasmodium falciparum infection, as detected by both microscopy and PCR, was associated with the IL-4 -590 T allele (P=0.005 and P=0.0005, respectively), whereas, no such associations were seen in the Dogon. Inter-ethnic differences in spleen rates, higher in the Fulani than the Dogon, were seen between T carriers (TT and CT) of both groups (P<0.0001). Parasite densities and number of concurrent clones did not vary between IL-4 genotypes within any of the studied groups. These results suggest an association between the IL-4 -590 T allele and P. falciparum prevalence within the Fulani but not the Dogon. No associations between IL-10 genotypes and studied malariometric indexes were observed in any of the two communities.

Immunogenetic control of antibody responsiveness in a malaria endemic area.

This study builds upon the established genetic control of antimalarial immune responses and prior association studies by using a family-based approach, transmission disequilibrium testing, to identify immune response genes that influence antibody responses to Plasmodium falciparum infection in an endemic Tanzanian population. Candidate polymorphisms are within the interleukin-1 (IL-1) gene cluster, the IL-10 promoter, Major histocompatibility complex class II and III, the 5q31-q33 region, and the T-Cell Receptor beta variable region. There was a significant association between the IL1RN alleles and total IgE. Weak evidence for association was present between polymorphisms in the IL10 promoter region and both anti-P falciparum IgE and IgG4 antibodies.

Multiclonal asymptomatic Plasmodium falciparum infections predict a reduced risk of malaria disease in a Tanzanian population.

Protective immunity to malaria is acquired after repeated exposure to the polymorphic Plasmodium falciparum parasite. Whether the number of concurrent antigenically diverse clones in asymptomatic infections predicts the risk of subsequent clinical malaria needs further understanding. We assessed the diversity of P. falciparum infections by merozoite surface protein 2 genotyping in a longitudinal population based study in Tanzania. The number of clones was highest in children 6-10 years and in individuals with long time to previous anti-malarial treatment. Individual exposure, analysed by circumsporozoite protein antibody levels, was associated with parasite prevalence but not with the number of clones. The risk of subsequent clinical malaria in children free of acute disease or recent treatment was, compared to one clone, reduced in individuals with multiclonal infections or without detectable parasites, with the lowest hazard ratio 0.28 (95% confidence interval 0.10-0.78 Cox regression) for 2-3 clones. The number of clones was not associated with haemoglobin levels. A reduced risk of malaria in asymptomatic individuals with multiclonal persistent P. falciparum infections suggests that controlled maintenance of diverse infections is important for clinical protection in continuously exposed individuals, and needs to be considered in the design and evaluation of new malaria control strategies.

Multiple genotypes of the merozoite surface proteins 1 and 2 in Plasmodium falciparum infections in a hypoendemic area in Iran.

In Iran, malaria transmission mainly occurs in south-eastern regions through both Plasmodium falciparum and P. vivax. The genetic diversity of P. falciparum isolates was analysed in 108 patients attending the regional hospital in Chabahar District, using the molecular markers msp1 and msp2. Multiple genotypes were detected in 87% of patients and the mean numbers of msp1 and msp2 genotypes were 2.51 (95% CI: 2.29-2.73) and 2.61 (95% CI: 2.39-2.83) respectively. Various allelic types of msp1 and msp2 were found, with msp2 3D7/IC type detected in 94% of infections. Plasmodium falciparum infections in south-east Iran appear to have a higher genetic diversity than expected for an area of low transmission. A situation of higher transmission in this area may be emerging, possibly because of reduced efficacy of first-line treatments.

Short report: Rapid DNA extraction from archive blood spots on filter paper for genotyping of Plasmodium falciparum.

The practical advantages of sampling and storing blood on filter paper for analyses of human and pathogen genes highlight the need for reliable, sensitive, and cost-effective DNA extraction methods. We describe a new Tris-EDTA (TE) buffer-based method for extraction of DNA from blood dried on filter paper. The method was evaluated against the commonly used methanol and Chelex methods, regarding polymerase chain reaction detection of Plasmodium falciparum parasites from samples stored for 1-2 years. The sensitivity of detection was dependent on the parasite density and type of filter paper. For 3MM Whatman filter paper, the sensitivity was 100%, 73%, and 93% for the TE, methanol, and Chelex methods, respectively. For the longer stored 903 Schleicher & Schuell filter paper, the sensitivity was 93%, 73%, and 0%, respectively. This rapid, simple, and inexpensive extraction method generated superior results from archived specimens compared with the two standard methods and may represent a useful tool in molecular epidemiologic studies.

Different antibody- and cytokine-mediated responses to Plasmodium falciparum parasite in two sympatric ethnic tribes living in Mali.

The Fulani are known to be less susceptible to Plasmodium falciparum malaria infections and to have lower parasitaemia despite living under similar malaria transmission intensity compared with other ethnic tribes. The aim of the present study was to examine whether the Fulani were more polarised towards Th2 as reflected by higher numbers of malaria-specific IL-4- and IL-10-producing cells and lower numbers of IFN-gamma- and IL-12-producing cells as compared to their neighbour ethnic tribe, the Dogon of Mali. Total IgE and both anti-malaria IgE and IgG antibodies were measured by ELISA and the numbers of IL-4-, IFN-gamma-, IL-10- and IL-12-producing cells were enumerated using enzyme-linked ImmunoSpot assay (ELISPOT). Numbers of parasite clones were detected by polymerase chain reaction (PCR). The study was performed outside the transmission period and all individuals included were asymptomatic. The results revealed that the Fulani were less parasitised, had fewer circulating parasite clones in their blood, had significantly higher anti-malaria IgG and IgE antibodies and higher proportions of malaria-specific IL-4- and IFN-gamma-producing cells compared to the Dogon. The higher antigen-specific production of IL-4 among the Fulani was statistically significant both before and after adjustment for level of spontaneous cytokine production, while greater IFN-gamma production only attained statistical significance after adjustment for spontaneous levels. Taken together, the association of higher anti-malarial IgE and IgG antibodies and increased numbers of specific IL-4- and IFN-gamma-producing cells compared to the ethnic sympatric tribe, the Dogon, may assist in explaining the lower susceptibility to malaria observed in the Fulani.

Elevated anti-malarial IgE in asymptomatic individuals is associated with reduced risk for subsequent clinical malaria.

Immunological characteristics were assessed for prospective risk of clinical malaria in a longitudinally followed population in a holoendemic area of Tanzania. Baseline characteristics including crude Plasmodium falciparum extract-specific IgE and IgG; total IgE; and parasitological indices, e.g. number of P. falciparum clones, were investigated among 700 asymptomatic individuals. Cox regression analysis estimated the risk of succumbing to a new clinical episode during a 40 weeks follow up. High anti-P. falciparum IgE levels were associated with reduced risk of acute malaria in all age groups independently of total IgE levels. Statistically significant reduced odds ratio of 0.26 (95% CI, 0.09-0.72, P=0.010) and 0.44 (95% CI, 0.19-0.99, P=0.047) for the two highest fifths, respectively was observed after adjustment for age, sex, total IgE, numbers of parasite clones per infection and HIV-1 seropositivity. In contrast, high levels of malaria specific IgG or total IgE were not associated with reduced risk to succumb to a new clinical episode. A protective effect of asymptomatic multiclonal P. falciparum infections was also confirmed. For the first time, anti-malarial IgE levels in asymptomatic individuals in endemic area are found to be associated with reduced risk for subsequent malaria disease. Specific IgE antibodies may play role in maintaining anti-malarial immunity, or indicate other aspects of immune function relevant for protection against malaria.