Immunogenicity and safety studies of an inactivated vaccine against Rift Valley fever.

Rift Valley fever (RVF) is an emerging transboundary, mosquito-borne, zoonotic viral disease caused by a single serotype of a virus belonging to the Phenuiviridae family (genus Phlebovirus). It is considered an important threat to both agriculture and public health in endemic areas, because the virus, transmitted by different mosquito genera, leads to abortions in susceptible animal hosts especially sheep, goat, cattle, and buffaloes, resulting in severe economic losses. Humans can also acquire the infection, and the major sources are represented by the direct contact with infected animal blood, aerosol, consumption of unpasteurized contaminated milk and the bite of infected mosquitoes. Actually, the EU territory does not seem to be exposed to an imminent risk of RVFV introduction, however, the recent outbreaks in a French overseas department and some cases detected in Turkey, Tunisia and Libya, raised the attention of the EU for a possible risk of introduction of infected vectors. Thus, there is an urgent need to develop new therapeutic and/or preventive drugs, such as vaccines. In our work, we studied the immunogenicity of an inactivated and adjuvanted vaccine produced using a Namibian field strain of RVF virus (RVFV). The vaccine object of this study was formulated with Montanide Pet Gel A, a polymer-based adjuvant that has been previously reported for its promising safety profile and for the capacity to elicit a strong immune response. The produced inactivated vaccine was tested on six sheep and the level of IgM and IgG after the immunization of animals was evaluated by a commercial competitive ELISA, in order to assess the immunogenicity profile of our vaccine and to evaluate its potential use, as an alternative to the attenuated vaccines commercially available, in case of Rift Valley fever epidemic disease on EU territory. Following the administration of the second dose, 35 days after the first one, all animals seroconverted.

Production of monoclonal antibodies binding to the VP7 protein of African horse sickness virus.

Monoclonal antibodies (MAbs) against AHSV were produced by immunising BALB/c mice with AHSV serotype 9 and six clones able to recognize specifically the VP7-AHSV with a strong reactivity were selected. The specificity of the MAbs was assessed in i-ELISA against a commercial VP7-AHSV and in immunoblot against a home-made VP7-AHSV, expressed by a Baculovirus expression system; potential cross-reactions with related orbiviruses (Bluetongue virus and Epizootic Haemorrhagic Disease virus) were investigated as well. One of the six MAbs selected, MAb 7F11E14, was tested in direct immunofluorescence and reacted with all nine AHSV serotypes, but didn't cross-react with BTV and EHDV. MAb 7F11E14 was also used to develop a competitive ELISA and was able to detect AHSV antibodies in the sera of AHS infected animals.

Preliminary results on innocuity and immunogenicity of an inactivated vaccine against Peste des petits ruminants.

Peste des petits ruminants (PPR) virus belongs to the family Paramyxoviridae and represents a major threat to small livestock industry. In recent years, outbreaks of PPR have occurred in Turkey and North Africa. In endemic areas, disease prevention is accomplished using live­attenuated vaccines. However, the use of live vaccines in non­endemic regions, such as Europe, is not approved by Veterinary Authorities. In these regions inactivated vaccines are then the only viable alternative. In this study an inactivated vaccine (iPPRVac) was formulated with either a water­in­oil emulsion (ISA 71 VG) or with delta inulin adjuvant, alone (AFSA1) or combined with a TLR9 agonist oligonucleotide (AFSA2). These formulations were then tested for immunogenicity on rats. The iPPRV formulation with AFSA2 adjuvant induced 100% seroconversion in rats after 2 injections and was subsequently evaluated in goats. Five goats were immunised twice subcutaneously, 36 days apart with iPPRVac + AFSA2. The immunised goats all seroconverted to PPR by day 9 and remained seropositive until the end of the experimental period (133 days). These data indicate that the rat model is useful in predicting vaccine responses in goats and that inactivated vaccine, when formulated with a delta inulin adjuvant, represents a promising alternative to live attenuated vaccines for PPR vaccination campaigns in non­endemic areas.

Rift Valley Fever Virus among Wild Ruminants, Etosha National Park, Namibia, 2011.

After a May 2011 outbreak of Rift Valley fever among livestock northeast of Etosha National Park, Namibia, wild ruminants in the park were tested for the virus. Antibodies were detected in springbok, wildebeest, and black-faced impala, and viral RNA was detected in springbok. Seroprevalence was high, and immune response was long lasting.

First External Quality Assessment of Molecular and Serological Detection of Rift Valley Fever in the Western Mediterranean Region.

Rift Valley fever (RVF) is a mosquito-borne viral zoonosis which affects humans and a wide range of domestic and wild ruminants. The large spread of RVF in Africa and its potential to emerge beyond its geographic range requires the development of surveillance strategies to promptly detect the disease outbreaks in order to implement efficient control measures, which could prevent the widespread of the virus to humans. The Animal Health Mediterranean Network (REMESA) linking some Northern African countries as Algeria, Egypt, Libya, Mauritania, Morocco, Tunisia with Southern European ones as France, Italy, Portugal and Spain aims at improving the animal health in the Western Mediterranean Region since 2009. In this context, a first assessment of the diagnostic capacities of the laboratories involved in the RVF surveillance was performed. The first proficiency testing (external quality assessment--EQA) for the detection of the viral genome and antibodies of RVF virus (RVFV) was carried out from October 2013 to February 2014. Ten laboratories participated from 6 different countries (4 from North Africa and 2 from Europe). Six laboratories participated in the ring trial for both viral RNA and antibodies detection methods, while four laboratories participated exclusively in the antibodies detection ring trial. For the EQA targeting the viral RNA detection methods 5 out of 6 laboratories reported 100% of correct results. One laboratory misidentified 2 positive samples as negative and 3 positive samples as doubtful indicating a need for corrective actions. For the EQA targeting IgG and IgM antibodies methods 9 out of the 10 laboratories reported 100% of correct results, whilst one laboratory reported all correct results except one false-positive. These two ring trials provide evidence that most of the participating laboratories are capable to detect RVF antibodies and viral RNA thus recognizing RVF infection in affected ruminants with the diagnostic methods currently available.

African horse sickness outbreaks in Namibia from 2006 to 2013: clinical, pathological and molecular findings.

African horse sickness (AHS) is a vector­borne viral disease of equids, endemic in Sub­Saharan Africa. This article reports the clinic­pathological and laboratory findings observed in the framework of passive surveillance during the AHS outbreaks which occurred in Namibia between 2006 and 2013. This study was conducted in the framework of the collaboration among the Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise (Teramo, Italy), the Namibian Ministry of Agriculture Water and Forestry, and the Namibian National Veterinary Association. A total of 92 horses were investigated, showing different clinical form of AHS: peracute/acute (n = 43), sub­acute (n = 21) and mild AHS fever (n = 19). Clinical data were not available for 9 horses, because they were found dead. Pathological findings have been recorded for 35 horses. At necropsy, pulmonary and subcutaneous oedema, haemorrhages and enlargement of lymph nodes were mainly observed. Diagnosis was confirmed by laboratory testing, AHS virus (AHSV) was isolated from 50 horses and the identified serotypes were: 1, 2, 4, 6, 7, 8, and 9. The phylogenetic analysis of the S10 genome sequences segregated the Namibian AHSV strains in the same clusters of those circulating in South Africa in recent years. The description of AHS clinical, pathological, and laboratory features of AHS provided in this article is of value for differential diagnosis and control of AHS, especially in areas currently free from this disease.

Production and characterization of monoclonal antibodies against horse immunoglobulins useful for the diagnosis of equine diseases.

Monoclonal antibodies (MAbs) against horse IgG were produced by immunizing Balb/c mice with purified horse IgG and were characterized in indirect ELISA versus purified immunoglobulins from donkey, cow, buffalo, sheep, pig, and chicken. Three MAbs (1B10B6C9, 1B10B6C10, 1B10B6E9) reacted only with horse and donkey IgG and IgM and, in western blotting, were specific for the Fc fragment of equine IgG. MAb 1B10B6E9 was used in chemiluminescent immunoblotting assay for the diagnosis of dourine and in indirect immunofluorescence assay (IFA) for the diagnosis of African horse sickness and dourine.

Urine protein profile of IgA nephropathy patients may predict the response to ACE-inhibitor therapy.

This study was aimed at the search of urinary biomarkers which might help to predict the clinical response of IgA nephropathy (IgAN) patients to angiotensin converting enzyme inhibitors (ACEi). First, we studied the urinary proteome of 18 IgAN patients (toward 20 healthy controls) who had been chronically treated with ACEi by using 2-D PAGE coupled to nano-HPLC-ESI-MS/MS analysis. We identified 3 proteins, kininogen (p = 0.02), inter-alpha-trypsin-inhibitor heavy chain 4 (35 kDa fragment) (p = 0.02) and transthyretin (p<0.0001), whose urinary excretion was different in IgAN patients' responders when compared to those who had not responded to ACEi. A reduction of daily proteinuria >50% and a stable renal function over time were used to classify patients as responders. Then, we adopted immunoblotting to confirm the predictive power of one of the above proteins, kininogen, in 20 patients with biopsy-proven IgAN, before starting any therapy. Thus, we confirmed that very low levels of kininogen urine excretion were indeed predictive of an inadequate or absent clinical response to ACEi therapy of IgAN patients, after 6-month follow-up. Concluding, the analysis of urine proteome of IgAN patients generated a set of proteins which distinguished subjects responsive to ACEi from those unresponsive to the inhibition of renin-angiotensin system (RAS).

Functional characterization of a novel Cx26 (T55N) mutation associated to non-syndromic hearing loss.

Mutations of the GJB2 gene, encoding connexin 26, are the most common cause of hereditary congenital hearing loss in many countries and account for up to 50% of cases of autosomal-recessive non-syndromic deafness. By contrast, only a few GJB2 mutations have been reported to cause an autosomal-dominant form of non-syndromic deafness. Here, we report a family from Southern Italy affected by non-syndromic autosomal dominant post-lingual hearing loss, due to a novel missense mutation in the GJB2 gene, a threonine to asparagine amino acid substitution at codon 55 (T55N). Functional studies indicated that the mutation T55N produces a protein that, although expressed to levels similar to those of the wt counterpart, is deeply impaired in its intracellular trafficking and fails to reach the plasma membrane. The mutation T55N is located at the apex of the first extracellular loop of the protein, a region suggested to play a role in protein targeting and a site for other two mutations, G59A and D66H, causing dominant forms of deafness.