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Mumps is a highly contagious viral disease that can be prevented by vaccination. In the last decade, we have encountered repeated outbreaks of mumps in highly vaccinated populations, which call into question the effectiveness of available vaccines. Animal models are crucial for understanding virus-host interactions, and viruses such as mumps virus (MuV), whose only natural host is the human, pose a particular challenge. In our study, we examined the interaction between MuV and the guinea pig. Our results present the first evidence that guinea pigs of the Hartley strain can be infected in vivo after intranasal and intratesticular inoculation. We observed a significant viral replication in infected tissues up to 5 days following infection and induction of cellular and humoral immune responses as well as histopathological changes in infected lungs and testicles, without clinical signs of disease. Transmission of the infection through direct contact between animals was not possible. Our results demonstrate that guinea pigs and guinea pig primary cell cultures represent a promising model for immunological and pathogenetic studies of the complex MuV infection.

Neutralizing antibodies against mumps and measles virus are considered a correlate of protection against these diseases. Measurement of neutralizing antibodies is mostly performed using plaque reduction neutralization assay or 50 % cell culture infective dose (CCID50) neutralization assay, but there are attempts for measuring neutralizing antibodies using enzyme-linked immunosorbent assay (ELISA) which is simpler, but the literature data regarding its convenience are diverse. The role of complement and antibodies in neutralizing capacity of sera is not completely defined. Here, CCID50 neutralization assay and ELISA were used to determine the neutralization capacity against mumps and measles virus in human sera and therapeutic immunoglobulins (IVIGs). Results showed no correlation of neutralization titers obtained by CCID50 neutralization assay and IgG content obtained by ELISA for mumps or measles in human sera. Data showed some neutralization activity against measles virus and quite high against mumps virus of naïve guinea pig serum and that its addition increases neutralization capacity of IVIG and human sera against mumps and measles viruses. Heat inactivation of human sera reduced neutralization capacity against measles to small extent, and substantially against mumps virus. There is a significant impact of complement in measurement of neutralization capacity against mumps virus.

Background: Antivenoms are the only validated treatment against snakebite envenoming. Numerous drawbacks pertaining to their availability, safety and efficacy are becoming increasingly evident due to low sustainability of current productions. Technological innovation of procedures generating therapeutics of higher purity and better physicochemical characteristics at acceptable cost is necessary. The objective was to develop at laboratory scale a compact, feasible and economically viable platform for preparation of equine F(ab')2 antivenom against Vipera ammodytes ammodytes venom and to support it with efficiency data, to enable estimation of the process cost-effectiveness. Methods: The principle of simultaneous caprylic acid precipitation and pepsin digestion has been implemented into plasma downstream processing. Balance between incomplete IgG breakdown, F(ab')2 over-digestion and loss of the active drug's protective efficacy was achieved by adjusting pepsin to a 1:30 substrate ratio (w/w) and setting pH at 3.2. Precipitation and digestion co-performance required 2 h-long incubation at 21 °C. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. In vivo neutralization potency of the F(ab')2 product against the venom's lethal toxicity was determined. Results: Only three consecutive steps, performed under finely tuned conditions, were sufficient for preservation of the highest process recovery with the overall yield of 74%, comparing favorably to others. At the same time, regulatory requirements were met. Final product was aggregate- and pepsin-free. Its composition profile was analyzed by mass spectrometry as a quality control check. Impurities, present in minor traces, were identified mostly as IgG/IgM fragments, contributing to active drug. Specific activity of the F(ab')2 preparation with respect to the plasma was increased 3.9-fold. Conclusion: A highly streamlined mode for production of equine F(ab')2 antivenom was engineered. In addition to preservation of the highest process yield and fulfillment of the regulatory demands, performance simplicity and rapidity in the laboratory setting were demonstrated. Suitability for large-scale manufacturing appears promising.

The hyperimmune horse plasma (HHP), prepared through active immunisation of horses with an antigen of interest, is the most common starting material for antitoxin (animal antibody-based therapeutics) production. Precise IgG quantification in plasma is a prerequisite for accurate estimation of the purification process efficiency. Although immunoglobulins from HHP have been purified for over a century, there is still no in vitro method for precise and accurate determination of IgG content in HHP. For this reason, the purification process efficiency has been assessed by antibody activity measurements, mostly performed in vivo. Here we describe the development of a precise and accurate in vitro immunoassay for IgG quantification in HHP. We showed and highlighted that any difference in composition of IgG population between the standard and the sample, with respect to both IgG subclass distribution and antigen-specific IgG content, leads to inaccurate IgG quantification. We demonstrated that caprylic acid precipitation as the method for IgG isolation from horse plasma renders the composition of IgG population unchanged. This very efficient, fast, simple and inexpensive method was used to prepare internal, sample- specific reference IgG for each plasma sample, which was tested simultaneously to a respective plasma sample. Deviation of IgG quantity determined by ELISA for each sample-specific reference from its nominal value was used for correction of the results of respective plasma sample, which led to accurate and precise IgG quantification as shown by method validation. The here presented novel concept of sample- specific correction of immunoassay results could be widely applicable and easily introduced in different immunoassays for more accurate and precise plasma IgG quantification.

Immunization programs have implemented live attenuated mumps vaccines which reduced mumps incidence ≥97%. Some of the vaccine strains were abandoned due to unwanted side effects and the genetic marker of attenuation has not been identified so far. Our hypothesis was that non-infectious viral particles, in particular defective interfering particles (DIPs), contribute to neuroattenuation. We showed that non-infectious particles of the mumps vaccine L-Zagreb attenuated neurovirulence of wild type mumps virus 9218/Zg98. Then, we attenuated recent wild type mumps virus MuVi/Zagreb.HRV/28.12 in Vero cells through 16 passages but already the fifth passage (p5) showed accumulation of DIPs and attenuated neurovirulence in a newborn rat model when compared to the second passage (p2). Sequence analysis of the p2 and p5 revealed a single mutation in the 5′ untranslated region of the HN gene. Analysis of the expression level of the HN protein showed that this mutation does not affect the expression of the protein. We conclude that the passages of MuVi/Zagreb.HRV/28.12 in Vero cells for only three passages accumulated DIPs which attenuate neurovirulence. These findings reveal DIPs as a very promising and general neuroattenuating factor which should be considered in the rational design of the new mumps vaccine.

Dear Editor,The article by Lamb et al. [1] gives a valuable review of the current needs for antivenom treatment of snakebite envenoming in Europe, the current practices in envenomation treatment ac...

A high molecular mass metalloproteinase with alpha-fibrinogenolytic activity, termed VaF1, was purified from nose-horned viper (Vipera ammodytes ammodytes) venom. Subcutaneous injection of 9mug of VaF1 did not induce bleeding in rats. Nevertheless, invitro it degraded collagen IV, nidogen and fibronectin, components of the extracellular matrix, although with low efficacy and narrow specificity. VaF1 would be expected to exert anti-coagulant action, due to its hydrolysis of fibrinogen, factor X, prothrombin and plasminogen, plasma proteins involved in blood coagulation. The enzyme is a single-chain glycoprotein with a molecular mass of 49.7kDa, as determined by mass spectrometry, and multiple isoelectric points centred at pH 5.8. The complete amino acid sequence of the precursor of VaF1 was deduced by cloning and sequencing its cDNA. Composed of metalloproteinase, disintegrin-like and cysteine-rich domains, VaF1 is a typical P-IIIa subclass snake venom metalloproteinase. Although it possesses a collagen-binding sequence in its disintegrin-like domain, VaF1 displayed no effect on collagen-induced platelet aggregation invitro. Two consensus N-glycosylation sites are present in the sequence of VaF1, however, the extent of its glycosylation is low, only 5.2% of the total molecular mass. Interestingly, in standard experimental conditions VaF1 is not recognised by antiserum against the whole venom, so it can contribute to post-serotherapy complications, such as ineffective blood coagulation, in the envenomed patient.

The hyperimmune horse plasma (HHP), prepared by active immunisation of horses with an antigen of interest, is the most common starting material for antitoxin (animal antibody-based therapeutics) production. Precise IgG quantification in plasma is a prerequisite for accurate estimation of the purification process efficiency. Although immunoglobulins from HHP have been purified for more than a century, we still lack in vitro method for precise and accurate determination of IgG content in HHP. In such situation, the purification process efficiency has been assessed by antibody activity measurements, performed in vivo. The development of a precise and accurate in vitro immunoassay for IgG quantification in HHP will be presented. It is based on a novel concept of sample-specific correction of immunoassay results. We discovered that any difference in composition of IgG population between the standard and the sample, with respect to both IgG subclass distribution and antigen-specific IgG content, leads to inaccurate IgG quantification. We demonstrated that caprylic acid precipitation as the method for IgG isolation from horse plasma renders the composition of IgG population unchanged and was used to prepare internal, sample-specific reference IgG for each plasma sample, which was tested simultaneously to a respective plasma sample. The application of the novel method for the estimation of the active principle yield in the process of IgG purification from HHP will be demonstrated. Also, the impact of subclass changes due to different purification processes on the quality of the final product will be discussed.

Antivenoms obtained from hyperimmune animal plasma have been the only specific and effective therapy against snakebites. Design of the ideal process preceding their commercial scale production should be guided by the tendency to refine IgGs or their antigen- binding fragments from residual plasma proteins in the most straightforward way, providing final product of retained potency and reduced side-effects inducing potential. Here we report simple, feasible and economically viable downstream processing strategy for Vipera ammodytes-specific antivenom preparation that has been guided by the imperative of constant maintenance of F(ab’)2 fragments in solution, as a precautionary measure against their conformational or structural change due to precipitation or binding to chromatographic support. Thus, protection of the active principle from possible degradation and/or aggregation was ensured. Also, for the first time caprylic acid fractionation of horse plasma was successfully co- performed with the pepsin digestion step. Developed mode employing simultaneous precipitation of contaminating proteins together with Fc fragment removal from IgGs resulted in F(ab’)2- based product that was additionally polished by anion-exchange chromatography in the flow-through mode, under conditions that prefer binding of processing by- product traces, especially pepsin. Such compressed manufacturing procedure yielded over 70% of active principle, as properly and reliably estimated by several in vitro methods. The final preparation was distinguished by high purity and preserved in vivo neutralisation efficacy in comparison to the starting plasma pool.

In order to perform their function, proteins frequently interact with other proteins. Various methods are used to reveal protein interacting partners and affinity chromatography is one of them. Snake venom is composed mostly of proteins, and various protein complexes in the venom have been found to exhibit higher toxicity levels than respective components separately. Complexes can modulate envenomation activity of a venom and/or potentiate its effect. Our previous data indicate that the most toxic components of the Vipera ammodytes ammodytes (Vaa) venom isolated so far – ammodytoxins (Atxs) are contributing to the venom’s toxicity only moderately, therefore we aimed to explore whether they have some interacting partner(s) potentiating toxicity. For screening of possible interactions immuno-affinity chromatography combined with identification by mass spectrometry was used. Various chemistries (epoxy, carbonyldiimidazole (CDI), ethylenediamine (EDA)) as well as protein G functionality were used to immobilize antibodies on monolith support, a Convective Interaction Media (CIM) disk. Monoliths have been demonstrated to better suite separation of large biomolecules. Using such approach several proteins were indicated as potential Atx-binding proteins. Among these, the interaction of Atxs with a Kunitz-type inhibitor was confirmed by far-Western dot-blot and surface plasmon resonance (SPR) measurement. It can be concluded that affinity chromatography on monolithic columns combined with mass spectrometry identification is a successful approach for screening of protein interactions and it resulted with detection of the interaction of Atx with Kunitz-type inhibitor in Vaa venom for the first time.

Hemorrhage is the most potent manifestation of envenomation by Vipera ammodytes ammodytes (V. a. ammodytes) venom in man. A detailed description of the venom components contributing to this effect is thus medically very important. We have characterized a novel component, termed here VaH3, as a potently hemorrhagic snake venom metalloproteinase (SVMP). Its proteolytic activity and overall stability depend on the presence of Zn(2+) and Ca(2+) ions. The molecular mass of this slightly acidic molecule, determined by MALDI/TOF analysis, is 104 kDa. Chemical reduction and S-carbamoylmethylation result in a single monomer of 53.7 kDa. N-deglycosylation decreased this mass by 4.6 kDa. The complete amino acid sequence of VaH3 was determined by protein and cDNA sequencing, showing that each of the identical glycoprotein subunits comprise a metalloproteinase, a disintegrin-like domain and a cysteine-rich domain, VaH3 belongs to the P-IIIc class of SVMPs. It shows strong sequence similarity to vascular endothelial cell apoptosis-inducing reprolysins. Anti-ammodytagin antibodies strongly cross-reacted with VaH3 and completely neutralized its hemorrhagic activity in rat, despite the fact that the two hemorrhagic P-III SVMPs from V. a. ammodytes venom do not share a very high degree of amino acid sequence identity. In spite of its narrow proteolytic specificity, VaH3 rapidly cleaved some basal membrane and extracellular matrix proteins, such as collagen IV, fibronectin and nidogen. Moreover, it also hydrolyzed plasma proteins involved in blood coagulation. It is an effective α-fibrinogenase that cleaves prothrombin and factor X without activating them. The degradation of these proteins likely contributes to the hemorrhagic activity of VaH3. A three-dimensional model of VaH3 was built to help explain structure-function relationships in ADAM/ADAMTS, a family of proteins having significant therapeutic potential and substantial sequence similarity to VaH3.

Meadow vipers (Vipera ursinii) form a group of five subspecies, but their distribution area is highly fragmented and covers Europe, Western and Central Asia. Karst viper, Vipera ursinii macrops (Vum) inhabits high mountain grasslands in Croatia, Bosnia and Herzegovina, Serbia, Macedonia, Montenegro and northern Albania. In Croatia it is a highly threatened viper species that appears only in five isolated localities. Scientific literature on V. ursinii deals only with its morphology, ecology and distribution range due to the species' conservation problems, while its venom composition and properties have not been investigated. Meadow vipers are medically less significant than other Vipera species. A majority of envenoming generally displays mild and negligible local symptoms only, that spontaneously resolve for a couple of days without any medical treatment or antivenom therapy. This may be associated with the short length of their flanges and a very low amount of injected venom, which cannot cause serious systematic symptoms. Here we investigate for the first time the composition and biological activity of the Vum venom composition and biological activity. The Vum venom is less lethally toxic in mice than the V. a. ammodytes venom, however the pattern of mice dying indicates the presence of a strong neurotoxic component. Interestingly, a two- dimensional gel electrophoresis revealed a lack of basic phospholipases, which are known neurotoxic components of Vipera venoms. Western blot of nonreduced Vum venom with anti- ammodytoxin antibodies (anti-Atx) gave no signal. Mass spectrometric identification of SDS-PAGE bands showed the lack of ammodytoxins, as well. This was finally proven by ELISA, in which Vum venom coated wells were not recognized by anti-Atx antibodies. Taken together, Vum venom might be a good starting material for the discovery of a novel neurotoxic component in Vipera venoms.

In clinical practice it is difficult to differentiate between V. berus and V. ammodytes venomous bites. In the past this was not a concern, but due to the current shortage in Viperfav™ and European viper venom antiserum availability, V. a. ammodytes venomous bites have recently been treated with ViperaTAb®, which is a pharmaceutical formulation containing a monospecific ovine Fab fragments against the venom of V. berus. OBJECTIVE: To evaluate ViperaTAb® in V. a. ammodytes envenomations. MATERIALS AND METHODS: This is a prospective case series of three consecutive patients envenomed by V. a. ammodytes snakebite treated with ViperaTAb®. V. ammodytes venom, neurotoxic ammodytoxins, and Fab fragment levels were determined in serum samples and a pharmacokinetic analysis of the antivenom Fab fragments was carried out. RESULTS: Three patients bitten by V. a. ammodytes with extensive local swelling, neurological symptoms and recurrent thrombocytopenia were treated with ViperaTAb®. V. ammodytes venom was detected in serum of all three patients. Ammodytoxins were detected in the serum of only the most severely envenomed patient who developed neurological symptoms. In the presented moderate cases, a dose of 8 mL of ViperaTAb® reduced swelling and improved systemic effects, such as thrombocytopenia. However, this dose of ViperaTAb® was not effective in the most severely envenomed patient with the highest serum values of V. ammodytes venom. In this case ViperaTAb® did not stop local swelling and it had no effect on neurological signs. ViperaTAb®'s systemic clearance, distribution and elimination half- lives were 4.3-13.4 mL/h/kg, 1.2-3.2 h and 14.1-55.4 h, respectively. CONCLUSIONS: In patients envenomed by V. a. ammodytes venom, ViperaTAb® reduces moderate swelling and temporarily improves systemic effects, except neurological symptoms. ViperaTAb® application induces a decrement of V. ammodytes venom level in the blood, but did not affect serum concentration of neurotoxic ammodytoxins in the one patient with measurable concentrations.

Antivenoms obtained from hyperimmune animal plasma, mostly equine or ovine, are the only specific therapeutics effective for rapidly counteracting post-snakebite pathophysiological manifestations. There are various well established refinement processing strategies that have been implemented into commercial scale antivenom production. However, optimisation of manufacturing process yielding safe, effective and available immunotherapeutics is still of great concern. Here, we report simple, feasible and economically viable fractionation protocol for preparation of V. a. ammodytes-specific antivenom. Hyperimmune equine plasma pool was fractionated in only few simple and easily scalable purification steps. The purification process can be stopped at different steps, depending on the desired final antivenom product: whole IgG molecules or F(ab')2 fragments, both fulfilling regulatory requirements for product purity. Particular emphasis was put on quantification of each purification step in terms of IgG or F(ab')2 yield determined by several in vitro methods. Throughout the procedure, IgG molecules or F(ab')2 fragments were constantly kept in the solution, preventing their precipitation and binding to chromatography columns which can lead to aggregation. Firstly, unwanted plasma proteins, mostly albumin, were precipitated by caprylic acid (CA). Ultrafiltered IgG-rich and CAdepleted supernatant was used for pepsin digestion. The final product, F(ab')2 fragments, was polished by ionexchange chromatography on CIM QA disk under conditions which prefer binding of pepsin and byproducts of enzymatic digestion exclusively.

The venom of Vipera ammodytes ammodytes (Vaa), like the venoms of other Viperinae snakes, is largely haemorrhagic and necrotising, and only to a lesser extent neurotoxic to humans. The components most extensively studied so far, and most probably involved in generating the observed pathologies, are haemorrhagins (H), members of the metalloproteinase group of enzymes, and neurotoxic ammodytoxins (Atxs), that belong to the secretory phospholipases A2. Rabbit antisera were prepared containing functional antibodies specific for each class of pathology-inducing venom constituents and for both classes together. The involvement of these antibodies in neutralising the toxicity of whole Vaa venom was assessed using the ED50 assay in mice. This assay is the only regulatorily approved assay for estimating anti-venom potency and as such has the task to quantify the active compound neutralising venom-induced pathology of the anti-venom. Fully functional anti-Atx antibodies were shown to be responsible for neutralising the portion of venom toxicity, while anti-H antibodies were not protective in this assay. Thus, the mouse ED50 assay, intended to measure the active principle of the anti-venom, does not measure antibodies specific for Vaa venom haemorrhagins, and consequently does not fulfil its primary task from the regulatory point of view.

Ammodytagin, a hemorrhagic Zn 2+ -dependent metalloproteinase from Vipera ammodytes ammodytes ( Vaa ) venom, is a glycosylated heterodimer of 108 kDa, as determined by MALDI mass spectrometry. Partial amino acid sequencing by Edman degradation and MS/MS analysis identified sequences belonging to metalloproteinase, disintegrin-like and cysteine-rich domains, which in addition to its heterodimeric nature allows classification into the P-IIIc group of snake venom metalloproteinases (SVMPs). Only few members of that group have been described so far. Ammodytagin possesses potent azocaseinolytic activity which can be inhibited by Na 2 EDTA, Zn 2+ and DTT. It cleaves insulin B-chain, hydrolysing it at positions Gln 4 –His 5 , His 10 –Leu 11 and Tyr 16 –Leu 17 . Furthermore, ammodytagin acts as a strong hemorrhagin in both rats and mice. Investigation of a substrate specificity revealed that the hemorrhagic activity of the novel SVMP might be the result of its involvement in cleavage of basal membrane components and depletion of fibrinogen, prothrombin and factor X in blood circulation. Finally, antiserum raised against ammodytagin was able to completely neutralise the hemorrhagic activity of the whole venom, suggesting it might be one of the key molecules towards which effective Vaa specific antivenom should be directed.

It was confirmed in this study that Vipera ammodytes ammodytes venom samples with a higher content of ammodytoxins (Atxs), basic neurotoxic phospholipases A2, are more lethal, exposing Atxs as one of the major toxic components in the long‐nosed viper venom. In addition, we recently correlated the ability of the venom to produce highly protective antiserum in rabbits with the amount of Atxs in the venom. Here, we developed a rapid, robust, and highly reproducible chromatographic method, based on HPLC separation on Convective Interaction Media to assess the quantity of Atxs in a particular venom sample in order to predict its potential to induce highly protective antiserum in immunized animals.

Background Despite continuing research efforts, determinants of mumps virus virulence are still largely unknown. One of consequences of this is difficulty in striking a balance between efficacy and safety of live attenuated mumps vaccines. Among mumps vaccine strains associated with occurrence of postvaccinal aseptic meningitis is L-Zagreb, developed by further attenuation of vaccine strain L-3. Starting from an archived L-Zagreb sample with suboptimal neuroattenuation score, we isolated different viral variants and compared their genetic and phenotypic properties, in investigation of neurovirulence markers. Methods Six different L-Zagreb variants were isolated by plaque purification. Their neurovirulent status was determined by rat-based neurovirulence test; population structure was determined by deep sequencing. Results We isolated one well neuroattenuated viral variant, two marginally neuroattenuated, and three insufficiently neuroattenuated. No genetic markers of neurovirulence could be identified. None of variants had detectable amounts of defective interfering particles. Two characteristics set insufficiently neuroattenuated variants apart from less-neurovirulent ones: elevated variability level in regions 1293−3314, 5363−7773 and 9382−11657, and/or elevated number of mutations present in frequencies ≥1%. The most neurovirulent variants possessed both of these features. Conclusions Distinctive heterogeneity profiles were obtained for insufficiently neuroattenuated L-Zagreb variants. No markers that would discriminate between marginally and well neuroattenuated variants were identified. The findings of this study may serve as a guideline during development of an improved L3/L-Zagreb vaccine strain.

In the envenomation caused by a bite of Vipera ammodytes ammodytes, the most venomous snake in Europe, hemorrhage is usually the most severe consequence in man. Identifying and understanding the hemorrhagic components of its venom is therefore particularly important in optimizing medical treatment of patients. We describe a novel high molecular mass hemorrhagin, VaH4. The isolated molecule is a covalent dimer of two homologous subunits, VaH4-A and VaH4-B. Complete structural characterization of A and partial characterization of B revealed that both belong to the P-III class of snake venom metalloproteinases (SVMPs), comprising a metalloproteinase, a disintegrin-like domain and a cysteine-rich domain. However, neither VaH4-A nor VaH4-B possess the Cys174 involved in the inter-subunit disulphide bond of P-III SVMPs. A three-dimensional model of the VaH4 dimer suggests that, Cys132 serves this function. This implies that dimers in the P-III class of SVMPs can be formed either between their Cys132 or Cys174 residues. The proteolytic activity and stability of VaH4 depend on Zn2+ and Ca2+ ions and the presence of glycosaminoglycans, which indicates physiological interaction of VaH4 with the latter element of the extracellular matrix (ECM). The molecular mass of VaH4, determined by MALDI/TOF mass spectrometry, is 110.2 kDa. N-deglycosylation reduced the mass of each monomer by 8.7 kDa. The two possible N-glycosylation sites in VaH4-A are located at completely different positions from those in homodimeric P-IIIc VaH3 from the same venom, however, without any evident functional implications. The hemorrhagic activity of this slightly acidic SVMP is ascribed to its hydrolysis of components of the ECM, particularly fibronectin and nidogen, and of some blood coagulation proteins, in particular the alpha-chain of fibrinogen. VaH4 is also significant medically as we found it cytotoxic against cancer cells and due to its substantial sequence similarity to ADAM/ADAMTS family of physiologically very important human proteins of therapeutic potential.

Antivenom raised against the venom of nose-horned viper, Vipera ammodytes (V. a.) ammodytes (European viper venom antiserum, Zagreb antivenom), contains neutralising equine F(ab′)2 fragments that are clinically successful against homologous venom, but also against the venoms of several others medically important European snakes due to its paraspecific action. In this work we demonstrated that Zagreb antivenom is preclinically effective in neutralising lethal toxicity and hemorrhagicity of venoms of Armenian mountain snakes – Montivipera raddei and Macrovipera lebetina obtusa as well. In order to better understand the biochemical basis of the observed paraspecificity, the ability of anti-V. a. ammodytes serum to recognise and neutralise proteinases of the two venoms was also investigated. Anti-V. a. ammodytes serum showed surprisingly low capacity to inhibit metalloproteinases of both venoms included in the study, probably due to weak immunorecognition of their P-I representatives. Also, it completely failed to abolish enzymatic action of serine proteinases from Macrovipera lebetina obtusa venom. Relevance of such finding is yet to be established.

VaSP1, a serine proteinase from Vipera ammodytes ammodytes venom, is a glycosylated monomer of 31.5 kDa, as determined by MALDI mass spectrometry, showing multiple isoelectric points between pH 6.5 and pH 8.5. Partial amino acid sequencing of VaSP1 by Edman degradation and MS/MS analysis identified sequences which allowed its classification among the so-called snake venom serine proteinase homologues, members of the peptidase S1 family, however being devoid of the canonical catalytic triad. Only few representatives of this group have been identified so far with just two of them characterised in detail at the protein level. Despite substitution of His57 with Arg, VaSP1 possesses proteolytic activity which can be inhibited by Pefabloc, benzamidine, Zn 2+ ions, DTT and trypsin inhibitor II, a Kunitz/BPTI group member. It hydrolyses N α -benzoyl-Phe-Val-Arg- p -NA, exhibiting Michaelis–Menten behaviour with K m = 48.2 μM and V m = 0.019 nM s −1 . The pH for optimal activity on tested substrate is around 9.0. VaSP1 also cleaves insulin B-chain, digesting it at positions His 10 -Leu 11 , Ala 14 -Leu 15 and Tyr 16 -Leu 17 . Furthermore, the novel serine proteinase is active towards wide array of proteins involved in haemostasis where its degradation of fibrinogen, fibrin, prothrombin, factor X and plasminogen in vivo probably results in depletion of coagulation factors in blood circulation. The possibility that VaSP1 possesses anticoagulant properties has been further indicated by its ability to prolong prothrombin time and activated partial thromboplastin time.

Vipera ammodytes is the most venomous European snake, whose venom has been used as antigen for immunization of antivenom-producing animals. Same as venom of any other snake, it is a complex mixture of proteins, peptides and other compounds which biochemical and pharmacological variability has been demonstrated at interspecies and intraspecies level. In this work we demonstrated intraspecific variability between 8 venom production batches using both the conventional and the new methodology. Moreover, in contrast to the literature on different venoms' variability, for the first time we were able to select those biochemical differences that are related to and give information on the venom's toxicity and immunogenicity. We have shown that methods quantifying ammodytoxin (the most toxic compound identified so far in the Vipera ammodytes ammodytes venom) content of the venom clearly distinguish between high and low immunogenic venoms.

Venoms are complex mixtures of proteins, peptides and other compounds whose biochemical and biological variability has been clearly demonstrated. These molecules have been used as antigens for immunization of anti-venom-producing animals (horses or sheep). Ammodytoxins (Atx) are potently neurotoxic compounds, and the most toxic compounds isolated so far from the Vipera ammodytes ammodytes (Vaa) venom. Recently we have shown that the level of antibodies specific to Vaa venom's most toxic component, ammodytoxin A (AtxA), (anti-AtxA IgG) in Vaa venom immunized rabbit sera highly correlated to the venom toxicity– neutralization potential of these sera. Here we investigated whether Atx content of Vaa venom could influence the outcome of immunization procedure. The novel ELISA was developed for precise determination of Atx content and Atx was quantified in venom samples used for immunization of rabbits. We clearly showed that animals immunized with the venom containing lower amount of Atx produced sera with significantly lower venom toxicity–neutralizing power and, vice versa, animals immunized with venoms containing higher amount of Atx produced sera with higher venom toxicity–neutralizing ability. Thus, the content of Atx in Vaa venom is a relevant parameter of its suitability in the production of highly protective Vaa anti-venom.

The contribution of antibodies directed against the two main toxic groups of proteins in the Vipera ammodytes ammodytes venom, haemorrhagic metalloproteinases (H) and neurotoxic sPLA2s (Atxs), to the overall protective efficacy of the whole venom antisera was investigated. Using ELISA assays we established a high correlation between the protective efficacy of the whole venom antisera in mice and their anti-Atxs antibody content. As the haemorrhage is the prevailing toxic effect of the venom in human, the lack of correlation also with anti-H IgG content exposed that the mouse model might not be optimal to evaluate the neutralizing potential of the venom-specific antisera for human therapy. We further revealed that Atxs and structurally very similar but non-toxic AtnI2 from the venom are not immuno cross-reactive.

Peptidoglycan monomer (PGM) is an adjuvant active molecule with potential for use in human and veterinary vaccine. PGM's action is short-lived in mammals hence its effects might be limited. Novel PGM-containing oil-based formulations have been developed recently by incorporation of PGM into Montanide ISA720 and ISA206 adjuvants with the aim to prolong and improve immunostimulating activities of PGM. In the present work we studied the efficacy of such novel adjuvant formulations using two different antigens, ovalbumin and snake venom respectively. Novel formulations were also tested in two experimental models, mice and rabbits. In rabbits the incorporation of PGM into oil-based adjuvants led to overall improvement of antigen-specific IgG response. However in the mouse model, under experimental conditions used, it was not possible to distinguish differences in antigen-specific IgG response among several strong oil-based adjuvant formulations.

Peptidoglycan monomer (PGM) is adjuvant active molecule in experimental mice, although its adjuvanticity is much lower in comparison to potent adjuvants. The novel adjuvant formulations were developed by incorporation of PGM into Montanide ISA 206 and Montanide ISA 720 adjuvants, with the aim to enhance its adjuvanticity by protecting it from the fast degradation and metabolic clearance. Adjuvanticity of the novel adjuvant formulations was tested in rabbits for induction of protein-specific antibodies. Both novel adjuvants ISA206(PGM) and ISA720(PGM) were significantly stronger than Montanide adjuvants themselves, and also significantly more potent than Complete Freund Adjuvant. Montanide ISA 720 was shown as much better carrier of PGM, since the novel ISA720(PGM) adjuvant was significantly stronger adjuvant than the ISA206(PGM).

Because of neurotropic and neurovirulent properties of mumps virus, neurovirulence testing of live attenuated vaccine is required by most national regulatory organisations. Such testing is preformed in monkeys (Ph.Eu. 01/2008:20618-Test for neurovirulence of live virus vaccines). But results obtained from these tests do not necessarily distinguish among the neurovirulent strain from those that are not. As a part of an international collaborative study we investigated the neurovirulence of three vaccine strains (JL5, Urabe AM9 and L-Zagreb) and two wild–type mumps viruses isolated in Croatia (9218/Zg98 and MuVi/Zagreb.HRV/28.12) by the neurovirulence assay in newborn rats. The results obtained by alternative assay on two vaccine strains (JL5 and Urabe AM9) correspond to the results obtained by the National Institute for Biological Standards (NIBSC) in the United Kingdom and the Food and Drug Administration in the United Station. Strain L-Zagreb also showed reduced neurovirulent properties as expected because it is a vaccine strain. Wild-type mumps virus isolates showed high neurovirulence. These results indicate as that the test in newborn rats is suitable for assessing the neurovirulence of mumps viruses. The test is robust, reproducible and follows the 3R principles.

Continuous quality control of venoms and antivenoms is necessary for a successful production of the only specific treatment of victims of venomous snake bites in Europe. Control is based on two in vivo tests: (a) the venom lethal toxicity assay (determination of median lethal dose of venom or LD50) and (b) test for determining the neutralization potency (effectiveness) of produced antivenoms (determination of mean effective antivenom dose or ED50). Both these tests, which cause suffering, pain and death of the experimental animals and also require a large number of animals, were identified by ECVAM (European Centre for Validation of Alternative Methods) as assays that are necessary to be replaced with alternative methods. Snake venoms are complex mixtures of more than a hundred mostly proteins and peptides, and non-protein compounds with different biochemical and pharmacological effects. Such complex mixtures are used as an antigen for animal immunization in antivenom production. For the development of in vitro tests, it is necessary to identify the components that contribute to the venom’s overall toxicity or specificity of antibodies that effectively neutralize them. So far several molecules, mainly from the family of metalloproteinases (haemorrhagins, the dominant cause of the human envenomation pathology) and phospholipase A2 (ammodytoxins, the most toxic molecules in the venom known to date) have been isolated and characterized from the European vipers’ venom. However, there were no literature data for their immunogenicity. Our study showed that ammodytoxin content, determined by two developed biochemical methods, HPLC and the "sandwich" ELISA, highly correlated with the lethal toxicity of the whole viper venom. Further we examined the role of ammodytoxin (Atx)- and haemorrhagin (H)-specific antibodies in the venom lethal toxicity neutralization. The results showed that functional anti-Atx antibodies were only partially involved in the neutralization of the venom toxicity. On the other hand, functional anti-H antibodies did not provide protection at all. Developed methods for anti-Atx or anti-H determination, due to only limited involvement of the mentioned antibodies in venom toxicity neutralization, cannot be a substitute for the in vivo assays. Deeper insight into snake venom composition at the molecular level and the participation of the particular components in the venom toxicity has yet to be done before solving this task. On the other hand, Atx content determination in different venom batches could be a good screening method in the selection of the best antigen for immunisation.