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In the COVID-19 pandemic, we have witnessed an impressive speed in the development of effective vaccines approved for human use, based on clinical studies comparing their efficacy to placebo. A head-to-head comparison of the neutralizing antibody (NAb) responses elicited by four vaccines used in Europe during 2021 (BNT162b2, mRNA-1273, ChAdOx nCoV-19, Ad26.COV2.S) and their comparison to NAb responses in convalescents showed that while the amount was comparable, NAbs induced by natural infection were of superior quality. The highly scattered immune responses elicited by the four vaccines were equalled by booster mRNA vaccination, reaching the highest, plateau level of NAb at the population level. Equally high NAbs were induced in convalescents after a single injection of either vaccine. The use of wild-type virus as a challenge in the NAb determination revealed that NAbs produced by disease were better activators of the complement system than NAbs induced by vaccination. The contribution of spike protein-specific IgGs to the SARS-CoV-2 neutralization was lower in convalescents compared to vaccinees, indicating that those who recovered from COVID-19 were armed with antibodies of additional specificities and/or classes that contributed to virus neutralization. Such findings point to the need to re-examine the correctness of certain epidemiological measures applied in the current epidemic. We also highlight the importance of a standardized approach in assessing humoral immunity by the usage of the most comprehensive assays as early as possible in future epidemics.

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.

COVID-19 presentations range from cold-like symptoms to severe symptoms with the development of acute respiratory distress syndrome (ARDS). We report on a severe COVID-19 patient who was mechanically ventilated and who developed ARDS and bacterial infection. Because of rapid clinical deterioration and the exhaustion of other treatment options, the family and attending physicians requested a compassionate use of adult allogeneic bone marrow-derived mesenchymal stem cells (MSC) in addition to commonly used immunosuppressive, antiviral, and supportive therapy. The clinical course is discussed thoroughly, with a special emphasis on the safety and effect of MSC therapy. Compassionate MSC treatment, given in three rounds, affected ARDS regression. The patient was discharged from the intensive care unit after 31 days and from hospital after 49 days in a good general condition. MSC treatment was not associated with any side effects and was well tolerated in a three-week period; therefore, it should be studied in larger trials and considered for compassionate use

In the COVID-19 pandemic, we witnessed an impressive speed in the development of effective vaccines that have been approved for use in humans on the basis of the clinical studies comparing their efficacy to placebo. Head-to-head comparisons of several approved vaccines are rare, as well as comparisons of immune responses generated by vaccination to the ones created by the disease. Due to lack of appropriate standardization of assays that evaluate immune response, it is difficult to compare results from different studies. However, such analyses are inevitable in order to properly assess the correctness of epidemic measures applied in an effort to combat the pandemic. Here we report on a head-to head comparison of the first four vaccines authorized for emergency use in Europe (BNT162b2, mRNA-1273, AZD1222 or Ad26.COV2.S) in their ability to induce neutralizing antibody response (NT). All four deliver genetic information for the full-length spike protein into the host cells and induce both humoral and cellular branch of spike-specific immune response. Thus, NT response could serve as an indicator of the quality of the overall immune response induced by these vaccines. Well standardized wild-type neutralization assay1 enabled reliable comparison of antibody response induced by vaccines to the response induced in convalescents. By analysing the response in 113 vaccinees (≥18 per vaccine) we observed that the highest NTs were induced by mRNA vaccines, followed by AZD1222 and finally Ad26.COV2.S. In comparison to convalescents, these NTs were significantly higher, comparable and significantly lower, respectively. NT decline was significantly slower in convalescents. Better complement-activating properties of antibodies developed in natural infection as well as lower anti-spike IgG content in convalescents’ in comparison to vaccinees’ sera of comparable SARS-CoV-2 neutralisation power indicate that convalescents are armed with antibodies of additional specificities and/or classes, that contribute to SARS-CoV-2 neutralisation. 1Ravlić et al. Front. Immun. 2022 ; https://doi.org/10.3389/fimmu.2022.816159

Intratumoral oncolytic virotherapy may have promise as a means to debulk and downstage inoperable tumours in preparation for successful surgery. Here we describe the pioneering case of a 50-year-old virologist with locally recurrent muscle-invasive breast cancer who was able to proceed to tumour resection after receiving multiple intratumoral injections of viruses, first an Edmonston Zagreb measles vaccine strainMeV) then vesicular stomatitis virus Indiana strain (VSV). The intratumoral virus therapy was well tolerated and led to transient pseudoprogression followed by partial tumour remission. Frequent imaging studies and regular clinical observations documenting size, consistency and mobility of the injected tumour demonstrate that both MeV and VSV contributed to the overall favourable response. Two months after the start of virus injections, the shrunken tumour was no longer invading the skin or underlying muscle and was surgically excised. The excised tumour showed strong lymphocytic infiltration, with increase in both CD20-positive B cells and CD8-positive T cells, as well as macrophages. PD-L1 expression was detected in contrast to the PD-L1 negative phenotype before the treatment. The patient completed one-year trastuzumab adjuvant therapy, remains well and recurrence free 22 months post-surgery. Although an isolated case, the study provides a strong rationale for the formal testing of oncolytic virotherapy also in patients with early stage cancer.

In the COVID-19 pandemic, we witnessed an impressive speed in the development of effective vaccines that have been approved for use in humans on the basis of the clinical studies comparing their efficacy to placebo. Head-to-head comparisons of several approved vaccines are rare, as well as comparisons of immune responses generated by vaccination to the ones created by the disease. Due to lack of appropriate standardization of assays that evaluate immune response, it is difficult to compare results from different studies. However, such analyses are inevitable in order to properly assess the correctness of epidemic measures applied in an effort to combat the pandemic. Here we report on a head-to head comparison of the first four vaccines authorized for emergency use in Europe (BNT162b2, mRNA-1273, AZD1222 or Ad26.COV2.S) in their ability to induce neutralizing antibody response (NT). All four deliver genetic information for the full-length spike protein into the host cells and induce both humoral and cellular branch of spike protein-specific immune response. Thus, NT response could serve as an indicator of the overall immune response induced by these vaccines. Well standardized wild-type neutralization assay1 enabled reliable comparison of antibody response induced by vaccines to the response induced in convalescents. By analysing the response in 113 vaccinees (≥18 per vaccine) we observed that the highest NTs were induced by mRNA vaccines, followed by AZD1222 and finally Ad26.COV2.S. In comparison to convalescents, these NTs were significantly higher, comparable and significantly lower, respectively. NT decline was significantly slower in convalescents. Better complement-activating properties of antibodies developed in natural infection as well as lower anti-spike IgG content in convalescents’ in comparison to vaccinees’ sera of comparable SARS-CoV-2 neutralisation power indicate that convalescents are armed with antibodies of additional specificities and/or classes, that contribute to SARS-CoV-2 neutralisation.

COVID-19 vaccines prevent severe forms of the disease, but do not warrant complete pro-tection against breakthrough infections. This could be due to suboptimal mucosal immu-nity at the site of virus entry, given that all currently approved vaccines are administeredvia the intramuscular route. In this study, we assessed humoral and cellular immuneresponses in BALB/c mice after intranasal and intramuscular immunization with adenovi-ral vector ChAdOx1-S expressing full- length Spike protein of SARS-CoV-2. We showed thatboth routes of vaccination induced a potent IgG antibody response, as well as robust neu- tralizing capacity, but intranasal vaccination elicited a superior IgA antibody titer in thesera and in the respiratory mucosa. Bronchoalveolar lavage from intranasally immunizedmice efficiently neutralized SARS-CoV-2, which has not been the case in intramuscularlyimmunized group. Moreover, substantially higher percentages of epitope- specific CD8 Tcells exhibiting a tissue resident phenotype were found in the lungs of intranasally immu-nized animals. Finally, both intranasal and intramuscular vaccination with ChAdOx1- Sefficiently protected the mice after the challenge with recombinant herpesvirus express-ing the Spike protein. Our results demonstrate that intranasal application of adenoviralvector ChAdOx1-S induces superior mucosal immunity and therefore could be a promis-ing strategy for putting the COVID-19 pandemic under control.

Intratumoral oncolytic virotherapy may have promise as a means to debulk and downstage inoperable tumours in preparation for successful surgery. Here we describe the pioneering case of a 50-year-old virologist with locally recurrent muscle-invasive breast cancer who was able to proceed to tumour resection after receiving multiple intratumoral injections of viruses, first an Edmonston Zagreb measles vaccine strain (MeV) then vesicular stomatitis virus Indiana strain (VSV). The intratumoral virus therapy was well tolerated and led to transient pseudoprogression followed by partial tumour remission. Frequent imaging studies and regular clinical observations documenting size, consistency and mobility of the injected tumour demonstrate that both MeV and VSV contributed to the overall favourable response. Two months after the start of virus injections, the shrunken tumour was no longer invading the skin or underlying muscle and was surgically excised. The excised tumour showed strong lymphocytic infiltration, with increase in both CD20-positive B cells and CD8-positive T cells, as well as macrophages. PD-L1 expression was detected in contrast to the PD-L1 negative phenotype before the treatment. Although an isolated case, the study provides a strong rationale for the formal testing of oncolytic virotherapy also in patients with early stage cancer.

We would like to use the opportunity to discuss some of thesafety concerns of convalescent COVID- 19 plasma, raised by Mung-munpuntipantip and Wiwanitkit in their letter. We agree with the authors that risk- benefit analyses for the use of convalescentplasma are of great importance and contribute to the improvementof patient safety.

Objectives: The patients with hematological malignancies are a vulnerable group to COVID-19, due to the immunodeficiency resulting from the underlying disease and oncological treatment that significantly impair cellular and humoral immunity. Here we report on a beneficial impact of a passive immunotherapy with convalescent plasma to treat a prolonged, active COVID-19 infection in a patient with a history of nasopharyngeal diffuse large B-cell lymphoma treated with the therapy inducing substantial impairment of particularly humoral arm of immune system. The specific aim was to quantify SARS-CoV2 neutralizing antibodies in a patient plasma during the course of therapy. Materials and methods: Besides the standard of care treatment and monitoring, neutralizing antibody titers in patient's serum samples, calibrated according to the First WHO International Standard for anti- SARS-CoV-2 immunoglobulin (human), were quantified in a time- dependent manner. During the immunotherapy period peripheral blood flow cytometry immunophenotyping was conducted to characterize lymphocyte subpopulations. Results: The waves of clinical improvements and worsening coincided with transfused neutralizing antibodies rises and drops in the patient's systemic circulation, proving their contribution in controlling the disease progress. Besides the patient's lack of own humoral immune system, immunophenotyping analysis revealed also the reduced level of helper T-lymphocytes and immune exhaustion of monocytes. Conclusion: Therapeutic approach based on convalescent plasma transfusion transformed a prolonged, active COVID-19 infection into a manageable chronic disease.

BACKGROUND: Passive immunotherapy is the century- old practice of administering antibodies from an exposed convalescent or vaccinated person to a patient susceptible to the disease in question. Experience from prior outbreaks with other coronaviruses (SARS-CoV-1) shows that such convalescent sera contain neutralizing antibodies (NAb) against relevant virus and that their use was beneficial in the treated patients. Collection of COVID-19 convalescent plasma (CCP) at Croatian Institute of Transfusion Medicine (CITM) started in July 2020 and first unit for clinical use was issued in December. Clinicians in Croatia started using CCP in second wave of pandemics, mostly for patients with haematological malignancies. Such patients are not able to fight the SARS-CoV-2 infection by producing their own antibodies. CCP with individually checked levels of NAb, as an external source of antibodies, showed promising results in improving condition of these patients during SARS-CoV-2 infection. AIMS: To present the establishment of relevant methodology to properly evaluate SARS-CoV-2 neutralization capacity testing and to assess the correlation of donor disease severity and demographic characteristics with antibody titre level. METHODS: Each donor had a documented history of laboratory-confirmed SARS- CoV-2 infection. All plasma was donated by recovered and healthy COVID- 19 patients and collected by apheresis. In the beginning of the collection, titre of NAb was measured with the SARS-CoV-2 neutralization assay for quantification of SARS-CoV-2 NAb on Vero E6 cell suspensions and with home working stocks of SARS-CoV-2 virus prepared from a clinical isolate of the Laboratory working stock label SARS-CoV-2 297/20 Zagreb virus. In the meantime, the regression analysis for Vidas SARS-CoV-2 IgG test was done. This resulted in high correlation coefficient with neutralization assay and the regression equation has been used for the estimation of cut-off NAb level. High titre was determined as ED50/ml ≥ 1200. Data were collected from national transfusion IT system (e-Delphyn) and CCP donor checklist. Chi-squared, Kruskal- Wallis and Mann- Whitney tests were performed in MedCalc software for statistical analysis. RESULTS: We collected data for donations during the eight months period (28th July 2020 to 28th March 2021). 256 CCP units have been collected from 162 apheresis procedures. The donors’ age median was 36 (18-62) years. 30.4 % procedures were collected from women. The NAb median level of CCP for clinical use was 2560 (274 - 145, 218). The most common blood type was A (43%), followed by O (33%), B (12%) and AB (12%). COVID-19 symptoms in donors varied: asymptomatic (2.5%), mild (75%), medium (19.4%) and severe (3.1%). 75.4% of all donations had high NAb titre. Titre level did not statistically differ between genders (P = 0.262, Z = 1.12), severity of symptoms (P = 0.072, χ2 = 6.99) nor blood types (P = 0.842, χ2 = 0.83). Severity of disease did not statistically differ between genders (P = 0.23, χ2 = 4.26) nor blood types (P = 0.803, χ2 = 5.34). SUMMARY/CONCLUSIONS: 3/4 of collected CCP had high NAb titre. In rare cases, when the CCP supplies were scarce, physicians required issuing of CCP with lower titres. NAb titre was not dependent on gender, symptom severity nor blood type. Application of serological test performed in CITM highly correlated with the neutralization assay and allowed quicker issuing of CCP for clinical use which is very important for due time therapy of COVID-19 patients.

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.

Immunogenicity testing in animals is a necessary preclinical assay for demonstration of vaccine efficacy the results of which are often the basis for the decision whether to proceed or withdraw the further development of the novel vaccine candidate. However, in vivo assays are rarely, if at all, optimized and validated. Here we clearly demonstrate the importance of in vivo assay (mumps virus immunogenicity testing in guinea pigs) optimization for gaining reliable results and the suitability of Fractional factorial design of experiments (DoE) for such a purpose. By the use of DoE with resolution IV (2IV(4-1)) we clearly revealed that the parameters significantly increasing assay sensitivity were interval between animal immunisations followed by the body weight of experimental animals. The quantity (0 versus 2%) of the stabilizer (foetal bovine serum, FBS) in the sample was shown as non-influencing parameter in DoE setup. However, the separate experiment investigating only the FBS influence, and performed under other parameters optimally set, showed that FBS also influences the results of immunogenicity assay. Such finding indicated that (a) factors with strong influence on the measured outcome can hide the effects of parameters with modest/low influence and (b) the matrix of mumps virus samples to be compared for immunogenicity must be identical for reliable virus immunogenicity comparison. Finally the three mumps vaccine strains widely used for decades in the licenced vaccines were for the first time compared in an animal model, and results obtained were in line with their reported immunogenicity in human population supporting the predictive power of the optimized in vivo assay.

L-Glutamine (L-Gln) instability in liquid media has been a well-known fact. Also, negative effect of ammonia, one of the L-Gln degradation products, on viability of many cell cultures and on different viruses’ growth has been described. However, negative effects of ammonia have been reported in doses excessively exceeding those that could be generated in regularly used liquid culture media due to L- Gln spontaneous breakdown (bellow 2 mM). Traditional virus vaccine production processes have been established and registered involving L-Gln containing media use. Eventual culture media replacement in the regular production process belongs to the major regulative changes that require substantial financial expenses. The aim of this study was to evaluate the effect of Minimum Essential Media with Hanks salts (MEM-H) storage on its relevant biological functions during virus vaccine production process, in relation to L-Gln decrease. Our results show a cell-type dependent effect of spontaneous L-Gln degradation during media storage. They also suggest that for cell cultures used in measles, mumps, and rubella virus production the media retain their functionality in respect to cell viability or virus growth over certain window of time despite L-Gln degradation.

Protein thermal shift assay (TSA) has been extensively used in investigation of protein stabilization (for protein biopharmaceutics stabilization, protein crystallization studies or screening of recombinant proteins) and drug discovery (screening of ligands or inhibitors). This work aimed to analyze thermal shift assay results in comparison to protein polymerization (multimerization and aggregation) propensity and test the most stabilizing formulations for their stabilization effect on enveloped viruses. Influence of protein concentration, buffer pH and molarity was tested on three proteins (immunoglobulin G, ovalbumin, and albumin) and results showed that each of these factors has an impact on determined shift in protein melting point Tm, and the impact was similar for all three proteins. In case of ovalbumin, molecular dynamics simulations were performed with the goal to understanding molecular basis of protein’s thermal stability dependence on pH. Effect of three denaturing agents in a wide concentration range on Tm showed nicely that chemical denaturation occurs only at the highest concentrations. Results showed similar effect on Tm for most formulations on different proteins. Most successful formulations were tested for enveloped virus stabilizing potential using cell culture infectivity assay (CCID50) and results showed lack of correlation with TSA results. Only weak correlation of Tm shift and protein polymerization measured by SEC-HPLC was obtained, meaning that polymerization cannot be predicted from Tm shifts.

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.

Viral particles are used in medical applications as vaccines or gene therapy vectors. In order to obtain product of high purity, potency and safety for medical use purification of virus particles is a prerequisite, and chromatography is gaining increased attention to meet this aim. Here, we report on the use of ion-exchange and hydrophobic interaction chromatography on monolithic columns for purification of mumps virus (MuV) and measles virus (MeV). Efficiency of the process was monitored by quantification of infective virus particles (by 50 % cell culture infective dose assay) and total virus particles, and monitoring of their size (by Nanoparticle Tracking Analysis). Ion-exchange chromatography was shown to be inefficient for MuV and best results for MeV were obtained on QA column with recovery around 17 %. Purification of MuV and MeV by hydrophobic interaction chromatography resulted in recoveries around 60 %. Results showed that columns with small channels (d = 1.4 µm) are not suitable for MuV and MeV, although their size is below 400 nm, whereas columns with large channels (6 µm) showed to be efficient and recoveries independent on the flow rate up to 10 mL/min. Heterogeneity of the virus suspension and its interday variability mostly regarding total-to-infective particle ratio was observed. Interestingly, a trend in recovery depending on the day of the harvest was also observed for both viruses, and it correlated with the total-to-infective particle ratio, indicating influence of the virus sample composition on the chromatography results.

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.

Vipera berus berus (Vbb) is the most widely distributed and Vipera ammodytes ammodytes (Vaa) the most venomous viper in Europe. In particular areas of the Old continent their toxic bites constitute a considerable public health problem. To make the current envenomation therapy more effective we have analysed the proteome of Vbb venom and compared it with that of Vaa. We found the proteome of Vbb to be much less complex and to contain smaller levels of particularly snaclecs and sPLA2s. Snaclecs are probably responsible for thrombocytopenia. The neurotoxic sPLA2s, ammodytoxins, are responsible for the most specific feature of the Vaa venom poisoning − induction of signs of neurotoxicity in patients. These molecules were not found in Vbb venom. Both venoms induce haemorrhage and coagulopathy in man. As Vaa and Vbb venoms possess homologous P-III snake venom metalloproteinases, the main haemorrhagic factors, the severity of the haemorrhage is dictated by concentration and specific activity of these molecules. The much greater anticoagulant effect of Vaa venom than that of Vbb venom lies in its higher extrinsic pathway coagulation factor-proteolysing activity and content of ammodytoxins which block the prothrombinase complex formation. Biological significance Envenomations by venomous snakes constitute a considerable public health problem worldwide, and also in Europe. In the submitted work we analysed the venom proteome of Vipera berus berus (Vbb), the most widely distributed venomous snake in Europe and compared it with the venom proteome of the most venomous viper in Europe, Vipera ammodytes ammodytes (Vaa). We have offered a possible explanation, at the molecular level, for the differences in clinical pictures inflicted by the Vbb and Vaa venoms. We have provided an explanation for the effectiveness of treatment of Vbb envenomation by Vaa antiserum and explained why full protection of Vaa venom poisoning by Vbb antiserum should not be always expected, especially not in cases of severe poisoning. The latter makes a strong case for Vaa antiserum production as we are faced with its shortage due to ceasing of production of two most frequently used products.

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.

The ammodytoxins (Atxs) are neurotoxic phospholipases which occur in Vipera ammodytes ammodytes (Vaa) snake venom. There are three Atx isoforms, A, B, and C, which differ in only five amino acid positions at the C-terminus but differ substantially in their toxicity. The objective of this study was to establish an analytical method for unambiguous identification of all three isoforms and to use the method to assess a procedure for purification of the most toxic phospholipase, AtxA, from the venom. Isolation procedure for AtxA consisted of isolation of Atx-cross-reactive material (proteins recognized by anti-Atx antibodies), by use of an affinity column, then cation exchange on CIM (Convective Interaction Media) disks. The purification procedure was monitored by means of reversed-phase chromatography (RPC) and mass spectrometry (MS). Although previous cation exchange of the pure isoforms enabled separate elution of AtxA from B and C, separation of AtxA from Atxs mixture was not accomplished. RPC was not able to separate the Atx isoforms, whereas an MS based approach proved to be more powerful. Peptides resulting from tryptic digestion of Atxs which enable differentiation between the three isoforms were successfully detected and their sequences were confirmed by post-source decay (PSD) fragmentation. Separation of Atx isoforms by ion-exchange chromatography is most presumably prevented by Atxs heterodimer formation. The tendency of Atxs to form homodimers and heterodimers of similar stability was confirmed by molecular modeling.