Viral Safety Issues in the Production and Manufacturing of Human Immunoglobulin Preparations from Equine Plasma/Serum.

The current Russian and foreign pharmacopoeias either do not provide any information about existing types of viral diseases in horses or do not present it in full. Data of modern domestic and foreign literature was used to prepare the most complete list of viruses that cause equine diseases including 36 infectious agents, 25 of which are pathogenic for humans, 13 of the 25 of which are widespread throughout Russia. Information is provided on the magnitudes of the disease incubation periods (which are most often within one month), the external clinical signs of these diseases (which can also be asymptomatic), and the maximum possible concentrations of viruses in the blood of horses with these diseases (which can reach 8 log conventional units/mL of blood). This information is offered for use in critical production stages of heterologous immunoglobulin drugs for medical use to assure viral safety.

Ensuring Viral Safety of Equine Immunoglobulins during Production.

Equine blood plasma/serum and intermediates must be monitored for the presence of live viruses pathogenic in humans during production of equine immunoglobulins. Information concerning low-cost and simple methods for the detection of live horse viruses pathogenic and non-pathogenic to humans was gained using data of modern domestic and foreign literature. These methods are based on cultivation of these viruses on sensitive biosystems. The presented information can be used to set up blood plasma/serum control of horses at different stages of immunoglobulin production, i.e., when taking blood from horses during their quarantine period, when collecting blood from immunized horses, and before bottling the medicinal intermediate in the primary package.

[Results of preclinical study of inactivated subunit adsorbed monovalent influenza vaccine "PANDEFLU"].

AIM: To perform preclinical study of subunit monovalent adsorbed inactivated influenza vaccine "PANDEFLU" [strains A/California/7/2009 (HIN1)v]. MATERIALS AND METHODS: Preclinical study of acute toxicity on experimental animals (assessment of vaccine's toxic effects on organs and body systems; pathomorphologic study of organs and tissues after administration of the vaccine; assessment of its influence on hematologic indicators). RESULTS: It was shown that administration of the vaccine did not lead to death of animals as well as to decrease of body mass or development of pathologic, focal sclerotic changes in parenchymal cells and visceral stroma; the vaccine did not negatively change hematologic and biochemical indicators of blood. Results of necropsy and histological study after acute administration of the vaccine in standard dose did not lead to irritation, inflammation or destruction of tissues in the place of inoculation. The vaccine was apyrogenic and did not have local irritating and allergenic effects. Status of animals after acute inoculation of the vaccine demonstrated its good tolerability and safety in doses exceeding standard human doses more than tenfold. CONCLUSION. Performed research demonstrated absence of contraindications for conduction of clinical trials of "PANDEFLU" vaccine on limited contingent of volunteers.

[Indication of the influenza virus in clinical samples using immunoenzyme and lanthanide immunofluorescence analyses]. / Indikatsiia virusa grippa v klinicheskikh obraztsakh s pomoshch'iu immunofermentnogo i lantanidnogo immunofliurestsentnogo analizov.

A comparative study of the potentials of enzyme-immunoassay (EIA) and lanthanide immunofluorescent assay (LIFA) for influenza virus detection in nasopharyngeal aspirates and nasopharyngeal washings was carried out. Monospecific and polyclonal antisera to hemagglutinin polyclonal antisera to matrix protein as well as polyclonal and monoclonal antisera to nucleoprotein were used. The nasopharyngeal aspirates were shown to contain virus-specific antigens in the amounts sufficient for influenza virus detection with polyclonal and monoclonal antibodies. There was a good correlation between chromophore results by EIA and fluorescence level in LIFA. The sensitivity of the test systems used (EIA and LIFA) was shown to be insufficient for the detection of virus-specific material in the nasopharyngeal washings. Besides, there was no correlation between the results obtained by different test systems. It was concluded that nasopharyngeal aspirates should be collected for rapid influenza diagnosis by EIA and LIFA.

The influence of the isolation technique of influenza virus nucleoprotein on its antigenic properties.

ELISA has been used to study the antigenic properties 1. of influenza virus nucleoprotein (NP-1) isolated from virions with the help of preparative polyacrylamide gel electrophoresis (PAGE); 2. of virion ribonucleoprotein (NP-2), and 3. of NP structures prepared by dissociation of ribonucleoprotein into RNA and protein in sucrose gradient containing NaCl (NP-3). The investigation of immunologic cross-reactivity has shown complete identity of NP-2 and NP-3 and their striking difference from NP-1. In contrast to NP-2, NP-3 was not contaminated by other virus antigens, it was a good immunogen and could be used for preparation of monospecific antisera of high titre. NP-1 did not induce a high antibody response,however, like NP-2 and NP-3, it retained its capacity to react with antisera to native virus. Owing to its simple production and high yield, this protein can be used in serodiagnosis for testing the antibody level against NP-protein in convalescent sera.

[Possibility of detecting the matrix protein of the influenza virus in intact and disrupted virions by immunoenzyme analysis and immune electron microscopy]. / Issledovanie vozmozhnosti vyiavleniia matriksnogo belka virusa grippa v intaktnykh i razrushennykh virionakh s ispol'zovaniem immunofermentnogo analiza i immunnoi élektronnoi mikroskopii.

ELISA and immune electron microscopy were used to study possible causes of the detection of antigenic reactivity of influenza virus matrix protein in purified virus suspension directly adsorbed on polystyrene. No interaction of antibody to M protein with the surface of virus and subvirus particles was observed. The process of virus sorption on polystyrene for a long period was shown not to lead to disruption of intact virus particles, and the detection of the internal matrix protein in preparations of purified influenza virus was due only to the presence of partially or completely destroyed virions in the viral suspension.

[Isolation of the internal proteins of the influenza virus by preparative polyacrylamide gel electrophoresis for obtaining monospecific antisera]. / Vydelenie vnutrennikh belkov virusa grippa metodom preparativnogo élektroforeza v poliakrilamidnom gele dlia polucheniia monospetsificheskikh antisyvorotok.

The principal possibility of isolation of internal proteins (M and NP) of influenza type A (H1N1 and H3N2) and B viruses by SDS-PAG preparative electrophoresis and preparation of monospecific antisera to these proteins was demonstrated. The resulting preparations may be used for testing the biological objects by enzymeimmunoassay.