Selection of Domestic Cell Lines for the Creation of Diagnostic and Preventive Preparations against Enterovirus 71.

We studied the sensitivity of domestic proprietary human and animal cell lines from the collection of M. P. Chumakov Federal Scientific Center for Research and Development of Immuneand-Biological Products to infection with different enterovirus 71 strains. A cell system based on domestic proprietary permanent cell line 4647 was for the first time used for reproduction of four enterovirus 71 strains (BrCr, 42266, 42934, and 43374). It was shown that strain 4647 is the optimal cell substrate for enterovirus 71 reproduction. The titers of enterovirus 71 for all four strains considerably (by 2 lgTCID50/ml and more) increased during sequential passages in permanent cell line 4647. The prospects of using permanent cell line 4647 for creation of diagnostic and preventive preparations against 71 was demonstrated.

[Use of continuous human and animal cell lines for the production of viral vaccines].

History of development of safety criteria for continuous human and animal cell lines approved for manufacture of immunobiologic preparations. It was noted that current WHO documents recommend mandatory use of respective WHO's reference cell cultures (Vero-10-87 for continuous cell lines, and Wi-38 or MRC-5 for diploid cell lines) during attestation of new cell cultures proposed for the manufacturing of immunobiologic preparations. Examples of practical use of continuous cell lines (CCLs) for production of viral vaccines on industrial scale are described. On the basis of modern data most important principles were formulated which should be considered to provide safety and efficacy of vaccines produced on the CCLs.

[The design and practical use of vaccines for the preventions of pressing viral infections].

The paper presents data on the design and practical medical use of vaccines for the prevention of urgent viral infections, including those against poliomyelitis (oral poliomyelitis vaccine), tick-borne encephalitis, hepatitis A, rabies, and enteroviris 71. It shows the basic lines in the improvement of manufacture and biological control of vaccines and in the development of a new vaccine for prevention of hemorrhagic fever with the renal syndrome.

Evaluation of the new control methods for oral poliomyelitis vaccine.

In the draft recommendations for production and control of OPV the WHO proposed new control methods: (i) mutant analysis with PCR and restriction enzyme cleavage (MAPREC) assay that allows evaluation of poliovirus population heterogeneity at the molecular level; (ii) neurovirulence (NV) test using transgenic mice susceptible to polioviruses and (iii) control of the seed lots for the presence of the simian virus 40 (SV40) DNA sequence. This paper is focused on our experience in the practical implementation of the new methods at the Institute of Poliomyelitis and Viral Encephalitides (IPVE). Using methods based on PCR we have demonstrated that working seed viruses used by IPVE for OPV production are free from SV40 DNA sequences. Our experience on the conduction of the OPV type 3 control using TgPVR21 mice NV test (seven vaccine lots) and the MAPREC assay (more than 150 samples of single harvests and monovalent bulks) showed that these methods may be used instead of the monkey NV test, because they could not pass the vaccine failed monkey NV test. The necessity for single harvests control is discussed.

Poliomyelitis in Russia in 1998-1999.

After introducing surveillance for poliomyelitis and AFP cases in the Russian Federation in 1998, 740 AFP cases have been registered in 1998-1999, and 18 of that number were considered as vaccine-associated paralytic poliomyelitis (VAPP). Of 18 cases 11 were classified as VAPP of vaccine recipients and confirmed by virus isolation; from two of the vaccine recipients virus was not isolated, and five were poliomyelitis cases in contact non-vaccinated children. In all the cases the disease was characterised with the typical clinical picture with residual pareses and paralyses. One case was fatal. Vaccine virus type 3 has been isolated from all the vaccine recipients. The MAPREC test has shown that the quality of monovaccine type 3 bulks used for vaccinating these children did not differ from the quality of other bulk vaccines produced by the Chumakov Institute of Poliomyelitis. Patients surveyed for gammaglobulin were positive. Polioviruses type 1 isolated from two of the contact cases had changed antigenic properties and were recombinants of types 1 and 2.

The mutagenic effect of measles virus on cell cultures of different origin.

Differences between strains of measles virus in their mutagenic effect on cells of different animals and man were found. The mutagenic activity of measles virus strains was species-specific. In the process of spontaneous transformation the sensitivity of cells to the mutagen changed.

[Chromosome nucleolus organizer regions of the African green monkey]. / Iadryshkoobrazuiushchie raiony khromosom afrikanskoi zelenoi martyshki.

The silver staining Ag-I for detection of nucleolar organizer regions (NOR) in the African green monkey chromosomes was used. NOR were situated on the secondary constrictions of one pair of homologous marker chromosomes and consisted of two parts. A block of condensed chromatin was seen in the middle of the despiralized portion corresponding to the secondary constriction. The data suggest that the stalks of satellites of chromosomes, rather than satellites themselves, are regions of nucleolar organizer.

[Damaging effect of 2 simian viruses on mammalian cell chromosomes]. / Povrezhdaiushchee deistvie dvukh virusov obez'ian na khromosomy kletok mlekopitaiushchikh.

A green monkey myxovirus (SV5) causes chromosome breaks in embryonal diploid fibroblasts of man, cotton rats, striped hairy-footed hamster, and in cells of one heteroploid line of newborn lamb kidney. Adenovirus SV15 is mutagenic for all cultures under study (diploid strains of human and cotton rat embryo fibroblasts and three continuous kidney cell lines from newborn lambs). The mutagenic activity of these viruses may be manifested without their undergoing a complete cycle of replication.

[Donor immunization with an inactivated concentrated purified vaccine against tick-borne encephalitis to obtain immune blood preparations]. / Immunizatsiia donorov inaktivirovannoi kontsentratrirovannoi ochishchennoi vaktsinoi protiv kleshchevogo éntsefalita dlia polucheniia immunnykh preparatov krovi.

Vaccination of seronegative and seropositive donors with a concentrated purified vaccine (CV) against tick-borne encephalitis induced antibody synthesis in a concentration sufficient for preparation of donor immunoglobulin with antibody titres of 1:320 to 1:1280 by HI test. The resulting preparation showed a high antiviral activity both in vivo and in vitro against antigenically different strains of tick-borne encephalitis virus. The immune plasma derived from CV-immunized donors had a therapeutic effect in patients with tick-borne encephalitis. The optimal schedules for immunization of donors are discussed.

[Use of molecular biological method (MAPREC-test) for evaluating residual neurovirulence and genetic stability of oral poliovirus vaccine type 3]. / Ispol'zovanie molekuliarno-biologicheskogo metoda (MAPREC-testa) dlia otsenki ostatochnoi neirovirulentnosti i geneticheskoi stabil'nosti oral'noi poliovirusnoi vaktsiny tipa 3.

MAPREC test (mutant analysis by polymerase chain reaction and restriction enzyme cleavage), evaluating the content of neurovirulent mutants with nucleotide substitution of U for C in position 472 in the 5'-nontranslated RNA region, in a virus population was used for a retrospective analysis of 3 specimens of inoculated viruses and 55 lots of type 3 oral poliovirus vaccine (OPV) prepared at M. P. Chumakov Institute of Poliomyelitis, Russian Academy of Medical Sciences, during recent 6 years. The mean content of neurovirulent mutants in inoculated viruses was 0.26 +/- 0.06%, in monovaccines 0.34 +/- 0.04% (the maximum allowable content is 1%). The method is well reproducible and detects about 0.1% mutants in the population. MAPREC test can be used instead of currently used residual virulence control on monkeys, as it detects viral preparations which were not tested on monkeys and does not reject high-quality vaccines. The protocol of using this method in combination with other methods for neurovirulence and genetic stability control of type 3 OPV is discussed.

[Experimental study of the ability of different strains of vaccinia virus to induce autoantibody formation]. / Eksperimental'noe izuchenie sposobnosti razlichnykh shtammov virusa vaktsiny indutsirovat' obrazovanie autoantitel

The intensity of autoantibody production to red blood cell lysate in guinea pigs inoculated with smallpox vaccine prepared from different strains was studied at 7, 14, 21 and 28 days after vaccine inoculation. The dynamics of autoantibody production in the same animals as well as the influence of altered responsiveness of the animals pre-treated with dyphtheria toxin on the intensity of autoantibody production were determined. The autoantibody-inducing activity correlated with reactogenicity of the strains and was most marked in the white cloned variant of the Tashkent strain, slightly lower in the L-IVP strain being intermediate in this criterion and insignificant in poorly reactogenic EM-63 and B-51 strains. In the animals with altered responsiveness the autoimmune responses were increased. The results of this study were tested statistically.

[Concentrated purified vaccine against tick-borne encephalitis prepared by means of zonal ultracentrifugation. Development of the preparation]. / Kontsentrirovannaia ochishchennaia vaktsina protiv kleshchevogo éntsefalita, prigotovlennaia metodom zonal'noiogo ul'tratsentrifugirovaniia. Razrabotka preparata.

A concentrated and purified (lyophilized) preparation with a high immunological activity was obtained from formalin-inactivated tissue culture suspensions of tick-borne encephalitis (TBE) virus. Its high immunogenicity was demonstrated in mouse protection tests against fatal challenge with TBE and in the studies of antibody production in monkeys of two species. The preparation was shown to contain no infectious TBE virus, to be safe for small laboratory animals and monkeys. Trials of the preparation in 19 volunteers demonstrated it to be well tolerated and to induce virus-neutralizing antibody production in man. It also sensitized lymphoid cells when inoculated subcutaneously with Al2O3. These experimental materials indicate the possibility of developing a highly potent vaccine against tick-borne encephalitis by concentration and purification of TBE particle suspensions.

[Isolation of adeno-associated virus type 4 from a culture of green monkey kidney cells]. / Vydelenie adenoassotsiirovannogo virusa tipa 4 iz kul'tury kletok pochek zelenykh martyshek.

In the process of biological control of uninfected green monkey kidney (GMK) cell cultures a thermostable hemagglutinating agent designated No. 5056 was isolated alongside with adenovirus-SV. By its antigenic properties the 5056 strain was identified as adeno-associated virus type 4 (AAV-4). In control of 574 specimens of GMK culture batches, 40 AAV-4 strains were isolated in the presence of a helper adenovirus. Some biological properties of the isolates and their resistance to certain physical and chemical treatments were studied. Two isolates of the satellite virus were examined in the electron microscope. A correlation between the rate of AAV-4 isolation from GMK cultures and the presence of complement-fixing antibody to AAV-4 in monkey sera was observed.

[Cultivation of the rabies virus in the continuous kidney cell line 4647 from the green marmoset]. / Kul'tivirovanie virusa beshenstva v linii perevivaemykh kletok pochki zelenoi martyshki 4647.

Continuous cells of green monkey kidney 4647 were found to be highly sensitive to three variants of the Vnukovo-32 vaccine strain of rabies virus preliminarily adapted to the primary line of Syrian hamster kidney cells. The virus litre increased with passages in the 4647 cell culture. The resulting virus variants had sufficient immunogenic potency. The time course of virus replication at a temperature of 32 degrees C was studied. The heteroploid cell line 4647 was found to be promising for manufacture of medical and veterinary cultural rabies vaccines.

[Comparison of methods for the large-scale culture of cells and viruses]. / Sravnenie metodov krupnomasshtabnogo kul'tivirovaniia kletok i virsuov.

The paper presents the results of experiments on propagation of primary, secondary, and continuous diploid and heteroploid human and animal cells in 2 different systems for large-scale propagation: in perfusion tank with Rashig rings and in tanks with microcarriers. Both methods of large-scale cultivation produce higher cell yields than the traditional cultivation methods. A yield of tick-borne encephalitis virus per 1 cell in the perfusion cultivator was 12 times as high as in roller cultures. Poliomyelitis virus titres were practically equal with both methods of large-scale cultivation and with the conventional method (7.73 lg PFU/ml in GMKC on microcarriers). The method of cultivation on microcarriers is more acceptable and advantageous as it gives higher yields of cells necessary for growth of poliomyelitis virus.

[Cytolar microcarriers for the cultivation of surface-dependent cells]. / "Tsitolar"--mikronositeli dlia kul'tivirovaniia povekhnostno--zavisimykh kletok.

The results of trials of one of the microcarriers (MC) developed by the authors, Cytolar, synthesized from denaturated collagen are presented. Both primary chick embryo cell and green monkey kidney cell cultures and diploid fibroblast-like human embryo cells were successfully cultivated on these MC. The properties of the developed MC were not inferior to those of the best known analogues.

[Creation of an experimental cell culture vaccine against distemper using cell line 4647]. / Sozdanie éksperimental'noi kul'tural'noi vaktsiny protiv chumy plotoiadnykh s ispol'zovaniem kletok 4647.

Experiment in susceptible animals demonstrated the effectiveness and safety of several samples of a distemper vaccine prepared in cell line 4647. Preparations of the vaccine virus variants generated in line 4647 after 10 passages at 34 degrees C, 37 degrees C and 40 degrees C and 20 passages of rapidly multiplying virus (34 degrees C) possessed the protective activity when used for immunization in a dose of 1000 PFU/animal. The results obtained in observations of the animals and examinations of sera for antibodies did not differ from those of control tests of "Vakchum".

[Several characteristics of new strains of diploid green marmoset fibroblasts]. / Nekotorye kharakteristiki novykh shtammov diploidnykh fibroblastov zelenykh martyshek.

Two strains of diploid fibroblasts were derived from green monkey fetal lungs and their main properties were studied. These strains were shown not to differ principally from the well studied human diploid fibroblasts. The derived cultures are free from contaminating viruses. The titers of attenuated poliomyelitis virus strains achieved in green monkey diploid cells are comparable to those in primary cultures of green monkey kidneys. The possiblity of using green monkey diploid fibroblasts in virological practice is discussed.

[Cultivation of primary animal cells on microcarriers of various types]. / Vyrashchivanie pervichnykh kletok zhivotnykh na mikronositeliakh razlichnogo tipa.

Primary cells of chick embryos (CEC), new-born piglet kidneys (NPK), and green monkey kidney (GMK) obtained by the conventional method of tissue trypsinization were shown to grow satisfactorily on microcarriers of various types. CEC formed a confluent monolayer on Supperbead particles within 38--48 hours when seeded in a dose of 3 x 10(5) cells/ml and on DEAE-Sephadex A-50 within 60--72 hours when seeded in a dose of 4 x 10(5) cells/ml. In both instances, with microcarriers used in a concentration of 1 mg/ml the number of CEC by the end of the cultivation period reached 0.9 million cells/ml. Primary NPK and GMK cells formed monolayers not on all microcarrier particles, nevertheless the number of cells in both cultures increased 2--2 1/2-fold in 7 days. The optimal seed concentration of cells depended both on the quality of the primary cell suspension and on the microcarrier type. The monolayer of CEC cells formed on DEAE-Sephadex A-50 particles persisted for 7--14 days.

[RAMT cell line as a substrate for the propagation of vaccinal viral strains]. / Liniia kletok RAMT kak substrat dlia razmnozheniia vaktsinnykh shtammov virusov.

Properties of the PAMT cell line which are deficient in hypoxanthine guanyl phosphoribosiltransferase were studied. The PAMT cell line derived from African green monkey kidney tissue may be identified by selective nutrient media, contains no contaminants, is not tumorigenic, is susceptible to a wide range of viruses. Titres of poliomyelitis, tick-borne encephalitis, and carnivore plague viruses were similar in cell cultures grown in the medium with 10% serum and in those adapted to growth in the medium containing 1% serum. The properties of PAMT cell line allow it to be used in manufacture of killed virus vaccines.