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.

[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.

[A cultured concentrated inactivated vaccine against tick-borne encephalitis studied during the immunization of children and adolescents]. / Izuchenie kul'tural'noi kontsentrirovannoi inaktivirovannoi vaktsiny protiv kleshchevogo éntsefalita pri immunizatsii detei i podrostkov.

The word deals with the results obtained in the study of the reactogenicity and immunological activity of concentrated and inactivated tissue-culture tick-borne encephalitis vaccine, manufactured by the Chumakov Institute of Poliomyelitis and Viral Encephalitides, in the immunization of children and adolescents. The vaccine proved to be moderately reactogenic and exhibited pronounced immunological activity. In 91.5% of the immunized children the fourfold increase of the antibody level was observed. On the basis of the data obtained in this study the tick-borne encephalitis vaccine was recommended for use in medical practice for the prophylaxis of tick-borne encephalitis among children and adolescents.

Viral safety of biological products in WHO policy.

The production and quality control of biological products are regulated by the different national and international requirements. One of the many functions of WHO is to develop an international consensus on basic criteria for the acceptability of biological products. These include general and international requirements for the production of individual biological products (vaccines, interferons, blood and blood products, etc.). Production and control of biologicals includes control of substrates, products during manufacture, and the final lots. By means of the application of these international requirements and guidelines, biological products can be manufactured with high quality, safety--especially viral safety--and efficacy.

[Immunogenicity and stability of a Soviet inactivated cultured vaccine against Japanese encephalitis]. / Immunogennost' i stabil'nost' otechestvennoi kul'tural'noi inaktivirovannoi vaktsiny protiv iaponskogo éntsefalita.

Six immunologically active vaccine batches inducing a specific antibody to Japanese encephalitis (JE) virus were obtained in serial manufacture of the preparation. In HI tests, the minimal antibody titre was 1:80, the maximal 1:320, neutralization index 1g was 3.7 to 5.2. The data on the stability of the antigenic potency of the vaccines in relation to the duration of storage at 4 degrees-6 degrees C are presented (the follow-up period 3 years). A certain relationship was found between the antigenic potency of the preparation and the titre of the initial infectious tissue culture virus. Also, a definite correlation was found between the initial immunogenic potency of the vaccines and their stability in storage. After 3 years of storage, three vaccine lots remained antigenically active, namely those which after manufacture had induced antihemagglutinins in titres 1:160 to 1:320. The antigenic activity of 6 vaccine batches prepared from the production strain Peking-1 (Nakayama serotype) was studied against the predominant strain of Jagar-10 serotype. All the freshly prepared vaccine batches were found to induce production of antihemagglutinins to both serotypes of JE virus, whereas virus-neutralizing antibodies were found only to the test strain Nakajama-NIH homologous to the vaccine Peking-1 strain. After 1 year of storage, four vaccine batches lost their capacity to induce production of antihemagglutinins to Jagar-01 strain, two batches induced antibody in low titres. This fact should be considered in evaluation of postvaccination immunity status in humans.

[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".

Vaccines: obstacles and opportunities from discovery to use.

Since smallpox, vaccine-development programs have varied, yet common elements exist. Investment in vaccine research and development depends on epidemiology, market size, funding, and liability. Fear of liability inhibits innovation, adaptation of existing methods, and regulatory decision-making. Vaccine evaluation proceeds from animal and in vitro studies to controlled human clinical trials. Successive stages provide the information base necessary for decisions on vaccine approval for general use, but sociopolitical forces can affect the process. The World Health Organization establishes nonbinding international norms for vaccine acceptability that are adopted by many countries. Concerns for purity and safety need to be addressed along with cost and vaccine availability. For example, replacement of brain tissue-derived rabies vaccine in developing countries with a continuous cell line-derived vaccine is possible if purity is balanced in relation to safety. Substantially reducing the toll of infectious diseases will require cooperation and common-sense decisions.

[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.

[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.

Recombinants between attenuated and virulent strains of poliovirus type 1: derivation and characterization of recombinants with centrally located crossover points.

Recombinants with a centrally located crossover point were selected from crosses between poliovirus type 1 strains and intertypic (type 3/type 1) recombinants. Two such recombinants were characterized in some detail. In one of them (v1/a1-6), the 5' half of the genome was derived from a virulent type 1 strain, while the 3' half came from an attenuated type 1 strain. The genome of the other recombinant (a1/v1-7) had the reverse organization, with the 5' and 3' halves being derived from the type 1 attenuated and virulent strains, respectively. As deduced from the RNase T1 oligonucleotide maps, the a1/v1-7 genome also had a relatively short centrally located insert of the poliovirus type 3 origin. Both recombinants exhibited ts phenotypes. The RNA phenotypes of the recombinants corresponded to that of the parent donating the 3' half of the genome, v1/a1-6 and a1/v1-7 expressing RNA- and RNA +/- characters, respectively. Despite being a ts RNA- virus, v1/a1-6 proved to be neurovirulent when injected intracerebrally into Cercopithecus aethiops monkeys, although it exhibited a somewhat diminished level of pathogenicity as compared to its virulent type 1 parent. Recombinant a1/v1-7 behaved as an attenuated strain. These data supported our previous conclusion drawn from the experiments with intertypic poliovirus recombinants that the attenuated phenotype of poliovirus depends largely on the structure of the 5' half of its genome, although mutations of the 3' half may alleviate the virulence of the virus to a degree.