Serological and virological assessment of oral and inactivated poliovirus vaccines in a rural population in Kenya.

A study was carried out in a rural community in Kenya to compare the humoral and intestinal immunity provided by three doses of oral poliovirus vaccine (OPV) and two or three doses of enhanced-potency inactivated poliovirus vaccine (IPV). The immunization series was started at 8-12 weeks of age and the interval between doses was 2 months. In children with low levels of maternal antibodies (i.e., those most at risk), the first dose of either vaccine stimulated antibody response. Children with high levels of maternal antibodies responded to the first dose of OPV, but not to that of IPV. Subsequent doses led to increases in the mean antibody titres with both vaccines. After three doses of OPV, the proportion of children with antibody titres of greater than or equal to 1:8 was 92% for type 1 virus, 98% for type 2, and 90% for type 3. After two doses of IPV the proportion of children with antibody titres of greater than or equal to 1:8 was 94%, 88%, and 97% for type 1, type 2, and type 3, respectively; after three doses of IPV, 100% of children had antibodies greater than or equal to 1:8 for types 1 and 3, and 98% for type 2. Intestinal immunity was tested with a challenge dose of type 1 OPV, but the dose used was too small to detect a significant difference between the vaccines.

Comparison of serum and salivary antibodies in children vaccinated with oral live or parenteral inactivated poliovirus vaccines of different antigen concentrations.

A new antigen-rich inactivated poliovirus vaccine (IPV) in ordinary (IPV1), double (IPV2) and quadruple (IPV4) antigen concentrations was given in 2 doses to 6 and 18 week old Pakistani infants. The immune responses to poliovirus types 1 and 3 were compared to those in infants given three doses of oral poliovirus vaccine (OPV) at 6, 12 and 18 weeks of age. Enzyme-linked immunosorbent assay, ELISA, was used to estimate IgG and IgA in serum and secretory IgA (SIgA) in saliva. Two to three years later, a follow-up of the serum antibody response was carried out in the same infants using a microneutralization test. Serum IgG antibody responses to poliovirus type 1 antigen after two doses of IPV1, IPV2 and IPV4 were not significantly higher than the response after three doses of OPV at 21 weeks of age (p greater than 0.05). The serum IgG responses to poliovirus type 3 were similar to those against type 1 in all the groups. Mean neutralizing antibody titres to poliovirus type 1 was significantly higher in the IPV2 group than the rest of the groups (p less than 0.01). For type 3, these titres were highest but not significantly, in the IPV4 group (p greater than 0.05). This study shows that two doses of a new antigen-rich IPV can give similar immediate serum antibody responses as OPV but higher late responses. SIgA antibodies in saliva were more efficiently induced by OPV after three doses than after 2 doses of IPV (p less than 0.05).

Immunologic memory induced at birth by immunization with inactivated polio vaccine in a reduced schedule.

One hundred forty-one healthy newborns were immunized 24 hours after birth with one dose of inactivated polio vaccine (IPV) of enhanced potency. Following the administration of a second vaccine dose six months later, a considerable proportion of babies responded with neutralizing antibody (NA) to the three poliovirus types. The very rapid occurrence and high antibody titer were indicative of an anamnestic response. Twenty-one infants who still had NA less than 1:4 to one-more poliovirus types after the second vaccine dose responded with very high NA values 7-10 days after a supplementary dose of IPV. It appears that IPV of enhanced potency administered at birth is apt to induce immunologic memory, which should provide the basis for protection against paralytic poliomyelitis in case of exposure to wild poliovirus later in life.

Measles virus fusion protein presented in an immune-stimulating complex (iscom) induces haemolysis-inhibiting and fusion-inhibiting antibodies, virus-specific T cells and protection in mice.

Immune-stimulating complexes (iscoms), which have recently been shown to be highly effective for the antigenic presentation of membrane proteins of viruses, were prepared with affinity-purified fusion (F) protein of measles virus (MV), using an adaptation of the standard method for iscom preparation. Immunization of monkeys with the F iscom preparation induced biologically active anti-F protein antibodies as was shown in haemolysis inhibition and cell-cell fusion inhibition tests. A whole MV iscom preparation, which also contained the haemagglutinin protein, induced not only also haemolysis-inhibiting antibodies, but, in contrast to the F iscom preparation, also haemagglutination-inhibiting and virus-neutralizing antibodies. In addition the F iscom preparation was shown to activate measles virus-specific T cells in mice. This was demonstrated by the generation of an MV-specific delayed type hypersensitivity response in F iscom-immunized animals and by the isolation of T cell clones specific for MV F protein with the T helper phenotype. Vaccination of mice with MV iscom or F iscom protected them from MV-induced fatal encephalopathy. The data concerning the immunogenicity of MV proteins presented in iscoms are discussed in relation to their potential for the development of an inactivated measles vaccine.

Poliomielitis paralitica en Guatemala / Paralytic poliomielytis in Guatemala

Durante la epidemia de poliomielitis paralítica que afectó a Guatemala entre 1982 y 1983, se llevó a cabo un estudio longitudinal de las características clínicas y virológicas de 133 niños con este diagnóstico ingresados en un hospital de la capital. La edad de los pacientes fluctuó entre los cuatro meses y los cinco años. El 68% no habían recibido ninguna dosis de la vacuna oral de poliovirus, mientras que al 6% se les habían administrado tres dosis. En 102 niños se aislaron poliovirus; en 92 de estos casos los virus excretados eran solo del tipo 1; en siete, del tipo 2; en uno, del tipo 3, y en dos niños, de los tipos 1 y 2. Se tomaron 66 cepas círicas para establecer la diferenciación intratípica y se halló que 55 de los 56 virus aislados del tipo 1 y el único del tipo 3 eran virus no análogos al de Sabin, mientras que las nueve cepas del tipo 2 eran análogas al virus de Sabin. Estas observaciones confirman la necesidad de la vacunación durante el primer semestre de vida y que el establecimiento de medidas adecuadas para controlar la transmisión de poliovirus del tipo salvaje produciría una notable disminución de la morbilidad causada por la poliomielitis

Inhibition of measles virus-induced cell-cell fusion with a monoclonal antibody directed against the haemagglutinin.

A neutralizing monoclonal antibody (C26-15) against the haemagglutinin (H protein) of measles virus was generated which caused cell-cell fusion inhibition in cultures of measles virus-infected cells. It was shown that this phenomenon coincided with a down-regulation of the expression of both the H protein and the fusion (F) protein. We also showed cell-cell fusion inhibition with a polyclonal rabbit serum directed against Tween-ether inactivated measles virus, which did not contain biologically active antibodies against the F protein. Cell-cell fusion inhibition caused by anti-H antibodies is distinct from cell-cell fusion inhibition induced by a direct interaction of anti-F antibody with the F protein in the membrane of infected cells. Since both mechanisms may also be involved in the in vivo situation, the exclusive role for the generation of anti-F antibody to prevent virus spread by cell-cell fusion in vivo is questioned. It is speculated that the observed down-regulation of both glycoproteins may lead to a less efficient killing of infected cells by cytotoxic T-lymphocytes, which may constitute an alternative explanation for the insufficient protection after vaccination with an inactivated measles vaccine.

Characterization and fibrin-binding properties of different molecular forms of pro-urokinase from a monkey kidney cell culture.

Culture fluid of a monkey kidney cell culture was harvested every two days, for a two week period, in order to obtain urokinase in the zymogen form. Pro-urokinase was isolated by immunoadsorption chromatography and gel filtered on Sephadex G-150, which resulted in three peaks with pro-urokinase activity. SDS-polyacrylamide gel electrophoresis showed that the first peak contained 55 kd pro-urokinase, aggregated with high molecular weight contaminants, whereas the second and third peaks consisted of almost pure 55 kd and 30 kd pro-urokinase, respectively. The latter form represented a relatively unknown and inactive precursor of low molecular weight urokinase, which was, like 55 kd pro-urokinase, activatable with plasmin. In comparison with tissue-type plasminogen activator, 55 kd and 30 kd pro-urokinase only bound weakly to purified fibrin clots and fibrin-sepharose columns. The extent of binding of the two pro-urokinases and their plasmin-activated forms to fibrin-sepharose decreased in the following order: 55 kd pro-urokinase 30 kd pro-urokinase 55 kd urokinase 30 kd urokinase. These results indicate that the two precursors exhibited stronger binding to fibrin-sepharose than the corresponding active enzymes, and the two 55 kd forms exhibited stronger binding than the corresponding 30 kd forms. This indicates the importance of both the zymogen nature and an intact NH2-terminal part of the molecules for binding to fibrin.

Vaccines: quantities, production and costs.

At a birth rate of about 150 million children per year, 300 to 400 million doses DTP-polio and 150 to 200 million doses of measles and BCG vaccines will be required annually for immunization of the world population against the target diseases as defined by the EPI of the World Health Organization. With the introduction of modern fermentation technologies production of these quantities is practically and economically feasible. The main problem is the administration of the vaccine to the target population. This may be achieved by application of a more condense and simplified vaccination schedule which has shown to be effective in clinical studies. Also, the costs of the immunization programmes may be considerably reduced in this way as the costs of administration form the major part of any immunization programme.

Large scale animal cell cultivation for production of cellular biologicals.

Through the developments in molecular biology the interest for large scale animal cell cultivation has sharply increased during the last 5 years. At our laboratory, four different cultivation systems were studied, all of which are homogeneous culture systems, as they lend themselves best for scaling up and for the control of culture conditions. The four different systems which were compared are: batch culture, continuous chemostat, continuous recycling and continuous perfusion culture system, both for cells growing in suspension and for anchorage dependent cells in microcarrier culture. Our results indicate that for the production of virus vaccines and cells the batch and recycling culture system are most suitable. Disadvantages of the continued chemostat culture system are: the system is only applicable for cells growing in suspension; relatively low concentrations of cells and cellular products are obtained. The continuous perfusion system appears to be very suitable for the production of cellular components and also for the production of viruses which do not give cell lysis.

Induction of long-term immunity to paralytic poliomyelitis by use of non-infectious vaccine.

Naturally acquired immunity to paralytic poliomyelitis, which is of long duration, is associated with serum antibody, immunological memory, or both. Persistence of circulating antibody is reassuring but not essential for long-term protection. Immunological memory induced by killed poliovirus vaccine is similar to that induced by infection, which is not a prerequisite for the induction of long-term immunity. Animal studies of experimental vaccines indicate that some antigenic components of poliovirus induce immunological memory without producing detectable antibody; killed poliovirus vaccine has the same effect in man. Killed vaccine containing 40, 8, and 32 D-antigen units of poliovirus types 1, 2, and 3, respectively, protects all recipients in both one-dose and two-dose schedules. This means that either schedule can be chosen to fit in with local immunisation programmes and thus reduce costs and increase population coverage.

[Biotechnology and vaccine manufacture]. / Biotechnologie en vaccinbereiding.

Modern biotechnology offers great possibilities for the large-scale manufacture of effective, safe and inexpensive vaccines. Improvements can also be expected in the manufacture of traditional vaccines, and there would appear to be prospect of producing vaccines against (tropical) parasitic disease for the first time. Close cooperation between university centres, research institutes and industry is essential to the development of biotechnology.

Inactivated poliovirus vaccine: current production methods and new developments.

The biotechnologic developments during the last decade have led to the production of inactivated poliovirus vaccine (IPV) on an industrial scale and at economically acceptable costs. Replacement of primary monkey kidney cells by subcultured monkey kidney, Vero, or human diploid cells as substrate for virus multiplication as well as the introduction of the microcarrier culture technique have made cell and virus cultivation in large fermentors of 100-1,000 liters feasible. Procedures for processing virus harvests into highly concentrated purified vaccines were developed; also, the safety and potency control tests were improved and simplified. It has been demonstrated that these more potent poliovirus vaccines, either alone or in combination with diphtheria-tetanus-pertussis vaccine, induce a high immunity with reduced vaccination schedules. The overall costs of vaccination will be reduced considerably in this way. In addition, the results of biochemical and immunologic studies indicate that neutralizing antibodies can be induced by the viral proteins alone. These findings open up promising perspectives for production of subunit poliovirus vaccines with use of recombinant DNA and synthetic antigen, a method that has already proved feasible for producing vaccine against foot and mouth disease. These new techniques may lead to a further reduction of production costs and will improve the safety of the vaccine.

Different secretory IgA antibody responses after immunization with inactivated and live poliovirus vaccines.

The influence on secretory IgA antibody levels in milk and saliva of vaccination with oral, live poliovirus vaccine ( OPV ) and inactivated poliovirus vaccine (IPV) was studied. IPV, especially the antigen-rich Dutch vaccine, more often induced increases in antibody titers in milk (50%) than did OPV (26%) (P less than .01). OPV more often decreased the antibody levels in milk (40%) than did IPV (10%) (P less than .01). It was striking that mainly high prevaccination titers were decreased. The increases of IgA antibody in saliva were less striking. IPV caused increases as often in milk as in saliva, whereas OPV more often induced increases in IgA antibody in saliva, but there was a poor correlation between the changes in antibody titers in milk and those in saliva.

Standardization of poliovirus neutralizing antibody tests.

Recently the Forum for Advancement of Immunization Research sponsored a Collaborative Study on Poliovirus Antibody Titration. Twenty laboratories from 12 countries participated. There were considerable differences in detail of test performance and test results among laboratories. The sensitivity of the tests varied over a 10-fold range (geometric mean titer from 21 to 288). The average coefficient of variation ranged from 4.5% to 13.5%. Tests performed at the Food and Drug Administration indicated that Hep-2 cells were more suitable than Vero cells for poliovirus titration by cytopathic effect end point or plaque assay. However, the cell lines were equally suitable for polio antibody titration by neutralization of cytopathic effect. A high degree of sensitivity and optimal reproducibility of antibody assay were observed in tests utilizing a medium to low dose of virus and serum-virus incubation overnight at 36 C. On the basis of current experience, a protocol is proposed for a test that provides high sensitivity and reproducibility and is practical for large-scale determinations of poliovirus antibody.

The evaluation of a physical method for the quantification of inactivated poliovirus particles and its relationship to D-antigenicity and potency testing in rats.

The use of a density gradient procedure for the quantification of intact, inactivated poliovirus particles in vaccine preparations is described. The procedure is both sensitive and highly reproducible and the results correlate with those of potency tests in rats and with D-antigen content as measured by ELISA. Because of the occasional ambiguity observed with D-antigen assays, it is suggested that the density gradient procedure will provide valuable additional information for the in vitro assessment of inactivated poliovirus preparations.