Potency tests of combination vaccines.

Combination vaccines differ from single-component vaccines in composition and in how they are manufactured, which poses significant challenges to implementing effective quality-control tests, including measurement of potency. Because each combination vaccine is unique, existing guidelines often fail to provide sufficient information to overcome the inevitable problems encountered when developing and implementing potency tests. Success depends on careful consideration of scientific and regulatory issues. Significant problems may occur if potential interactions between different components in the vaccine are not taken into account during product and test development. Thorough analysis of critical assay parameters and attention to scientific and statistical justifications for the test increase the likelihood of its acceptance. Practical approaches based on experience include rational design of validation studies, complete evaluation and documentation of the potency tests under the conditions in which they are to be applied, and establishing the relationship between production lots of vaccine and lots used in clinical trials.

Oral iodine supplementation does not reduce neutralizing antibody responses to oral poliovirus vaccine.

Iodine deficiency is a major cause of impaired mental development, goitre, and cretinism in many parts of the world. Because existing immunization programmes can be used to deliver oral iodized oil (OIO) to infants at risk, it was important to know whether OIO could adversely affect the antibody response to vaccines, such as trivalent oral poliovirus vaccine (OPV). A randomized, double-blind, placebo-controlled clinical trial was conducted in Subang, West Java, Indonesia, in which 617 eight-week-old infants received either OIO or a placebo (poppy-seed oil) during a routine visit for their first dose of OPV as part of the Expanded Programme on Immunization (EPI). The infants received two boosters of OPV at 4-week intervals after the first dose, and were followed up when 6 months old. Neutralizing antibody titres to poliovirus serotypes 1, 2, and 3 were compared in serum samples that were taken from 478 of these infants just before the first dose of OPV and at 6 months. It was found that oral iodized oil did not reduce the antibody responses to any of the three serotypes of OPV. These results indicate that oral iodine may safely be delivered to infants at the same time as oral poliovirus vaccine according to current EPI immunization schedules.

PIP: This is a randomized, placebo-controlled clinical trial on the effect of oral iodized oil (OIO) on the immune response to oral poliovirus vaccine (OPV). 617 8-week old infants were enrolled in the study conducted in Subang, West Java, Indonesia. Infants received either IOI--at which time they also received OPV and diphtheria-pertussis-tetanus vaccine--or a placebo (poppy seed oil) during their first Expanded Program on Immunization (EPI) contact for their first dose of OPV. After the first dose, 2 boosters of OPV were received by the infants at 4-week intervals, and there was a final follow-up evaluation when infants reached their 6th month. Serum samples were collected from each infant at enrolment and at follow-up. A total of 478 pairs of pre-immune and postimmune sera were collected for evaluation. Neutralizing antibody titers to poliovirus serotypes 1,2, and 3 were compared in serum samples. The range of measured neutralizing antibody activity was 0.01-14 IU for type 1, 0.05-49 IU for type 2, and 0.01-11 IU for type 3 poliovirus. After the immunization, 2 (0.4%), 1 (0.2%), and 16 (3.3%), respectively, of the infants had no detectable neutralizing antibodies to all 3 poliovirus serotypes. It was found that OIO did not reduce the antibody responses to any of the 3 serotypes of OPV but did improve infant survival in the same cohort. These findings indicate that oral iodine supplementation may be safely combined with the delivery of the first dose of OPV according to EPI schedules.

Poliomyelitis in intraspinally inoculated poliovirus receptor transgenic mice.

Mice transgenic with the human poliovirus receptor gene develop clinical signs and neuropathology similar to those of human poliomyelitis when neurovirulent polioviruses are inoculated into the central nervous system (CNS). Factors contributing to disease severity and the frequencies of paralysis and mortality include the poliovirus strain, dose, and gender of the mouse inoculated. The more neurovirulent the virus, as defined by monkey challenge results, the higher the rate of paralysis, mortality, and severity of disease. Also, the time to disease onset is shorter for more neurovirulent viruses. Male mice are more susceptible to polioviruses than females. TGM-PRG-3 mice have a 10-fold higher transgene copy number and produce 3-fold more receptor RNA and protein levels in the CNS than TGM-PRG-1 mice. CNS inoculations with type III polioviruses differing in relative neurovirulence show that these mouse lines are similar in disease frequency and severity, demonstrating that differences in receptor gene dosage and concomitant receptor abundance do not affect susceptibility to infection. However, there is a difference in the rate of accumulation of clinical signs. The time to onset of disease is shorter for TGM-PRG-3 than TGM-PRG-1 mice. Thus, receptor dosage affects the rate of appearance of poliomyelitis in these mice.

Characterization of mouse lines transgenic with the human poliovirus receptor gene.

Two mouse lines transgenic with the human poliovirus receptor gene (PVR), TGM-PRG-1 and TGM-PRG-3, were characterized to determine whether transgene copy number and PVR expression levels influence susceptibility to poliovirus. The mouse lines have been bred for more than 10 generations and the transgene was stably transmitted to progeny as determined by Southern blot hybridization and restriction fragment length polymorphism. The transgene copy number is 10 in the TGM-PRG-3 mouse line and one in the TGM-PRG-1 mouse line. Abundance of PVR RNA is on average three-fold higher in TGM-PRG-3 relative to TGM-PRG-1 tissues, and the abundance of the receptor molecule is three-fold higher in TGM-PRG-3 central nervous system tissues compared to TGM-PRG-1 tissues as determined by Western blot analysis. When TGM-PRG-1 and TGM-PRG-3 mice were inoculated intracranially with a neurovirulent type III poliovirus strain, they developed clinical symptoms and CNS lesions characteristic of human poliomyelitis. These results indicate that the PVR gene is expressed as a functional receptor in the CNS of both mouse lines rendering the mice susceptible to poliovirus infection. Even though the two mouse lines have different copy numbers of the transgene and different levels of PVR RNA and protein, they are similar in their susceptibility to poliovirus.

Inactivated poliovirus vaccine protects transgenic poliovirus receptor mice against type 3 poliovirus challenge.

Transgenic (Tg) mice expressing the human poliovirus receptor (PVR) were vaccinated with inactivated poliovirus vaccine (IPV) and evaluated for induced immunity against type 3 poliomyelitis. One injection of monovalent type 3 IPV elicited protective immunity against wild-type poliovirus. In contrast, 2 injections of trivalent IPV were required for protection. Neutralizing antibody response and protection were vaccine dose-dependent. Administration of polio-immune mouse plasma protected unimmunized mice, demonstrating that neutralizing antibody was sufficient for immunity. IPV heated to remove its D antigen component did not induce protection in Tg PVR mice. IPV derived from a wild-type poliovirus strain gave better protection against wild-type viral challenge than IPV derived from an attenuated poliovirus strain. The newly developed Tg PVR mouse-protection test may be useful in evaluating existing IPV potency tests and for attempts to improve formulations of trivalent IPV or combined vaccines for childhood immunization schedules.

Microevolution of type 3 Sabin strain of poliovirus in cell cultures and its implications for oral poliovirus vaccine quality control.

Screening for sequence heterogeneities in Sabin Type 3 strains of attenuated poliovirus demonstrated mutations that consistently accumulate to significant levels following 10 passages in cultures of primary African green monkey kidney (AGMK) cells or continuous cultures of Vero cells. Fourteen newly identified mutations were quantified by mutant analysis by PCR and restriction enzyme cleavage in passages and in batches of commercial vaccines made in AGMK and Vero cells from the Sabin original (SO) seed virus and from a seed virus rederived by RNA plaque purification (RSO or "Pfizer" seed). Nine of the 14 mutations were reproducibly observed in more than one series of passages. Although 5 other mutations were observed in only one set of passages each, their content gradually increased to a high percentage, suggesting that all the mutations that we found accumulated consistently. SO-derived samples accumulated more mutations than did RSO-derived ones, and the number of mutations and the rates of their accumulation were higher in Vero than in AGMK cells. While the rates of accumulation of most mutations were higher when passaging was performed at 37 degrees, a U-->C transition at nucleotide 5832 occurred faster at 34 degrees, the temperature used for vaccine production. Analysis of Type 3 oral poliovirus vaccine (OPV) monopools made by six manufacturers found only 5 of these newly identified mutations in vaccine batches (nucleotides 3956, 4935, 5357, 5788, and 5832). Some of the mutations were found in trace amounts (less than 0.1%) while others were present at up to 1.8% levels. The pattern of these mutations was characteristic for the type of seed virus and the cell substrate but demonstrated no correlation with results of the monkey neurovirulence test. Therefore the only mutation occurring in Type 3 OPV which contributed to neurovirulence in monkeys was the previously described reversion at nucleotide 472. Quantitation of reversion at nucleotide 472 can be utilized for assessment of acceptability of vaccine lots, while other mutations can be used for monitoring the consistency of vaccine production.

Genetic stability and mutant selection in Sabin 2 strain of oral poliovirus vaccine grown under different cell culture conditions.

Mutations that consistently accumulated in the attenuated Sabin 2 strain of poliovirus during propagation in cell cultures were identified by sequence heterogeneity assay and quantified by mutant analysis by PCR and restriction enzyme cleavage (MAPREC). Eight additional sites previously identified in stool isolates were also examined by MAPREC in the virus passages. The pattern of selectable mutations and the rate of their accumulation depended on the type and confluence of the cell culture and the temperature of virus growth. Five unstable genomic sites were identified in Sabin 2 virus passaged 10 times at 34 degrees in African green monkey kidney (AGMK) cells, with the mutations accumulating in the range 1 to 24%. Accumulation of these mutations did not appear to result in a loss of attenuated phenotype since the virus passaged under these conditions passed the monkey neurovirulence test (MNVT). The content of the 481-G revertant known to be related to neurovirulence in monkeys did not increase. Thus, our results suggest that upon growth of Sabin 2 virus in AGMK cells at 34 degrees, the key determinant(s) of attenuation remained stable, and the mutations that occurred did not affect monkey neurovirulence. In virus passaged 10 times at 37 degrees in AGMK cells, 4 unstable genomic sites were identified, in some of them accumulating up to 12% of the mutants. This virus sample severely failed the MNVT. Virus passaged in Vero cells at 34 and 37 degrees accumulated mutants at 7 and 14 genomic sites, respectively, including 481-G in both cases, with almost complete substitution of the original nucleotides at some of the sites. We tested 44 commercial monopools of Type 2 OPV and found out that all of them contained 481-G revertants in the range 0.4-1.1%. An increase in the 481-G revertants in passaged viruses to the level of 4% and above correlated with failure of these samples by the MNVT. Since the pattern of selectable mutations differed in viruses grown in the two cell cultures used in this study, specific mutation profiles should be determined for each cell substrate used for vaccine production to assess manufacturing consistency.

Consistent selection of mutations in the 5'-untranslated region of oral poliovirus vaccine upon passaging in vitro.

We have previously found that upon passaging type 3 oral poliovirus vaccine (OPV) in cell cultures the proportion of revertants at nucleotide 472 rapidly increases [Chumakov et al.: Proceedings of the National Academy of Sciences of the United States of America 88:199-203 1991]. Systematic study on the accumulation of these revertants showed that it was dependent on the multiplicity of infection and the temperature at which virus was grown. Revertants at position 472 of type 3 OPV accumulated faster in vaccines derived from Sabin Original (SO) substrain than from RNA-plaque purified (RSO) substrain. The rate of accumulation of 472-C revertants differed among cell lines and was higher in overgrown cell cultures suggesting that host factors are involved in the selection of mutants. We also found that accumulation of mutants occurred in vitro at position 480 in type 1 and position 481 in type 2 OPV, making the selection for revertants in domain F of the 5'-noncoding region a general phenomenon for all three Sabin strains. Assessment of the abundance of these mutants may be used for evaluation of the quality of OPV lots.

Quantitative aspects of the mutant analysis by PCR and restriction enzyme cleavage (MAPREC).

Quantitation of virus revertants by PCR and restriction enzyme cleavage may give nonlinear results and, in some cases, produce artifacts caused by nucleotide misincorporation and heteroduplex formation, occurring during PCR. Modifications of the procedure allowed us to overcome these problems and develop a highly sensitive and reliable method of mutant quantitation. This procedure can be used to assess the quality of live vaccines and to study heterogeneity of viral and bacterial populations.

Characterization of interleukin-1 production by microglia in culture.

The production of interleukin-1 (IL-1) by cultured neonatal rat microglia was studied using the D10 cell assay. The results show that IL-1 was secreted in response to lipopolysaccharide (LPS) in a dose- and time-dependent fashion. IL-1 production was specific to microglia and was not induced in astrocytes. Indomethacin, which is known to modulate the release of IL-1 from monocytes, had no effect on LPS-stimulated microglia. Aging of the microglia from two weeks to 4 weeks in culture, however, reduced the release of IL-1 in response to LPS. Our data indicate that microglia are a major source of IL-1 and that the release of IL-1 depends on the presence of inflammatory mediators such as LPS and the age of the culture.

Modulation of the effector functions of cytolytic T-lymphocytes with synthetic peptide inhibitors of protein kinases.

The hypothesis was tested that it is possible to influence cellular responses of intact cells using synthetic peptide substrates, pseudosubstrates, and inhibitors of protein kinases. Using cytotoxic T-cells (CTL), we demonstrate here that some basic amino acid-containing synthetic peptide substrates of protein kinases [e.g., of cGMP-dependent protein kinase (peptide PKG-S), synthetic peptide inhibitor of cGMP-dependent protein kinase (peptide PKG-I), and peptide corresponding to the tyrosine phosphorylation site in pp60src (peptide RR-src)] were strongly inhibitory in T-cell receptor (TCR) and T-cell growth factor, interleukin 2 (IL-2)-triggered proliferation of CTL. These peptides also inhibited other cellular responses of CTL. Peptides which contain basic amino acids, but do not have substrate specificity determinants for protein kinase, were not inhibitory. The inhibition with peptides is not due to their toxicity, since no cell death was observed by the trypan blue exclusion test and by lactate dehydrogenase release. Use of the granule exocytosis assay provided opportunities to clarify the mechanism of the peptide action. Tested peptides inhibited not only cell-surface ligand-induced CTL activation, but also affected cell-surface receptor-independent CTL activation (granule exocytosis and gamma-interferon secretion) induced by the synergistic action of the protein kinase C activator (PMA) and ionophore A23187. It was found that minor changes in amino acid composition or amino acid position in the synthetic peptides dramatically change their ability to affect lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormal production of interleukin-1 by microglia from trisomy 16 mice.

The production of interleukin-1 (IL-1) was examined in cultured CNS microglia obtained from trisomy 16 (Ts16) fetal mouse brain, a model system for studies relevant to Down syndrome (DS). When compared to microglia from their normal littermates, Ts16 microglia produced significantly higher levels of IL-1 activity both before and following stimulation with lipopolysaccharide (LPS). IL-1 release was stimulated by alpha/beta interferon (IFN) in the normal but not Ts16 microglial cultures. The overall level of IL-1 production in normal littermates, however, was still less than that seen in Ts16. Thus, microglia from Ts16 mice may function in an inappropriate manner and, if this abnormality occurs in vivo, may have wide ranging effects on a developing nervous system.

Modulation of cytolytic T lymphocyte functions by an inhibitor of serine/threonine phosphatase, okadaic acid. Enhancement of cytolytic T lymphocyte-mediated cytotoxicity.

A specific and potent inhibitor of protein phosphatases 1 and 2A, okadaic acid (OA), and its inactive analog, tetramethyl ether (OA-TME), were tested in the cytotoxicity and granule exocytosis assays of CTL activation. At low concentrations OA enhanced, whereas at higher concentrations OA inhibited, CTL responses. The Ag-specific and retargeted cytotoxicity, granule exocytosis induced by target cell (TC), anti-TCR mAb, or PMA and A23187, and conjugate formation with TC were inhibited by pretreatment of CTL with OA as expected if protein phosphatases and protein dephosphorylation were indeed involved in the TCR-mediated signal transduction and effector responses of CTL. Cytotoxicity and granule exocytosis were unaffected by pretreatment of CTL with OA-TME. The inhibitory effect of OA on the exocytic response of CTL induced by TC and anti-TCR mAb can be dissociated from the inhibition of the response to PMA and A23187, suggesting the involvement of a serine and/or threonine protein phosphatase in the early events of transmembrane signaling. At lower concentrations, OA, but not OA-TME, was able to enhance the Ag-specific cytotoxicity and TC-induced exocytosis from CTL clones. The enhancement of these TCR-mediated responses of CTL was observed only if the activation was induced by the Ag on the TC surface, because OA did not enhance either the anti-TCR mAb-induced exocytosis of granules from the CTL clone or lysis of the Ag-nonbearing TC by CTL in a retargeting assay. The biphasic character of the effects of OA on CTL-TC interactions suggests the existence of at least two functionally distinct phosphatases in CTL. The ability of OA to enhance the Ag-specific response is unique and indicates the presence of an inhibitory phosphoprotein phosphatase that should be considered as a participant in the down-regulation of the cell-cell interactions between CTL and TC. The inhibitory effects of OA on both TC-induced and anti-TCR mAb-triggered CTL responses at higher concentrations point to the importance of yet another phosphatase in the CTL-TC interactions and in the TCR-mediated transmembrane signaling. The use of OA may help to decipher the details of biochemical changes involved in T lymphocyte effector functions.

Extracellular ATP in T-lymphocyte activation: possible role in effector functions.

We hypothesized that cytolytic T lymphocytes (CTL) may utilize extracellular ATP (ATPo) during the effector phase of the CTL-target cell interactions and that CTL could be the source of ATPo. It is demonstrated here that incubation of CTL with activating ligands [Con A or monoclonal antibody (mAb) to the T-cell antigen receptor (TCR)] results in the extracellular Ca2(+)-independent accumulation of the ATPo. The addition of the ATP-degrading enzymes into the mixture of CTL and target cells results in a strong inhibition of the CTL-mediated, TCR-triggered lethal-hit delivery to the target cell. In a parallel control experiment, the employed enzymes did not affect target cell-induced, TCR-triggered exocytosis of granules from CTL. Thus, the removal of ATPo with enzymes does not interfere with the activation of CTL by the target cell but does block lytic events. Cloned helper T lymphocytes also accumulate ATPo after incubation with anti-TCR mAb or Con A, suggesting the possibility that ATPo, which acts in concert with ectoprotein kinases and/or purinergic receptors, may be of general use as a messenger in cellular interactions of T lymphocytes.

Ecto-ATPase activity in cytolytic T-lymphocytes. Protection from the cytolytic effects of extracellular ATP.

Addition of ATP or ATP analog to the incubation media is shown to result in cell death in experiments with different cultured cell lines as evidenced by the results of several independent assays, both in the absence or presence of extracellular Ca2+. Cytolytic T-lymphocyte (CTL) clone itself was not only resistant to cytolytic effects of ATP, but was able to "rescue" antigen-nonbearing 51Cr-labeled cells from lytic effects of extracellular ATP (but not from lytic effects of adenosine 5'-thiotriphosphate) when present during assay. To test whether the resistance of CTL to ATP is due to a high activity of ecto-ATPase, four independent assays of ATPase activity were utilized to demonstrate the presence and relatively high activity of the ecto-ATPase(s) on CTL surface. Studies of substrate specificity of CTL ecto-ATPase suggest that there is more than one nucleoside 5'-triphosphatase on the surface of CTL. The enzyme(s) activity is Ca2+- and Mg2+-dependent and in this respect is similar to recently described hepatic cells ecto-ATPase. We tested effects of known ATP-binding site-specific reagents fluorescein 5'-isothiocyanate (FITC) and 5'-fluorosulfonylbenzoyladenosine (FSBA) to find covalent modification procedures to be used in studies of functional role of ecto-ATPase. FSBA, but not FITC, inhibits lymphocyte ecto-ATPase but addition of ATP together with FSBA protects ecto-ATPase activity. Inactivation of CTL ecto-ATPase by pretreatment with FSBA makes CTL susceptible to lytic effects of extracellular ATP, as was hypothesized for the functional role of this enzyme in CTL.

Concanavalin A induces a cytoskeletal association of T200 molecules in T lymphocytes.

A recent report indicated that T200 molecules interact with elements of the cytoskeleton in BW5147 T lymphoma cells. We have confirmed the cytoskeletal association of T200 by examining nonionic detergent-soluble and detergent-insoluble fractions of murine T cell tumor cell lines, cloned cytotoxic T lymphocyte lines, and thymocytes. Concanavalin A (Con A)-treated and untreated cells were extracted with 0.5% Triton X-100 and the remaining insoluble material was extracted under conditions allowing actin depolymerization. In the absence of Con A treatment, little T200 could be recovered from the depolymerized insoluble fraction. However, in T cells treated with capping concentrations of Con A, a considerable amount of T200 was rendered insoluble in nonionic detergent, and T200 could be recovered from the insoluble fraction by a buffer which dissociates actin polymers. A lesser, but still significant, amount of T200 associated with the detergent-insoluble fraction of thymocytes treated with concentrations of Con A and succinyl Con A, which are mitogenic for T cells. We also found that in T cells treated with mitogenic concentrations of succinyl Con A, more T200 associated with cytoskeleton than did H-2 or LFA-1 molecules. Because T200 is such a predominant molecule on the surface of T cells, such translocations of the molecule may have a major impact on the physiology of the cell, especially if T200 functions as a protein tyrosine phosphatase as recent evidence by others suggests.

Effect of Ly 5 allotype on in vitro immune responses.

We have tested in vitro immune responses of several kinds to determine if Ly 5 allotype influences reactivity of murine splenocytes in processes thought to involve the T200 glycoprotein. Matings were established among C57BL/6J (B6) (Ly 5.1) and C57BL/6-Ly 5.2 (B6-Ly 5.2) congenic mice (both H-2b) to obtain sibling mice segregating for alloalleles of the Ly 5 system. F2 progeny of the three Ly 5 genotypes were tested for antibody-dependent cell-mediated cytotoxicity (ADCC), proliferation in allogeneic mixed lymphocyte culture (MLC), mitogen responsiveness, natural killer (NK) cell activity, and cytotoxic T-lymphocyte (CTL)-mediated cytotoxic activity. We observed that in Ly 5 segregant mice, higher CTL activity was associated with the Ly 5.2 type. No allotype effect was observed in MLC, mitogen responses, and NK cell-mediated cytolysis. In the parental and F1 animals, mice carrying the Ly 5.2 allele had significantly higher ADCC levels, though this effect was not seen in the segregants. Our results indicate that Ly 5 or some closely linked gene or genes influence CTL activity of murine splenocytes in vitro.