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.

Isolation of a biologically active soluble human interferon-alpha receptor-GST fusion protein expressed in Escherichia coli.

The cDNA encoding the extracellular domain of the human interferon-alpha (IFN-alpha) receptor (Uzé, G., Lutfalla, G., and Gresser, I. Cell 1990;60:225-234) lacking the signal peptide has been expressed in Escherichia coli as a fusion protein with glutathione S-transferase. The fusion protein represented 12% of total bacterial proteins and was found exclusively within cytoplasmic inclusion bodies. Inclusion body material was completely solubilized by 8 M urea; 20% solubilization was achieved by cell lysis in the presence of 0.45% cholamidopropyl dimethylammoniol-propane sulfonate and 1% Triton X-100. The soluble fusion protein was purified by gel filtration and affinity chromatography. Overall recovery of affinity purified fusion protein was approximately 100-200 micrograms/liter of cell culture. The affinity purified and refolded fusion protein exhibited the expected amino terminal sequence and M(r) of 68,000 on reduced sodium dodecylsulfate gel electrophoresis. The protein reacted with antibodies specific for the cloned IFN-alpha receptor and inhibited the antiviral and antiproliferative activities of recombinant IFN-alpha B. We have demonstrated that the fusion protein binds to IFN-alpha B and competes with the cell surface receptor for binding to this IFN-alpha species.

Purification and characterization of multiple components of human lymphoblastoid interferon-alpha.

Twenty-two components of human interferon-alpha (IFN-alpha) derived from Sendai virus-induced Namalwa cells were purified by sequential immunoadsorbent affinity chromatography using four monoclonal antibody affinity columns followed by ultrafiltration and reversed-phase high-performance liquid chromatography. The specific activity ranged from 0.2 to 2.6 x 10(8) IU/mg protein on Madin-Darby bovine kidney cells, 0.3 to 4.6 x 10(8) IU/mg protein on human WISH cells, and 10(4) to 7 x 10(5) units/mg protein on mouse L929 cells. The apparent molecular weights of the components ranged from 17,500 to 23,300 using nonreducing sodium dodecyl polyacrylamide-gel electrophoresis and 17,500 to 27,600 using reducing sodium dodecyl polyacrylamide-gel electrophoresis. The amino-terminal amino acid sequences were similar among the components as well as to those reported for the cloned human IFN-alpha genes (Pestka, S. (1986) Methods Enzymol. 119, 3-14). However, four components, f, i, l, and m, have amino-terminal amino acid sequences which appear to be unique when compared to those predicted from the cDNA clones. One component, pre-a, has a potential N-linked glycosylation site on the Asn of residues 2 through 4, Asn-Leu-Ser.

Regulation of interferon production by human monocytes: requirements for priming for lipopolysaccharide-induced production.

Macrophages are uniquely responsive to bacterial lipopolysaccharide (LPS) for activation of a number of host defense functions and production of bioactive mediators. One potentially important mediator produced by LPS-stimulated macrophages is interferon (IFN-alpha/beta). In contrast to murine observations, we have observed that freshly isolated human monocytes, purified by counter-current centrifugal elutriation, do not produce interferon in response to LPS. This is not due to a lack of response to LPS, as assessed by the induction of other monokines, or to an incapacity for IFN production, since IFN was inducible by poly-I,C treatment of monocytes in the absence of any other exogenous stimulus. However, human monocytes can be primed for the production of IFN in response to LPS if they are cultured in the presence of either granulocyte-macrophage colony stimulating factor (GM-CSF) or interferon-gamma (IFN-gamma). The IFN secreted is of the alpha subtype. Monocytes primed with GM-CSF or IFN-gamma also maintained LPS responses for production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1). M-CSF did not prime monocytes for LPS-induced IFN production, although it did enhance production of TNF-alpha and promoted monocyte survival. Northern analysis indicated that the induction of IFN-alpha by LPS was regulated primarily at the mRNA level. The highly regulated production of IFN-alpha by monocytes/macrophages has important implications for autocrine action of interferons in the activation and differentiation of these cells.

Antibodies to interferon-alpha 2 in patients treated with interferon-alpha 2 for hairy cell leukemia.

Some patients treated with interferon (IFN) have developed antibodies (ABs) to that IFN. We examined the incidence of such ABs in hairy cell leukemia (HCL) patients treated with IFN-alpha 2a and IFN-alpha 2b. In the initial enzyme-linked immunosorbent assay (ELISA) assays, the serum samples were tested against their corresponding IFNs. Of 73 evaluable patients treated with IFN-alpha 2b, 6 patients, who tested negative prior to treatment, were positive by ELISA following treatment with IFN. Two of the samples tested positive in the neutralization assay. Regarding the samples from patients treated with IFN-alpha 2a, 2 of the 44 patients were ELISA positive following IFN treatment (while being negative prior to treatment) and 2 others became positive in the neutralization assay following IFN treatment. Five serum samples with neutralizing activity were tested for their ability to bind to each of eight natural IFN-alpha species derived from human lymphoblastoid cells induced with Sendai virus; a different cross-reactivity profile was seen for each sample. In conclusion, a low incidence of neutralizing and nonneutralizing ABs against IFN-alpha 2 was observed in patients with HCL treated with recombinant DNA-derived IFN-alpha 2a or IFN-alpha 2b. Finally, ABs against these IFNs cross-react by ELISA with several naturally occurring IFN-alpha s.

Amino terminus is essential to the structural integrity of recombinant human interferon-gamma.

Species lacking either 8 or 10 residues at the amino terminus of recombinant human interferon-gamma (Hu-IFN-gamma) were generated by limited digestion with Staphylococcus aureus V8 protease. A crude digest, consisting predominantly of these species, were completely inactive in inducing antiviral activity and the expression of HLA-DR antigens on HL-60 cells. The NH2-terminal deletion fragments were separated from residual intact IFN-gamma and from smaller polypeptides by reverse phase high performance liquid chromatography (HPLC) at pH 2.2. Intact IFN-gamma, purified by HPLC and subsequently refolded by dilution in 0.1 M sodium phosphate buffer (pH 7.5, 0.1% bovine serum albumin) was similar to untreated IFN-gamma in terms of binding to its cell surface receptor and in inducing antiviral activity and the expression of HLA-DR molecules. Conversely, biological activity was not detected in purified fragments 8-139 and 10-139. Examination of fragments 8-139 and 10-139 by far-UV circular dichroism revealed that cleavage of 8-10 residues at the amino terminus accompanied a dramatic change in secondary structure (6% alpha-helical and 36% beta-sheet content) as compared to untreated or HPLC-purified IFN-gamma (66% alpha-helix and 0% beta-sheet content). In summary, these results indicate that the amino terminus contributes to the structural integrity of the IFN-gamma molecule.

Products of stimulated monocytes enhance the activity of interferon-gamma.

We have investigated the interaction of interferon-gamma (IFN-gamma) with monocytes or products of stimulated monocytes. We have shown that IFN-gamma does not stimulate IFN-alpha production in monocytes. Staphylococcus aureus (SAC) but not lipopolysaccharide (LPS) induced IFN-alpha secretion by monocytes. However, it was observed that supernatants of monocytes stimulated with IFN-gamma in combination with either LPS or SAC had higher levels of antiviral activity than supernatants of monocytes stimulated only with IFN-gamma. Moreover, the degree of enhancement of antiviral activity was dependent on the dose of either LPS or SAC used to stimulate the monocytes. Supernatants of monocytes stimulated with LPS or SAC enhanced the antiviral activity of IFN-gamma but not IFN-alpha. Thus, LPS- or SAC-stimulated monocytes produced a factor(s) that augmented the biological activity of IFN-gamma. To identify the factor within stimulated monocyte supernatants that was responsible for this enhancement, several monokines were added to IFN-gamma. Tumor necrosis factor (TNF) significantly increased the antiviral activity of IFN-gamma, although TNF by itself had no antiviral activity. Interleukin 1 (IL-1) or granulocyte-monocyte colony-stimulating factor (GM-CSF) did not enhance the activity of IFN-gamma. Our data indicate that the interaction between IFN-gamma and monocytes is bidirectional. Not only can IFN-gamma activate monocytes, but products of stimulated monocytes also enhance the biological activities of IFN-gamma.

Selective defects in cytomegalovirus- and mitogen-induced lymphocyte proliferation and interferon release in patients with acquired immunodeficiency syndrome.

To examine the defect in cellular immunity in patients with acquired immunodeficiency syndrome (AIDS), we studied in vitro lymphocyte proliferation and interferon (IFN) release in response to cytomegalovirus (CMV) antigen and Concanavalin A mitogen in 40 homosexual men with AIDS, 10 homosexual men with chronic lymphadenopathy syndrome, 7 healthy homosexual men, and 18 healthy heterosexual subjects of either sex. CMV serology by an enzyme-linked immunosorbent assay and viral cultures for CMV were performed. Lymphocytes of patients with AIDS showed impaired CMV-specific release of IFN but normal mitogen-induced IFN release. The defect was not attributable to CMV infection per se. Cell proliferation in response to both CMV antigen and mitogen was impaired in patients with AIDS who had opportunistic infections. The defect could not be attributed to CMV viremia. We concluded that impaired release of IFN in response to a viral antigen is characteristic of lymphocytes in patients with AIDS and that this defect is distinct from a defect in mitogenic responsiveness, which coexists predominantly in patients with opportunistic infections.

Poliovirus and polio antibody assay in HEp-2 and Vero cell cultures.

HEp-2 cell cultures were about three to 30 times more sensitive for poliovirus titration than Vero cells. Attenuated strains induced a complete cytopathic effect in HEp-2 but not in Vero cells. For polio antibody titration, HEp-2 and Vero cells were equally suitable. A high degree of sensitivity and reproducibility of virus neutralization was achieved in tests utilizing a low virus dose and serum-virus incubation overnight at 36 degrees C. Staining of infected trays with crystal violet obviated reading of viral CPE under the microscope and expedited the evaluation of larger-scale tests.