Murine monoclonal antibodies were produced which coimmunoprecipitated, under reducing conditions, 130,000and 55,000-dalton (Da) polypeptides from cells infected with human cytomegalovirus (CMV) strain AD169. A 92,000-Da species, possibly a biosynthetic intermediate, was also detectable. One of the monoclonal antibodies, 15D8, neutralized CMV AD169 only in the presence of guinea pig complement. A second monoclonal antibody, 14E10, coimmunoprecipitated the 130,000and 55,000-Da polypeptides but did not neutralize viral infectivity. By sequential immunoprecipitation, both monoclonal antibodies have been shown to recognize the same polypeptides. Monoclonal antibody 15D8 detected the 130,000-and 55,000-Da polypeptides in five of six clinical strains and three laboratory strains tested. The 14E10 monoclonal antibody detected the 130,000-Da protein in four of six CMV clinical isolates and in strain AD169 but did not immunoprecipitate any polypeptides from extracts of cells infected with either Towne or Davis laboratory strains. In kinetic studies, the synthesis of the 130,000-Da polypeptide preceded the appearance of the 55,000-Da polypeptide. In infected cells radiolabeled with a pulse of L-[35SImethionine, the isotope was initially detected in the 130,000-Da polypeptide but could be chased into the 55,000-Da polypeptide. These polypeptides exist in the intracellular and extracellular virus as disulfide-linked multimers. Extracellular virus contained a high-molecular-weight (greater than 200,000 Da) multimer composed entirely of 55,000-Da polypeptides. In extracts from infected cells an additional high-molecular-weight multimer was detected consisting of disulfidelinked 130,000-Da polypeptides.

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Studies were performed to define the nature of the Toxoplasma gondii antigens that are recognized by human immunoglobulin M (IgM) and IgG antibodies. Both IgM and IgG antibodies were found to be directed mainly against T. gondii membrane antigens in sera obtained from patients with acute toxoplasmosis. Treatment of the membrane preparation with DNase, RNase, or lipase had no apparent effect on the reactivity of the membrane antigens with IgM and IgG antibodies. Lipids isolated from tachyzoites were not recognized by either IgM or IgG antibodies. Exposure of T. gondii membranes to heat, proteolysis, or oxidation with sodium periodate decreased the reactivity of the membrane preparations with both IgM and IgG antibodies. A preparation of T. gondii proteins and polysaccharides were recognized by both immunoglobulin classes. T. gondii polysaccharides reacted with human IgG antibodies produced during both the acute and chronic phases of the infection. We concluded that, after infection with T. gondii, IgM and IgG antibodies are elicited in response to both protein and carbohydrate constituents of the invading parasite.

The inhibitory action of 9-(1,3-dihydroxy-2-propoxymethyl)guanine on the replication of human cytomegalovirus was studied. Three laboratory strains (AD-169, Towne, and Davis) and three early passage (<10) clinical isolates were all inhibited in yield inhibition assays. In cultures infected with AD-169, virus yields could be inhibited if the drug was added as late as 3 days after the replication cycle had begun. The effects of the drug were' fully reversible during the first 4 days of the viral replication cycle. Viral infectivity and viral DNA synthesis were reduced more than viral protein synthesis. Synergistic antiviral effects were observed with betacysteine, and to a lesser extent, with beta-serine recombinant interferons, but only over a narrow range of dose combinations.The novel guanosine analog, 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), is active in vitro against human cytomegalovirus (CMV) (1, 13, 24). Acycloguanosine and other nucleoside analogs have limited activity against human CMV (3,5,12,16,19,23 various concentrations was then added to the tissue cultures. When a multiplicity of infection (MOI) of 0.1 was used, the virus was allow'ed to replicate for 5 days; replication was continued for multiple cycles (10 to 12 days) if the MOI was <0.1. Infected cultures were harvested by scraping the cells into the supernatant, pooling duplicate wells, sonicating to disrupt the cells, and then freezing the cells at -70°C. For some experiments, the supernatant and intracellular virus were harvested separately. Samples were assayed for PFU of virus on HEL cells (21). Inhibition was determined by comparing the yields from drug-treated virus-infected cultures with those from untreated virus-infected cultures and was expressed as a percentage of control yields. For plaque reduction assays, the drug was incorporated into the semisolid overlay. For experiments in which the effects of IFNs and DHPG in combination were tested, cells were first treated overnight with IFN and then infected with CMV. Both drugs were included in the maintenance medium, either DMEM or DMEM with 1% agarose, during virus replication. To evaluate drug interactions, we calculated a combination index (CI) by the formula derived by Spector et al. (28): CI = ln(A) + ln(B) -ln(AB) -ln(VC), where A and B are the mean virus yields in the presence of antiviral agents A and B, respectively, AB is the virus yield in cultures treated with both agents, and VC is the virus yield in untreated controls. The CI ± standard error (SE) was calculated for each drug combination with data from independent experiments. When the CI was within 2 SEs of 0, the combined drug effects were additive. When the CI was greater than ±2 SE, the effects were synergistic; when the CI was less than ±2 SE, the effects were antagonistic. For plaque reduction assays, the drug interactions were assessed by the formula [(A)(B)]i [(AB)(VC)]. When the numerical result was >1, the effects were synergistic; when the numerical result was equal to 1, the effects were additive; and wh...

Western Blot analysis revealed that both IgM and IgG antibodies present in the sera of humans infected with Toxoplasma gondii recognize three major antigens with apparent m.w. of 32,000, 22,000, and 6000, respectively. In addition, IgG antibodies recognized at least 17 other antigenic components. After subcellular fractionation, enrichment of the three major antigens recognized by IgM and IgG antibodies by the membrane fraction was observed. Solubilization of membrane-enriched preparations with a mixture of sodium dodecyl sulfate and sodium deoxycholate did not reveal any new antigenic structures that reacted with IgM or IgG antibodies. Treatment of Toxoplasma lysate preparations and various fractions obtained after differential centrifugation with NaIO4 diminished the reactivity of the antigens with both IgM and IgG antibodies. Lipase treatment had no effect on the number or nature of antigens recognized by IgM antibody. Treatment with pronase and trypsin eliminated the 32,000 and 22,000 m.w. antigenic components detected by IgM antibodies, whereas such treatment had no effect on the 6000 m.w. component. Periodic acid-Schiff staining of polyacrylamide gels of Toxoplasma sonicates revealed the presence of three components corresponding to m.w. of 62,000, 45,000, and 6000, respectively. At least 15 components, including the 6000 m.w. component, directly bound concanavalin A.