Dengue Virions and Antigens in Brain and Serum of Infected Mice

The temporal relationships of the production of infectious dengue-2 virus and its antigens were investigated in intracerebrally infected suckling mice. Infectious virus, a slowly sedimenting noninfectious hemagglutinin (SHA), and a noninfectious soluble complement-fixing antigen (SCF) were found in the brain. Serum contained high concentrations of SCF antigen relative to infectivity when compared to SCF to infectivity ratios in the brain. Degradation of virions by Tween-80 and ether produced two antigens with sedimentation characteristics similar to the noninfectious antigens occurring naturally in infected tissues. However, the virionderived SHA differed from native SHA when examined by electron microscopy and by equilibrium centrifugation in cesium chloride. Virion-derived SCF (as well as the virions and both SHA antigens) was denatured by sodium lauryl sulfate (SLS) and 2-mercaptoethanol (2-ME), whereas native SCF retained its complement-fixing activity. SLS and 2-ME treatment of dengue-1 and dengue-2 sucrose-acetone antigens increased their serotypic specificity. The hemagglutinin present in sucrose-acetone antigens was predominantly native SHA.     

Complement-Fixation Test for Detection of Herpes-like Viruses in Cell Cultures of Burkitt''s Lymphoma

Although biopsies of Burkitt's tumors contained no detectable complement-fixing (CF) antigen or antigens, tumor cell lines contained CF antigen or antigens related to the presence of a herpes-like virus particle.     

RS virus components. I. Isolation and purification.

RS virus was centrifuged in zonal rotor on 55% sucrose cushion. Three layers were collected: light (RS-LL) containing complement-fixing antigen, medium (RS-ML) containing both complement-fixing and virus particle antigens, and heavy (RS-HL) containing antigen associated with the virus particle. RS-LL was chromatographed on Sephadex G-200 column. Two peaks were obtained, containing complement-fixing activity (RS-LL-1 and RS-LL-2). After sonication (20 Hz, 30 min) RS-ML and RS-HL also were chromatographed on Sephadex G-200 column. Two protein peaks were obtained from each layer (RS-ML-1 and RS-ML-2 from medium, and RS-HL-1 and RS-HL-2 from the heavy layer), corresponding to RS virus proteins.     

Biophysical studies of respiratory syncytial virus. I. Density of respiratory syncytial virus and associated complement-fixing antigens in cesium chloride density gradient

Coates, Helen V. (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), Ben R. Forsyth, and R. M. Chanock. Biophysical studies of respiratory syncytial virus. I. Density of respiratory syncytial virus and associated complement-fixing antigens in a cesium chloride density gradient. J. Bacteriol. 91:1263-1269. 1966.-Concentrated fluids from respiratory syncytial (RS) virus-infected tissue cultures (HEp-2 and BEK) were subjected to equilibrium sedimentation in cesium chloride. When two antigenically distinct strains of RS virus (Long and 18537) were tested, approximately 90% of the infectious virus was recovered in a sharp, symmetrical peak with a density of 1.22 to 1.24. In a similar study, unconcentrated virus had a density of 1.25 to 1.27. Two immunologically distinguishable complement-fixing antigens (antigens A and B) were detected at densities of 1.28 to 1.32 and 1.23 to 1.37. In addition, the existence of a third antigen (density of 1.22 to 1.30) was suggested. The possible origin of these antigens is discussed relative to the known properties of RS virus and the other myxoviruses.     

Preparation of La Crosse Virus Hemagglutinating Antigen in BHK-21 Suspension Cell Cultures

Hemagglutinating and complement-fixing antigens of La Crosse virus (California arbovirus group) were produced in serum-free suspension cultures of BHK-21/13S cells. The appearance and production of these antigens were correlated with the titer of infectious virus. No significant differences in antigen titers were produced by varying virus dose 10-fold. Hemagglutinin appeared 6 to 8 hr after inoculation and reached peak titer in 14 to 22 hr. Both beta-propiolactone and Tween 80-ether treatment inactivated infectious virus in the antigens. Unlyophilized antigen was stable at -60, 5 and 24 C for at least 117 days but not for 1 year. Lyophilized antigen was stable for at least a year, however, at -20 and 5 C. Cell culture-produced antigen was more sensitive than brain-produced antigen in detecting hemagglutination inhibition antibody in human sera.     

Characterizion of Mycoplasma pulmonis variants isolated from rabbits. II. Basis for differentiation of antigenic subtypes

The antigen composition of Mycoplasma pulmonis variants was studied by complement-fixation, agar-gel diffusion, and growth-inhibition tests. Two classes of complement-fixing antigens were demonstrated for M. pulmonis strains 47 and 63: (i) cross-related, heat-labile, water-soluble antigens, and (ii) high-titered, subtype-specific, heat-stable, water-soluble antigens. Lipid antigens prepared by organic solvent fractionation were low-titered antigens and showed little specificity. With the aid of agar-gel double-diffusion plates, the subtype-specific antigens were found to be precipitated by trichloroacetic acid and to be stable to periodate, but they were inactivated by pronase. Pronase-stable, periodate-labile precipitating antigens were observed as common components between the two variants. Antisera prepared with boiled antigens were found to be serologically active on gel diffusion but lacked neutralizing ability in growth-inhibition tests. Each of three strains of M. pulmonis (47, 63, ATCC 14267) could be identified as a variant because each strain possessed immunologically distinct heat-stable subtype-specific antigen(s).     

Expression of a New Complement-fixing Antigen reactive with Murine Sarcoma Virus Rat Antiserum in Rat Cells transformed by Polyoma Virus

WE reported accelerated transformation by DNA viruses (SV40 and polyoma) in rat embryo (RE) cells chronically infected with a C-type RNA virus^1,2. Recently we found in RE cells transformed by polyoma virus a new complement-fixing (CF) antigen detectable by rat antisera having broad reactivity with the various intraspecies and interspecies antigens of the RNA tumour viruses^3–8; this antigen, however, was distinct from the murine intraspecies and interspecies group-specific (gs) antigens both immunologically and by virtue of other properties. It is also distinct from the polyoma virion (capsid) and tumour (“T”) antigens.     

Stability of rubella complement-fixing antigens

Various types of rubella complement-fixing (CF) antigen preparations derived from infected BHK-21 cell cultures were examined for stability to certain chemical and physical agents. Antigens studied included: infected culture fluids concentrated 100-fold, ether-treated fluid concentrates, alkaline buffer extracts of infected cells, and large- and small-particle CF antigens separated by Sephadex gel filtration. All preparations were stable at -20 C for months, and were unaffected by three cycles of freezing and thawing. Ether treatment rendered antigens highly susceptible to thermal inactivation at 56 C; untreated antigens were inactivated slowly and showed a persistent fraction of activity even after 2 hr. Large-particle antigen was found to be slightly more susceptible than small-particle antigen to thermal inactivation and ether treatment. CF activity in alkaline buffer extracts was more labile than that in untreated or ether-treated fluid concentrates upon dialysis, Sephadex gel filtration, and sucrose density gradient centrifugation.     

Vertical toxoplasmosis in a murine model. Protection after immunization with antigens of Toxoplasma gondii incorporated into liposomes

Distinct Toxoplasma gondii antigens were entrapped within liposomes and evaluated for their ability to protect Balb/c mice against congenital transmission: soluble tachyzoite antigen (L/STAg), soluble tissue cyst antigen (L/SCAg), soluble tachyzoite plus tissue cyst (L/STCAg) or purified 32kDa antigen of tachyzoite (L/pTAg). Soluble tachyzoite antigen alone in PBS (STAg) or emulsified in Freund's Complete Adjuvant (FCA/STAg) was also evaluated. Dams were inoculated subcutaneously with these antigens 6, 4 and 2 weeks prior to a challenge with four tissue cysts of the P strain of T. gondii orally between 10 and 14 days of pregnancy. Significant diminution differences were observed between the frequency of infected pups born of the dams immunized with the antigens incorporated into liposomes and that of pups born of the dams immunized with antigen emulsified in FCA or non immunized group (p<0.05). There was a significant decrease in the number of pups born dead in the groups L/STAg, L/SCAg and L/pTAg when compared with pups from all other groups (p <0.05). All dams immunized with or without adjuvant showed an antibody response and a proliferation of T-cells. However, no correlation was found between immune response and protection against the challenge.     

Soluble antigens of vaccinia-infected mammalian cells. I. Separation of virus-induced soluble antigens into two classes on the basis of physical characteristics

Cohen, Gary H. (University of Pennsylvania, Philadelphia), and Wesley C. Wilcox. Soluble antigens of vaccinia-infected mammalian cells. I. Separation of virus-induced soluble antigens into two classes on the basis of physical characteristics. J. Bacteriol. 92:676-686. 1966-Infection of mammalian cells with members of the poxvirus group elicits production of a number of virus-induced, soluble antigens. Immunoelectrophoresis and immunodiffusion techniques employing soluble antigen preparations obtained from vaccinia virus-infected KB cells revealed at least seven well-defined immunoprecipitin bands. On the basis of fractionation and subsequent characterization of the soluble antigen mixture by gel filtration, calcium phosphate chromatography, isoelectric precipitation, disc electrophoresis, and ultracentrifugation studies, two distinct classes of virus-induced antigens differing markedly in molecular weight were recognized. A high molecular weight class (200,000 and greater) contained at least three virus-induced antigens; a low molecular weight class (50,000 to 100,000 range) contained at least four immunoprecipitins. Further separation of the antigens within the two groups was accomplished. The two classes were distinguished also by their ability to stimulate synthesis of virus-neutralizing antibody. Antisera prepared against the high molecular weight class proved effective in neutralizing vaccinia virus. In contrast, the low molecular weight antigens showed little, if any, ability to induce formation of neutralizing antibody.     

Preparation of Type 2 Herpes Simplex Virus Complement-Fixing Antigen

Comparable complement-fixing antigens of type 1 and type 2 herpes simplex virus were produced by extraction of infected African green monkey cells with 0.85% NaCl which was buffered at pH 9.0 with 0.05 m glycine-NaOH. The optimal antigen dilutions were higher in titrations against hyperimmune animal sera than in titrations against human sera. Complement-fixing antibody to type 2 herpes antigen was detected in 5 of 17 sera from healthy humans.     

Comparison of Various Methods for Preparation of Viral Serological Antigens from Infected Cell Cultures

In efforts to prepare more potent and sensitive viral serological antigens, several aspects of the production of antigens from infected cell cultures were studied. Antigens derived from whole, infected culture material and from the cellular and fluid phases were compared. Freezing and thawing, sonication, and alkaline buffer extraction were compared for effectiveness in releasing antigen from host cells. The effect of the multiplicity of infection on titers of viral antigens produced in cell cultures was studied. Generally, higher titered antigens were derived from the infected cells than from the culture fluids, but for certain viruses complement-fixing (CF) antigens derived from the culture fluids gave higher antibody titers than did cell-associated antigens. With each virus-host cell system studied, treatment with alkaline buffers extracted appreciable amounts of CF antigen from the host cells, but in some instances more antigen was released by freezing and thawing or by sonication. Extraction of infected cells with alkaline buffers was not a satisfactory method for preparation of hemagglutinating (HA) antigens for any of the viruses studied. The highest-titered HA antigens were produced from infected cells disrupted by freezing and thawing or sonication. The highest titered CF and HA antigens were produced from cell cultures infected at multiplicities of one or greater. Complement-fixing antigens produced by infecting cells in suspension and then planting had lower titers than antigens produced in parallel by infecting developed monolayers. Optimal methods are summarized for preparation of serological antigens to a variety of viruses of man.     

Characterization of Epstein-Barr virus antigens. I. Biochemical analysis of the complement-fixing soluble antigen and relationship with Epstein-Barr virus-associated nuclear antigen.

The Epstein-Barr virus-soluble (S) antigen extracted from RAJI cells was characterized by sucrose gradient centrifugation, gel filtration, and ion-exchange chromatography. The sedimentation coefficient was estimated to be 8.5S corresponding to a molecular weight of 180,000. The S antigen binds to DEAE-A25 ion exchanger from which it can be eluted with 0.3 M NaCl in Tris buffer (pH 7.2). All fractions which contained complement-fixing S antigen also inhibited the anticomplement immunofluorescence reaction as used to detect the Epstein-Barr virus-associated nuclear antigen. These results are consistent with the hypothesis that the S and Epstein-Barr virus-associated nuclear antigens are either a single antigen or that both activities are present on the same molecule.     

Immunological studies with an m-deficient histoplasmin skin-test antigen

Previous studies have shown that a single skin test to histoplasmin may induce complement-fixing antibodies or M precipitins (or both) to histoplasmin in histoplasmin-sensitive, but serologically negative, individuals. Ideally a skin-test antigen should be one which detects hypersensitivity without stimulating humoral antibodies. Histoplasmin skin-test antigens presently used contain both H and M antigens. The present study was undertaken to evaluate an histoplasmin skin-test antigen deficient in the M component but containing the H antigen. Thirty histoplasmin-hypersensitive subjects were bled prior to administration of the experimental skin-test antigen and at various time intervals thereafter. Only six of the thirty hypersensitive subjects showed serological responses. The sera of the six, however, only showed weak precipitin reactions, five showed M bands, and only one showed an M and an H band. None showed complement-fixation titers with either the yeast or mycelial antigens of Histoplasma capsulatum. Our data suggest that the use of a skin-test antigen purified to contain only H component would detect histoplasmin hypersensitivity without inducing antibodies and would eliminate false-positive serological reactions caused by the M component.     

The skin-reactive antigens of Mycoplasma pneumoniae.

Guinea pigs experimentally infected with Mycoplasma pneumoniae or immunized with the orgnaism in combination with Freund's complete adjuvant developed a delayed hypersensitive skin reaction following on intradermal injection of the M. pneumoniae antigen. The amount of protein necessary to produce the delayed skin reaction was as low as 0.01 mug. When the sonicated whole cells were extracted with aqueous acetone, the delayed skin reactivity was found mostly in the acetone insoluble (lipid-depleted) fraction. On the other hand, the lipid fraction which was isolated by a chloroform-methanol extraction of the acetone-soluble fraction and had a high titer of complement-fixing activity, exhibited little delayed skin reactivity. The lipid-depleted antigens as the whole cell antigens produced delayed skin reactivities in human patients.     

Developmental Sequence and Intracellular Sites of Synthesis of Three Structural Protein Antigens of Influenza A2 Virus

Specific antisera for hemagglutinin (HA) and neuraminidase antigens of influenza A(2) virus (A(2)E) were produced through the segregation of the two proteins in reciprocal viral recombinants of A(2)E and A(0)e viruses. Gamma globulin fractions of these specific antisera and of antiserum specific for the nucleoprotein (NP) antigen of A(0)e virus were conjugated with fluorescein isothiocyanate and employed to follow the synthesis of the three structural proteins in clone 1-5C-4 human aneuploid cells, with parallel measurement of serological and biological activity of the antigens by other techniques. In this system, NP antigen appeared first (at 3 hr) in the cell nucleus, whereas HA and neuraminidase appeared coincidentally, at 4 hr after infection, in the cytoplasm. The initial detectability of biological or complement-fixing activity of the proteins coincided with their demonstrability as stainable antigens. Late in infection, all three antigens were detected at the cell surface. Antibody specific for HA partially blocked the intracellular staining of neuraminidase and inhibited the enzymatic activity of both extracted and intact extracellular virus. These observations suggest the close intracytoplasmic proximity of the two envelope antigens and perhaps their initial association in a larger protein.     

Proteins of Vesicular Stomatitis Virus: II. Immunological Comparisons of Viral Antigens

The antigens of the nucleoprotein core and the coat of vesicular stomatitis virus (VSV) particles of the Indiana serotype were prepared and purified by sucrose gradient fractionation. Antibody was prepared separately to each of the two antigen fractions. By immunological procedures, it was shown that soluble antigens of VSV preparations sedimenting at 20S and in the leading edge of the 6S region are antigenically related to VP3, the protein of the virus core, whereas the 6S soluble antigen cross-reacts only with viral coat antibodies. These results confirm previous results obtained by polyacrylamide gel analysis of the antigens. It has further been demonstrated that the 6S antigen is a glycoprotein. Comparing antigens of the New Jersey and Indiana serotype showed that the coat antigens of virus particles and the 6S antigen are immunologically distinct in the two serotypes. In complement-fixation tests, the core antigens and the soluble 20S antigens from one serotype showed a cross-reaction with antiserum prepared against core proteins of the other serotype.     

Immunogenic complexes of the soluble surface antigens of Sh. sonnei]

The comparative study of the immunogenic properties of Sh. sonnei (phases I and II) soluble surface antigens obtained by the modified method of aqueous-saline extraction and Sh. sonnei (phase I) antigen obtained by Boivin's method was made with the use of the keratoconjunctival test in guinea pigs. The protective activity of a high molecular fraction obtained by the fractionation of phase I soluble surface antigens in Sepharose 4B was studied. Boivin's antigen, when used for immunization in optimum doses, was found to have pronounced protective properties, whereas phase II soluble surface antigens showed no protective activity. A high molecular fraction obtained from phase I soluble surface antigen was found to be the most immunogenic. Protective activity was largely connected with protein antigen. The question whether protein antigen was an independent protective antigen or whether it constituted a part of a complex which determined the protective activity of a high molecular fraction remained unsolved.     

Origin and Structure of the Group-Specific, Complement-Fixing Antigen of Rickettsia rickettsii

Rickettsia rickettsii was treated with ether and examined by negative-contrast electron microscopy. Group-specific complement-fixing antigen was seen to be originating from the cell wall. The antigen was composed predominately of round particles 10 to 60 nm in diameter. Intact R. rickettsii and antigen from ether-treated organisms were purified by density gradient centrifugation and analyzed by polyacrylamide gel electrophoresis. The whole rickettsial cell was composed of a minimum of 30 proteins which ranged in molecular weight from about 23,000 to 155,000. The "soluble" antigen contained nine proteins ranging in molecular weight from about 28,000 to 150,000.     

Biophysical Comparison of Two Canine Adenoviruses

The two canine adenoviruses, infectious canine hepatitis (ICH) virus and infectious canine laryngotracheitis (ICL) virus (also designated as Toronto A26/61 virus), were studied with respect to their morphology and the biological properties of their soluble components. The two viruses were found to be composed of soluble components similar to, and carrying biological activities corresponding to, those of the human adenoviruses after fractionation by rate zonal centrifugation and anion-exchange chromatography. The following soluble components were identified: hexons (carrying a common group-specific complement-fixing antigen), penton oligomers (complete soluble hemagglutinin), and penton and fiber monomers (incomplete soluble hemagglutinins). The latter were indicated by hemagglutination enhancement with selected antisera directed against human adenovirus soluble components. Elution characteristics of corresponding components of the two viruses in anion-exchange chromatography experiments were distinctly different. Electron microscopic examination of purified virions and soluble components revealed the fiber component of ICL virus to be 35 to 37 nm in length, and that of ICH virus to be 25 to 27 nm long.