H. Závadová

Absence of Cytomegalovirus DNA from Adenocarcinoma of the Colon

Biopsy specimens from 7 patients with adenocarcinoma of the colon and from 1 patient with Crohn’s disease were tested for the presence of cytomegalovirus (CMV) DNA. Using a reassociation kinetics test with an estimated sensitivity of 0.3 genome equivalents per cell, we failed to detect CMV DNA in any of the 8 specimens.

Hepatitis B virus proteins expressed by recombinant vaccinia viruses: influence of preS2 sequence on expression surface and nucleocapsid proteins in human diploid cells

SummaryFifteen vaccinia virus (VV) recombinants derived from VV strains Praha, LIVP and DD (i.e. Dryvax Wyeth vaccine - derived) and expressing genes for S, preS2-S or c antigens of hepatitis B virus (HBV) were tested in monkey CV-1 cells and human diploid LEP cells. The production of infectious virus was found to be alike in all the recombinants and parental viruses as well. However, several recombinants produced markedly lesser amounts of S and preS2 antigens in LEP cells than in CV-1 cells. This reduction was independent of the parental virus used. There was, however, a relationship between the production of preS2 in CV-1 cells and the production of S and preS2 antigens in LEP cells; in general, recombinants efficiently inducing preS2 antigen formation in CV-1 cells produced markedly reduced amounts of S and preS2 antigens in LEP cells. Reduction of HBV antigen production in LEP cells was not apparent in recombinants expressing only S or c antigens of HBV, and the production of c antigen by double recombinants was not influenced by simultaneous expression of preS2 and S. The various recombinants also differed in the ratio of S:preS2 antigen formation. This difference seemed to be associated with the length of the untranslated leader sequence preceding preS2 but not with the parental virus or cell type used. The titers of antibodies against S and preS2 antigens induced in mice immunized with different recombinants differed markedly. The differences in the ratio of S:preS2 antigen production in vitro were not reflected in vivo by S:preS2 antibody ratio.

A recombinant vaccinia virus expressing hepatitis B virus middle surface protein : restricted expression of HBV antigens in human diploid cells

SummarySeveral vaccinia virus recombinants inducing the synthesis of the middle surface (M) protein of hepatitis B virus (HBV) were constructed. One of them, denoted vl37, was examined in some detail. The virus replicated nearly to the same extent in various cell lines, viz. human embryo diploid fibroblast LEP and MRC-5 cells, rabbit embryo fibroblast REF cells, TK− rat RAT-2 cells, and green monkey CV-1 cells. However, the production of M protein was found considerably lower in the human LEP and MRC-5 than in the other cells examined. In addition, the kinetics of M formation were different in these two cell systems, LEP cells lagging significantly behind CV-1 cells. The low-level production of M protein in LEP cells was not increased by repeated vl37 passages in LEP cells, nor by a passage in a laboratory worker accidentally infected with the vl37 virus, nor by shortening the leader sequence preceding the translation initiation codon. The greater part of the M antigen was found to be cell associated, more so in the cells of human than monkey origin. From the major HBV S antigen (HBsAg) isolated from the plasma of chronically infected subjects, the antigen released by cell destruction differed by binding to polymerized human albumin. This property was utilized in ELISA to detect anti-preS2 antibody. Rabbits inoculated intradermally with the vl37 virus developed antibodies reactive in this assay as well as with a synthetic peptide corresponding in the amino acids 14–34 of the NH2terminus of the HBsAg preS2 region.

Priming effect of recombinant vaccinia virus coding for the middle hepatitis B surface antigen

SummaryRecombinant vaccinia virus expressing the middle hepatitis B virus surface antigen was incapable of inducing marked antibody response against the S and pre-S2 antigenic specifities in mice. However, mice immunized with this virus produced antibodies to both these antigens after the following administration of subtreshold doses of plasmatic hepatitis B surface antigen.

Preparation of antisera against the S antigen of influenza A virus by immunization of guinea pigs with internal S antigen.

Internal S-antigens obtained by ether destruction of different influenza A viruses were administered intraperitoneally to guinea pigs at three day intervals. Three to five doses evoked S-antibodies in the course of the second and third week after the beginning of immunization; at that time the sera were free of V-antibodies. In a minor proportion of animals (19 out of 94) V-antibodies were detected later, when S-antibodies were starting to decrease. The preliminary evidence obtained indicated that the development of V-antibodies in these sera was independant of the level of S-antibodies as well as of the number of S-antigen doses administered. The S-antisera prepared in the way described were found to be potent enough to be used in the fluorescent antibody technique.

Sensitivity to 1-adamantanamine as a marker in genetic studies with influenza viruses

Abstract The sensitivity of several influenza viruses to 1-adamantanamine (1-Adam) was studied using the plaque inhibition test in rhesus monkey kidney cells. Of the viruses tested, A2/Singapore-MK and A/WS-MK were found to be sensitive while A/NWS-D was resistant to the agent. Viruses differing in 1-Adam sensitivity and some other properties were used in recombination experiments. The results revealed that the resistance to 1-Adam could be transferred from one influenza virus to another. It was not possible to link sensitivity to 1-Adam with any other of the virus properties tested.

Influence of cis-Dichlorodiammineplatinum (II) on Growth of SV 40 Virus in Green Monkey Kidney Cells

Cis-dichlorodiammineplatinum (II) partially inhibits the replication of SV 40 virus in green monkey kidney cells. The results of both immunofluorescence and complement-fixation tests indicated that the production of the T antigen of SV 40 was unaltered, but V antigen production was suppressed. The partial block of V antigen synthesis was most marked in the early period after infection, subsequently it was less pronounced. Both infectious virus production and V antigen formation were most markedly inhibited in cells treated with DDP both before and after infection.

Synthesis and immunogenicity of hepatitis B virus envelope antigen expressed by recombinant vaccinia virus. Finding of retention signal in the C-terminal portion of the preS1 domain of subtype adyw

SummaryFive different recombinant vaccinia viruses expressing the envelope antigen of hepatitis B virus (HBsAg) under the control of the P7·5 promoter were constructed. Cell cultures infected with some of the recombinant viruses synthesized both middle (M) and major surface (S) protein of HBsAg. It was shown that the length of the nontranslated sequence preceding preS2-ATG influenced the extracellular or intracellular HBV antigen distribution and the preS2:S antigen ratio. Some recombinants synthesized an M protein that was enlarged by additional 35 amino acids of preS1 domain and was entirely retained within the infected cells. Antibody responses to the S and preS2 antigens in mice revealed significant differences in the immunogenicity of individual recombinants.

A vesicular stomatitis virus (cytomegalovirus) pseudotype and its use in neutralization tests.

Infection with vesicular stomatitis virus (VSV) of human diploid cells preinfected with the AD-169 strain of human cytomegalovirus (CMV) resulted in the formation of a VSV (CMV) pseudotype. Its formation was favored by increasing the bicarbonate content in doubly-infected cultures. The pseudotype was capable of infecting not only human but also rabbit cells. Pseudotype particles formed after infection with the tl 17 mutant of VSV, which carries a thermolabile lesion in its neutralization antigen, were more stable at 45 degrees than the original tl 17 virus. The pseudotype was used in the neutralization test with human sera. All sera positive for CMV antibody in the complement-fixation (CF) test were also reactive in the neutralization test. In addition, numerous sera negative for CMV antibody in the CF test neutralized the pseudotype.

Clinical reactivity and immuno-genicity of hemagglutinin influenza vaccine. II. Clinical reactions, hemagglutination-inhibiting and strain and type-specific complement-fixing antibody responses in infants.

The reactivity and immunogenicity of a polyvalent hemagglutinin influenza vaccine was studied in subjects aged 5–7 months. The vaccine was administered in three doses either subcutaneously (0.5 ml per dose) or intradermally (0.1 ml per dose) over a period of 6–7 months. Clinical reactions, both local and febrile, even after administering two or three doses, were quite mild. After two 0.5 ml doses of vaccine administered subcutaneously, most subjects possessed hemagglutination-inhibition (HI) antibodies to all the six vaccine viruses. In most subjects the antibodies dropped to und etectable levels in the course of four to five months. A booster effect was observed after the third vaccine dose. Intradermal administration of the vaccine (0.1 ml) was much less efficient. Even three doses of vaccine did not induce antibody development in most subjects. The antibody response in the complement-fixation (CF) test using strain-specific V antigens corresponded, in general, with the results of the HI test; however the CF antibodies to the A 2/Taiwan and B/Md viruses were found much less frequently than the corresponding HI antibody. Most of the infants vaccinated subcutaneously also developed antibodies reactive in the CF test with the soluble (S) antigens of influenza A and B. They were much less frequent in subjects immunized intradermally.