Cytogenetic evaluation of human endothelial cell cultures.

Cytogenetic evaluation of serially subcultivated human endothelial cells revealed significant differences between cultures derived from fetal umbilical cords and cultures derived from various vessel sites in adults. A rapid increase in the prevalence of polyploid cells, to levels of 100% in many cases, was detected in human umbilical vein endothelial cell cultures but not in endothelial cell cultures from adult vessels. Because the development of polyploidy has been viewed as one signpost of in vitro senescence, it may be that these in vitro observations of high levels of polyploidy are a reflection of the fact that umbilical tissue is at the end of its in vivo developmental lifespan when studied. Consistent karyotypic alterations also were observed in two clones from adult human abdominal aorta, even though these cultures exhibited low percentages of polyploid cells. Cultures of one clone exhibited a trisomy of chromosome 11, on which there are at least three onc gene loci, and a deletion of chromosome 13 through band q14. A loss of band 13q14 is a prezygotic chromosomal lesion known to predispose to retinoblastoma. In the other clone, two cell populations were observed, and each displayed a chromosomal abnormality. A trisomy of the long arm of chromosome 2 was noted in one cell population via a marker chromosome involving 2 and 14. The other cell population exhibited an abnormality of chromosome 2. Neither of these karyotypic alterations was detected in the parent culture from which the clones were derived. The results reported in this study have both practical and theoretical implications. The high incidence of polyploidy in serially cultivated umbilical cultures as well as the occurrence of chromosomal changes in umbilical and aortic cultures testify to the need for cytogenetic monitoring of cell cultures even though they are derived from presumably normal tissue. Cytogenetic changes in the endothelium may be important in atherogenesis and other pathologic states. The conversion of diploid endothelial cells into polyploid endothelial cells may provide a convenient model cell system for studying mechanisms of the development of polyploidy in cells and their relationship to in vitro senescence.

Clinical evaluation in healthy adults of a hepatitis B vaccine made by recombinant DNA.

A vaccine formulated from hepatitis B surface antigen (HBsAg) produced by a recombinant strain of the yeast Saccharomyces cerevisiae was administered to two groups of human volunteers composed of 37 healthy, low-risk adults. Each subject received a 10-micrograms dose of HBsAg at 0, 1, and 6 months. By one month, 27% to 40% of the vaccinees had antibody to HBsAg, and by three months 80% to 100% were antibody positive. Large boosts in titer followed the third dose at six months. The antibody formed is predominantly specific for the a determinant of HBsAg. There have been no serious reactions attributable to the vaccine. The most frequent complaint has been transient soreness at the injection site. As far as we know, this is the first reported use in man of a vaccine prepared by recombinant DNA technology.

Live attenuated varicella virus vaccine. Efficacy trial in healthy children.

We conducted a double-blind, placebo-controlled efficacy trial of the live attenuated Oka/Merck varicella vaccine among 956 children between the ages of 1 and 14 years, with a negative clinical history of varicella. Of the 914 children who were serologically confirmed to be susceptible to varicella, 468 received vaccine and 446 received placebo. The vaccine produced few clinical reactions and was well tolerated. There was no clinical evidence of viral spread from vaccinated children to sibling controls. Approximately eight weeks after vaccination, 94 per cent of the initially seronegative children who received vaccine had detectable antibody to varicella. During the nine-month surveillance period, 39 clinically diagnosed cases of varicella, 38 of which were confirmed by laboratory tests, occurred among study participants. All 39 cases occurred in placebo recipients; no child who received vaccine contracted varicella. The vaccine was 100 per cent efficacious in preventing varicella in this population of healthy children (P less than 10(-9).

Vaccine against human hepatitis B virus prepared from antigen derived from human hepatoma cells in culture.

Vaccine against human hepatitis B was prepared using antigen derived from hepatitis B carrier hepatoma cells grown in the interstices of a Diaflo hollow filter unit. Hepatitis B surface antigen (HBsAg) produced by these cells was purified by immune affinity chromatography, digestion with DNase and pepsin, and Sephadex G-150 separation. The Formalin-treated antigen was formulated in 20-micrograms dose on alum adjuvant with thimerosal added as a preservative. This cell culture vaccine was as potent as human plasma-derived vaccine as measured in a mouse potency assay. The vaccine proved safe in tests in chimpanzees and in human subjects who were in late stages of cancer of the central nervous system and who were receiving therapy for their condition. None of five subjects who received the vaccine developed untoward clinical reactions. Two of the subjects who received all three doses of vaccine developed antibody against HBsAg. Three persons, two given only the primary doses and one who was given all three doses but was lost to follow-up, demonstrated no response. The slow and relatively low antibody responses to the vaccine were similar to those in other immunosuppressed persons who were given vaccine of human plasma origin.

Human hepatitis B vaccine from recombinant yeast.

The worldwide importance of human hepatitis B virus infection and the toll it takes in chronic liver disease, cirrhosis and hepatocarcinoma, make it imperative that a vaccine be developed for worldwide application. Human hepatitis B vaccines are presently prepared using hepatitis B surface antigen (HBsAg) that is purified from the plasma of human carriers of hepatitis B virus infection. The preparation of hepatitis B vaccine from a human source is restricted by the available supply of infected human plasma and by the need to apply stringent processes that purify the antigen and render it free of infectious hepatitis B virus and other possible living agents that might be present in the plasma. Joint efforts between our laboratories and those of Drs W. Rutter and B. Hall led to the preparation of vectors carrying the DNA sequence for HBsAg and antigen expression in the yeast Saccharomyces cerevisiae. Here we describe the development of hepatitis B vaccine of yeast cell origin. HBsAg of subtype adw was produced in recombinant yeast cell culture, and the purified antigen in alum formulation stimulated production of antibody in mice, grivet monkeys and chimpanzees. Vaccinated chimpanzees were totally protected when challenged intravenously with either homologous or heterologous subtype adr and ayw virus of human serum source. This is the first example of a vaccine produced from recombinant cells which is effective against a human viral infection.

Production of purified hepatitis B surface antigen from Alexander hepatoma cells grown in artificial capillary units.

An artificial capillary system was devised for growth of hepatoma cells that yields very high titers of hepatitis B surface antigen (HBsAg). High yield of antigen was facilitated by slowing cellular metabolism through reduction of incubation temperature and addition of 0.1 mM caffeine. Deletion of serum from the medium did not reduce the yield of antigen. HBsAg prepared from the culture fluid by affinity chromatography and additional chemical and enzymatic steps was essentially pure and was indistinguishable from HBsAg prepared from infected human plasma. Preparation of HBsAg from the cell culture source presents advantages over that of human plasma and might be a source of HBsAg for vaccine preparation.

The preparation and safety of hepatitis B vaccine.

Preparation of hepatitis B vaccine in our laboratories consists of a series of steps that include initial concentration of surface antigen by ammonium sulphate precipitation, followed by isopycnic banding and rate zonal centrifugation in a K-II centrifuge. The partially purified antigen concentrate is digested with pepsin at pH 2 and the antigen is unfolded in 8 M urea solution followed by renaturation. After gel filtration, the antigen is treated with formalin in I :4000 dilution, adsorbed on to alum, and preserved with thimerosal. The final product contains essentially pure hepatitis B surface antigen. The process relies both on physical elimination of infectious virus particles and treatment with highly viral-destructive reagents in the pepsin (pH 2), urea and formalin steps. The process is known to be highly destructive of all known viruses tested and to include procedures that are known to be highly destructive of representatives of all known groups of animal viral agents. The three-step process in inactivation provides a fail-safe system for establishing safety of the product. Tests in more than 20000 persons, who are under surveillance, have shown no untoward effect and have confirmed the safety of the product.

Studies in chimpanzees of live, attenuated hepatitis A vaccine candidates.

Human hepatitis A virus was attenuated in virulence for chimpanzees by passage in FRhK6 and human diploid lung fibroblast cell cultures. A number of variants were developed by passage in cell cultures which showed different levels of virulence/attenuation for chimpanzees. These results were compared to those obtained with marmosets and reported previously. In general, most variants behaved similarly in the two animal types. Two chimpanzees which gave vaccine-like responses following inoculation with HAV cell culture variants were challenged with virulent HAV. Both animals were immune to HAV infection. These findings provide further evidence for the feasibility of developing live, attenuated vaccines against human hepatitis A.

Quality and safety of human hepatitis B vaccine.

Preparation of hepatitis B vaccine in our laboratories consists of a series of steps that include initial concentration of surface antigen by ammonium sulfate precipitation, followed by isopycnic banding and rate zonal centrifugation in a K-II centrifuge. The partially purified antigen concentrate is digested with pepsin at pH2 and the antigen is unfolded in 8M urea solution followed by renaturation. After gel filtration, the antigen is treated with formalin in 1:4000 dilution, adsorbed onto alum, and preserved with thimerosal. The final product contains essentially pure hepatitis B surface antigen. The process relies both on physical elimination of infectious virus particles and treatment with highly viral-destructive reagents in the pepsin, urea and formalin steps. The process is known to be highly destructive of all known viruses tested and to include procedures that are known to be highly destructive of representatives of all known groups of animal viral agents. The three-step process in inactivation provides a fail-safe system for establishing safety of the product. Tests in more than 20'000 persons, who are under surveillance, have shown no untoward effect and have confirmed the safety of the product.

Progress toward a live attenuated human hepatitis A virus vaccine.

Human hepatitis A virus (HAV) was first grown in cell cultures four and one-half years ago, enabling significant progress toward the development of HAV vaccines. Vaccine development in a number of laboratories has proceeded on three fronts: 1) live, attenuated vaccine of cell culture origin; 2) inactivated vaccine of cell culture origin; and 3) genetic recombinant vaccines. Our studies to date have focused most heavily on the development of a live, attenuated HAV vaccine, although we have also made a prototype killed HAV vaccine form infected marmoset liver which induced anti-HAV and solid immunity to infection in marmosets. HAV was attenuated in virulence for both marmosets and chimpanzees by serial passaging in fetal rhesus monkey kidney cells and human diploid embryonic lung fibroblasts. A number of variants were produced which showed different levels of virulence/attenuation in these animal models. Some variants showed desirable live vaccine-like properties (little or no induction of enzyme elevations; little or no liver histologic change; retention of anti-HAV induction capacity). Vaccinated animals were solidly immune to challenge with virulent HAV. Experimental vaccines have been prepared from several attenuated HAV variants and preliminary studies in humans are planned.

Comparison of enzyme-linked immunosorbent assay and neutralization techniques for measurement of antibody to respiratory syncytial virus: implications for parenteral immunization with live virus vaccine.

The sensitivity of an enzyme-linked immunosorbent assay (ELISA) to detect low levels of antibody to respiratory syncytial (RS) virus was compared with a tube dilution neutralization test (NEUT) on sera obtained from children who received a parenteral live RS virus vaccine. Among the children who developed antibody in response to live RS virus vaccine. ELISA was as sensitive as NEUT at detecting antibody increases. Some children who did not have detectable prevaccine ELISA antibody possessed NEUT antibody; these children were generally less than 12 months old, suggesting that they had low levels of maternal antibody. Low levels of NEUT or ELISA antibody were associated with the absence of antibody increases after injection of live RS virus vaccine. The quantity of antibody stimulated by this live RS virus vaccine was small compared with that which was stimulated by naturally acquired RS virus infection. We concluded that ELISA is a satisfactory test for determining antibody to RS virus in vaccine field trials, given the understanding that low levels of preexisting antibody are not detected in some instances.