IgE and IgG antibodies to beta-propiolactone and human serum albumin associated with urticarial reactions to rabies vaccine.

We examined the antibody response to a rabies vaccine doubly inactivated with 0.025% beta-propiolactone and 0.1% tri(n)butyl phosphate and stabilized with 2.5% human serum albumin. Antibodies were measured by using the following four antigen preparations: complete doubly inactivated rabies vaccine, rabies vaccine inactivated only with tri(n)butyl phosphate, beta-propiolactone and human serum albumin, and human serum albumin alone. The fluid phase of the preparation of beta-propiolactone and human serum albumin completely inhibited IgE binding to solid-phase vaccine. Of 21 subjects with urticarial reactions to a booster, 19 had IgE to doubly inactivated vaccine and to beta-propiolactone and human serum albumin. None of 27 immunized subjects without urticaria had detectable IgE. In paired pre- and postimmunization sera, IgE appeared in six of seven of the subjects with urticaria and in one of seven nonreactors. These sera did not contain a significant level of IgE to singly inactivated vaccine or to human serum albumin alone.

Sequence of the serotype-specific glycoprotein of the human rotavirus Wa strain and comparison with other human rotavirus serotypes.

Complementary DNA was synthesized from the double-stranded RNA of the Wa strain of human rotavirus and inserted into the bacterial plasmid pBR322. Clones which contained the gene that codes for the viral glycoprotein (VP7) were identified and the nucleotide sequence was determined. The gene was 1062 base pairs in length with an open reading frame which coded for 326 amino acids. Two potential glycosylation sites were found as well as two hydrophobic regions at the N-terminus of the polypeptide. The untranslated regions at the 5' and 3' ends were 48 base pairs and 33 base pairs long, respectively. Only one nucleotide at position 493 differed from the sequence of the Wa VP7 gene described by Richardson et al. (1984, J. Virol. 51, 860-862). A strong prokaryotic promoter sequence was also found between residues 434 and 462. A comparison of the amino acid sequence of the Wa strain (serotype 1) to the Hu/5 strain of human rotavirus (serotype 2) and SA11, the simian rotavirus (serotype 3), revealed a high degree of homology (79.1% and 83.1%, respectively) between the serotypes, suggesting that rotavirus serotypes are stable. The hydrophilic regions of VP7 of the three serotypes were identified and compared for homology. Four of these regions showed variation between serotypes.

Antibody responses to human diploid cell vaccine for rabies with and without human rabies immune globulin.

One hundred one volunteers with no exposure to rabies were given human diploid cell vaccine (HDCV) for rabies with or without 20 international units of human rabies immune globulin (HRIG)/kg of body weight to evaluate schedules for therapy with HDCV and HRIG after exposure. All of the volunteers who received three or more doses of HDCV alone or four or more doses of HDCV with HRIG developed high titers of neutralizing antibodies by day 35, which persisted for at least 60 days. By day 7, of the 61 volunteers given HRIG and HDCV, 53% had neutralizing antibodies by a mouse neutralization test and 67% had neutralizing antibodies by a rapid fluorescent focus inhibition test. Similar antibody levels were found in volunteers given HRIG alone, a finding which suggests that low or undetectable early titers after administration of HDCV and HRIG were due to inadequate HRIG dosage rather than any interaction between the passive antibody (HRIG) and the vaccine antigen. These results suggest that trials with 30 or 40 international units of HRIG/kg in combination with HDCV are warranted.

Responses to human diploid cell rabies vaccine: neutralizing antibody responses of vaccinees receiving booster doses of human diploid cell rabies vaccine.

Rabies neutralizing antibody levels were determined before and after administration of a booster-dose of Wyeth rabies vaccine (WRV) in persons immunized earlier with either duck embryo vaccine (DEV) or with WRV. Virtually all those receiving an initial 3-dose regimen of WRV (0, 7 and 21--28 days) still had neutralizing antibody one year later, but there was a decline in titer from 10--50 IU per ml at 35 days to about 1--3 IU. Only one-half of those receiving DEV as the primary vaccine had even detectable antibody one year later. All volunteers responded anamnestically to a single WRV booster given 8--12 months after either primary vaccine. Those given WRV initially had much higher antibody levels than those given DEV, but after the WRV booster antibody levels in all vaccinees remained high, even one year later.

Potency testing of diploid-cell rabies vaccine and correlation with immunogenicity.

Two procedures for evaluating the potency of diploid-cell rabies vaccine (WRV) were compared by mouse-serum neutralization (MNT) and RFFIT serological determinations, using NIH Reference Rabies Vaccine No. 182 as base. Mean antibody-induction potency value ratios in female CD1 mice for 12 WRV assays were 5.12 by MSN and 4.57 by RFFIT compared with a 2.84 Antigen Value by the standard 28-day NIH mouse neutralization tests on the same lots. The mean antibody-induction potency values for 12 control DEV preparations were 0.71 (MSN) and 0.81 (RFFIT) versus 0.51 Antigen Value by NIH test. The statistical parameters of all respective means were similarly compared. MRV potencies of the order of magnitude shown correlated well with superior human immunogenicity but not linearly over a ten-fold range of antigen values from about 1 to 10. MNT antibody responses were 15-30 I.U. per ml of serum to WRV in this range (3-dose immunization); corresponding overall RFFIT values were 8 to 15 I.U. per ml. Control immunizations with DEV preparations yielded immune responses approximately one order of magnitude lower under comparable conditions.

Specific chromosome changes associated with rabbit cell lines cultured in vitro.

Thirty-three rabbit cell lines were established from various fetal tissues of the inbred strain III of the New Zealand rabbit (Oryctolagus cuniculus). None of these lines exhibited senescence during a growth period of more than 2 years. Karyologic studies of most cell lines at 10 to 20 cell-passage intervals revealed that the karyotype stability of the rabbit cells in vitro was correlated with the organs from which the cell lines were derived. Thus, lines derived from cornea, spleen, and kidney tissues usually contained high frequencies of polyploidy in their early passages, whereas most of those derived from lung and skin were found to retain the normal diploid karyotype for much longer periods of time. One line derived from fetal lung tissue, designated Lung 16, remained diploid up to 100 passages. In late passages of the majority of all the lines studied, the cells became pseudodiploid, hyperdiploid, or polyploid. Among the pseudodiploid and the hyperdiploid cell lines, the chromosomal changes followed three basic patterns: (1) a gain of one or more telocentric chromosomes; (2) a loss of one telocentric chromosome plus a metacentric marker chromosome (M); or (3) a gain of a long telocentric marker chromosome with or without changes in the number of telocentric D chromosomes. By the G-banding technique, the telocentric chromosome involved in these three patterns was identified as the D-group chromosome 18 and the M marker chromosome as an isochromosome of 18. These results suggest that chromosomal rearrangement in rabbit cells involving trisomy of 18 may be responsible for the longevity of these cell lines cultured in vitro.

The development of rabbit diploid cell lines.

The authors describe briefly studies on the development of diploid cell lines from fetal tissues (origin: four inbred rabbits). Some lines have been maintained in continuous serial culture for over three years. The Lung 16 may represent a qualifying rabbit diploid cell line when used between cell passages 48 and 100; during this period it has shown a minimum deviation from the normal diploid rabbit karyotype; it supports the growth of rubella virus, is non-tumorigenic and might be useful as a substrate for human vaccine.

Clinical responses to T(n)BP-disrupted HDCS (WI-38) rabies vaccine.

Study of 10 batches of an antirabies vaccine made from purified, concentrated subunits (Pitman-Moore strain adapted to Wistar HDCS). One single dose of vaccine is insufficient; three or more doses induce high antibody levels. Among more than 2000 vaccines secondary reactions were rare and generally mild and transient. All the individuals vaccinated post-exposure remained without rabic symptoms. The vaccine was also particularly efficacious for booster doses.

Immunization schedules for the new human diploid cell vaccine against rabies.

Clinical and antibody responses of human volunteers to four different serial production lots of human diploid cell vaccine (HDCV) each with a different antigenic value are described. Three to four doses of HDCV administered over a period of 14 days produced high levels of virus neutralizing antibodies with an average titer up to 20 times higher than the titer elicited by four doses of duck embryo vaccine. Antibodies were still present one year after completion of vaccination. Only minimal differences in antibody response could be observed between groups receiving vaccines of different antigenic values. Untoward reactions to the vaccine were few. The possibility of using the vaccine as part of postexposure human prophylaxis against rabies is discussed.