Shedding of rotavirus after administration of the tetravalent rhesus rotavirus vaccine.

BACKGROUND: The tetravalent rhesus rotavirus vaccine (RV-TV) has been administered to several thousand children in multiple settings throughout the world. It has been proved safe and efficacious, resulting in an application for licensure in 1997. However, only limited information has been reported on viral shedding after RV-TV vaccination. METHODS: Stool specimens were collected between Days 3 and 5 after administration of each of 3 doses of RV-TV to 248 subjects 6 to 12 weeks of age in 8 centers across the United States. Rotavirus antigen was measured by an enzyme-linked immunosorbent assay to determine the number of subjects who shed after each dose. The relative quantities of vaccine strains shed were then determined by plaque purification and serotype analysis. RESULTS: Rotavirus shedding was detected in 125 (50.4%) subjects, and 19 shed after more than 1 dose. Although fewer subjects shed rotavirus after Dose 2 (14.5%), shedding after Doses 1 (26.0%) and 3 (22.5%) were comparable. After plaque purification and serotyping, most viruses shed (76.2%) were identified as G3 after Dose 1, but a major shift to G2 strains was found after Doses 2 (61.3%) and 3 (69.0%). CONCLUSIONS: Sequential RV-TV administrations caused no overall significant decrease in the number of vaccinees who experienced detectable shedding. A major shift in shedding was found from the serotype G3 vaccine strain (RRV) after the first dose of vaccine to the serotype G2 reassortant after Doses 2 and 3.

Attenuation of a human rotavirus vaccine candidate did not correlate with mutations in the NSP4 protein gene.

The NSP4 protein of a simian rotavirus was reported to induce diarrhea following inoculation of mice. If NSP4 is responsible for rotavirus diarrhea in humans, attenuation of a human rotavirus may be reflected in concomitant mutations in the NSP4 gene. After 33 passages in cultured monkey kidney cells, a virulent human rotavirus (strain 89-12) was found to be attenuated in adults, children, and infants. Nucleotide sequence analysis of the NSP4 protein gene revealed only one base pair change between the virulent (unpassaged) and attenuated 89-12 viruses, which resulted from a substitution of alanine for threonine at amino acid 45 of the encoded NSP4 protein. Because both threonine and alanine have been found at position 45 of NSP4 in symptomatic and asymptomatic human rotaviruses, neither amino acid in this position could be established as a marker of virulence. Therefore, attenuation of rotavirus strain 89-12 appears to be unrelated to mutations in the NSP4 gene.

Heparin potentiates endothelial cell growth factor stimulation of plasminogen activator synthesis by diploid human lung fibroblasts.

Endothelial cell growth factor (ECGF) stimulates the synthesis of t-PA and u-PA by confluent, diploid human lung fibroblasts, and this activity is potentiated considerably by heparin. In contrast, the malignant cell lines, Bowes melonoma and CALU-3, producers of t-PA and u-PA, respectively, are insensitive to ECGF. Studies with metabolic inhibitors and direct measurements of PA-specific mRNAs show that ECGF-mediated production of PA by human lung fibroblasts is dependent on de novo protein and RNA synthesis. The mechanism by which heparin potentiates this effect is thought to reside in its ability to prolong or strengthen the interaction of ECGF with cell surface receptors. The results raise the possibility that endogenous ECGF or related polypeptides (and heparin) may act to regulate PA synthesis by lung fibroblasts and possibly other responsive target cells in vivo.

Prostaglandin E inhibits the production of human interleukin 2.

Prostaglandins of the E type specifically inhibited the production of interleukin 2 (IL-2) by normal human lymphocytes, whereas PG synthetase inhibitors such as indomethacin and fentiazac raised IL-2 production above normal levels. Removal of adherent cells from mononuclear cell populations also resulted in enhanced IL-2 production. The resultant nonadherent cell population lost sensitivity to the enhancement effect of PG synthetase inhibitors, suggesting that a PGE-producing adherent cell plays a major role in the regulation of IL-2.

Development of a vaccine against experimental cholera and Escherichia coli diarrheal disease.

The results of the present investigation indicate a simple approach to the development of a single-vaccine formula which may ultimately be used to confer protection against both cholera and certain types of Escherichia coli diarrheal disease in humans and domestic animals. The design of the vaccine is based on the well-documented ability of cholera antitoxin to neutralize both cholera and heat-labile E. coli enterotoxins (CT and LT, respectively) and on the ability of killed E. coli to enhance the immune response to cholera toxoid and, possibly, to conventional cholera vaccine as well. Evidence presented shows that a parenterally administered E. coli vaccine, prepared from an LT-only enterotoxigenic strain, reproducibly elevated rabbit antitoxin responses to cholera toxoid and that such responses correlated with dramatic protection against live cholera vibrios and the homologous E. coli strain in the rabbit ligated loop model of diarrheal disease. The results show also that cholera vaccine acted to suppress the rabbits' immune response to the cholera toxoid and E. coli vaccine formula, even though all three antigens combined still provided significant protection against live organism challenge. On the basis of data presently available, the vaccine formula would be composed simply of cholera toxoid and E. coli vaccine, but may also include cholera vaccine. Since it has already been established that cholera toxoid and cholera vaccine are each safe for human use, additional vaccine development would require investigation of the safety of E. coli vaccine, alone and in combination with the other components.

Development of a purified cholera toxoid. III. Refinements in purification of toxin and methods for the determination of residual somatic antigen.

The addition of an ultrafiltration step to the purification procedures previously described for cholera toxin (Rappaport et al., (1974) permitted the preparation of highly purified antigenic toxoids essentially free of somatic antigen(s). The purity of such toxoids is established: (i) by the absence of more than about one part limulus amebocyte lysate (LAL)-positive endotoxin per 10(5) parts toxoid and (ii) by the inability of the toxoids to elicit a significant rise in rabbit vibriocidal antibody. The antigenicity of the toxoids is demonstrated by their ability to produce the same high levels of rabbit serum antitoxin as are produced by comparable toxoids containing small amounts of somatic antigen. The results also indicate that amounts of somatic antigen of the order of less than or equal to 1 mug/100 mug of toxoid do not exert an adjuvant effect on the toxoid, at least with respect to circulating antitoxin. Other data show that, where present, the ability of somatic antigen to elicit vibriocidal antibody is influenced by the immunization schedule employed and that a correlation exists between the LAL-determined endotoxin content of the toxoids and their ability to stimulate vibriocidal antibody. Somatic antigen-free toxoids, purified and tested by the refinements herein described, were prepared for use in the National Institutes of Health sponsored field trials, and data pertaining to their purity and antigenic properties are presented.

Activation of Heat-labile Escherichia coli enterotoxin by trypsin.

Trypsin-treated, cell-free filtrates derived from enterotoxigenic Escherichia coli, strain H197 (O78:H11), exhibited a fourfold or greater increase in heat-labile vascular permeability factor activity and a 10-fold or greater increase in the ability to stimulate secretion of growth hormone by cultured rat pituitary cells. In contrast, trypsin-treated filtrates were not different from untreated filtrates in their ability to elicit a secretory response in ligated rabbit intestinal loops. However, incubation of culture filtrate in ligated intestinal loops, or with rabbit intestinal fluid (in vitro), resulted in at least a twofold increase in permeability factor that did not occur in the presence of trypsin inhibitor or with heat-inactivated intestinal fluid. Moreover, trypsin inhibitor could reduce the secretory response to culture filtrate. These findings suggest that activation of heat-labile E. coli enterotoxin by host enzymes may play a role in the development of a full pathogenic effect.

Development of a purified cholera toxoid. I. Purification of toxin.

The enterotoxin from Vibrio cholerae is selectively concentrated from cell-free culture supernatant by co-precipitation with hexametaphosphate and is further purified by adsorption on aluminum hydroxide powder. The bulk of residual somatic antigen becomes insoluble upon lyophilization of the toxin preparation and is removed by centrifugation of the rehydrated material. Other contaminants are eliminated by treatment with activated carbon. Preparations of toxin, purified by this method, have been characterized by: (i) a single immunoprecipitin line against polyvalent antisera; (ii) homogeneity on acrylamide gels; (iii) specific activities on the order of 22 limit-of-bluing doses/mug; (iv) ultraviolet spectra characteristic of pure protein; and (v) overall yields on the order of 50%, irrespective of purification scale. Such preparations, however, have been shown to contain trace amounts of somatic antigen when they are intensively tested either for their ability to elevate serum vibriocidal antibody titers in immunized rabbits or for their ability to increase resistance of immunized mice to live vibrio challenge. In the latter test system, the level of residual somatic antigen per 50 mug of toxin (toxoid) antigen generally did not exceed 0.025% of the Division of Biological Standards reference vaccine, V. cholerae Inaba IN-12. Methods for elimination of this small amount of somatic antigen have been investigated and are discussed. The particular combination of purification steps which are presently described have been easily and reproducibly applied on a production scale to prepare gram amounts of toxin with a high degree of purity, even under a variety of initial conditions.

Development of a purified cholera toxoid. II. Preparation of a stable, antigenic toxoid by reaction of purified toxin with glutaraldehyde.

Evidence is presented which confirms that cholera toxoids obtained by reaction of purified toxin with Formalin possess the ability to partially reactivate both in vivo and in vitro. At the same time, conditions are presented for the preparation of stable, antigenic cholera toxoids by reaction of purified toxin with glutaraldehyde. Treatment of purified cholera toxin with approximately 200 mol of glutaraldehyde per mol of toxin at pH 7.8 reproducibly resulted in the preparation of toxoids which: (i) possessed less than 20 bluing doses per 100 mug; (ii) did not reactivate in vivo or in vitro; (iii) precipitated with, and neutralized antitoxin; (iv) elevated prolonged serum antitoxin in immunized rabbits; (v) protected immunized guinea pigs against toxin skin challenge; and (vi) lent themselves to enhanced antigenicity by means of an in situ adjuvant system which may be suitable for man. Acrylamide gel electrophoresis and molecular sieve chromatography of a series of glutaraldehyde-derived toxoids suggested that the reaction products consisted of monomeric and polymeric species and that the proportion of higher-molecular-weight species was determined by the relative concentrations of toxin and glutaraldehyde. The results suggested a relationship between complete and irreversible elimination of toxicity and the formation of higher-molecular-weight toxoids.