Ronald W. Ellis

The Efficacy in Navajo Infants of a Conjugate Vaccine Consisting of Haemophilus influenzae Type b Polysaccharide and Neisseria meningitidis Outer-Membrane Protein Complex

BACKGROUND AND METHODS Several conjugate vaccines against Haemophilus influenzae type b have been developed in the search for one that induces protection even in young infants. We evaluated the safety and efficacy of a conjugate vaccine that links the H. influenzae type b capsular polysaccharide to the outer-membrane protein complex (OMPC) of Neisseria meningitidis serogroup B. We conducted a double-blind, placebo, controlled trial in Navajo infants, who are at high risk for systemic infections caused by H. influenzae type b. The infants were randomly assigned to receive the first dose of vaccine or placebo at 42 to 90 days of age and the second at 70 to 146 days of age. RESULTS Of the infants in the trial, 2588 were assigned to receive the vaccine and 2602 to receive placebo. The mean follow-up was 269 days in the vaccine group and 267 days in the placebo group. Before the age of 18 months, there was 1 systemic H. influenzae type b infection in the vaccine group, as compared with 22 in the placebo group (P less than 0.001; point estimate of efficacy, 95 percent; 95 percent confidence interval, 72 to 99 percent). Of the 22 H. influenzae type b infections in the placebo group, 13 were meningitis. Among the children who received both doses, there was 1 H. influenzae type b infection in the vaccine group (n = 2056) and 14 in the placebo group (n = 2105) (P less than 0.001; point estimate of efficacy, 93 percent; 95 percent confidence interval, 53 to 98 percent). The single infection in the vaccine group occurred at 15 1/2 months of age in an infant with osteomyelitis. Between the first and second doses there were no H. influenzae type b infections in the vaccine group and eight in the placebo group (P less than 0.005; point estimate of efficacy, 100 percent; 95 percent confidence interval, 41 to 100 percent). CONCLUSIONS The H. influenzae type b OMPC vaccine, administered at 2 and 4 months of age, is safe and induces a high rate of protection against invasive disease caused by H. influenzae type b in infants under the age of 18 months. Protection begins after the first dose.

Modified cases of chickenpox after varicella vaccination : correlation of protection with antibody response

Four thousand forty-two healthy children and adolescents, ages 12 months to 17 years, were vaccinated with a single dose of live attenuated varicella vaccine (VARIVAX®; Merck Sharp and Dohme Research Laboratories) containing ∼1000 to 1625 plaque-forming units/dose during clinical trials conducted from 1987 to 1989. Clinical follow-up of vaccinees revealed that 2.1 and 2.4% of vaccinees developed modified cases of varicella in the first and second years, respectively, after vaccination. Most of those who developed varicella postvaccination had an attenuated illness, characterized by fewer lesions and a lower incidence of fever (≥100°F, oral) than after natural infection. The likelihood of developing varicella postvaccination decreased (P < 0.0001) as the 6-week postvaccination glycoprotein-based enzyme-linked immunosorbent assay titer increased. In addition the number of lesions in these cases tended to decrease (P = 0.07 for Year 1 and P = 0.02 for Year 2) as the 6-week glycoprotein-based enzyme-linked immunosorbent assay titer increased. Thus the 6-week postvaccination glycoprotein-based enzyme-linked immunosorbent assay titer can be used as a surrogate marker for protection from natural disease.

Expression and secretion in yeast of a 400-kDa envelope glycoprotein derived from Epstein-Barr virus.

The major envelope glycoprotein (gp350) of Epstein-Barr virus has been expressed and secreted in the yeast Saccharomyces cerevisiae as a 400-kDa glycoprotein. This is the first example of the secretion of such a large, heavily glycosylated heterologous protein in yeast. Since gp350 proved highly toxic to S. cerevisiae, initial cellular growth required repression of the expression of gp350. Using temperature- or galactose-inducible promoters, cells could be grown and the expression of gp350 then induced. After induction, the glycoprotein accumulated both intracellularly as well as in the culture medium. Only the most heavily glycosylated form was secreted, suggesting a role for N-linked glycans in directing secretion. The extent of O-linked glycosylation of the yeast-derived protein was similar to that of the mature viral gp350. N-linked glycosylation varied slightly depending upon culture conditions and host strain used and was more extensive than that associated with the mature viral gp350. Although there is no evidence that more than a single mRNA for the glycoprotein was expressed from the recombinant plasmid, variously sized glycoproteins accumulated in yeast at early stages after induction, probably reflecting intermediates in glycosylation. The yeast-derived glycoproteins reacted with animal and human polyclonal antibodies to gp350 as well as with a neutralizing murine monoclonal antibody to gp350, suggesting that this glycoprotein retains several epitopes of the native glycoprotein.

Production and immunological analysis of recombinant hepatitis B vaccine

The synthesis of the hepatitis B surface antigen (HBsAG) in cells of Saccharomyces cerevisiae and its subsequent isolation, purification and analysis is described. The final, purified HBsAg particle exhibits close structural and biochemical similarities to particles derived from the plasma of chronically infected humans. Particles of yeast and human origin have been found, by chimpanzee efficacy studies and by various in vitro analyses, to be immunologically equivalent. The antigenic expression of a determinant-specific epitopes, as measured by antibody binding to synthetic peptides, has also been shown to be equivalent.

New technologies for making vaccines

Technologies for making active vaccines fall into 3 general groups: live, subunit (killed or inactivated) and genetic. Each of these groups is further divisible into multiple categories, which include recombinant-derived antigens as well as native microorganisms and their components. In addition, there are new enabling technologies such as delivery systems and vectors which can be applied to these approaches. Most disease targets, whether infectious or noninfectious in origin, can be approached by the application of several different vaccine technologies, as can be tested during the discovery phase of research. The criteria for choosing early in a development program which of the vaccine technologies are likely to ultimately be most fruitful for a given application include: knowledge of the pathogenesis of the given infection/disease; technical feasibility; immunobiology and associated mechanisms; preclinical efficacy profile; anticipated clinical safety; regulatory; manufacturing; and marketing. All of these criteria should be considered together in making selections for an R&D program. This paper is reviewing the major vaccine technologies and relevant examples of how these criteria are used to make decisions in vaccine development.

Antibody assays suitable for assessing immune responses to live varicella vaccine

An enzyme-linked immunosorbent assay for antibodies to varicella-zoster virus (VZV), using purified viral glycoproteins as antigen (gpELISA), was compared with other assays for measuring vaccine-induced antibody responses. The gpELISA was more sensitive than conventional assays, proved highly specific for VZV and agreed well with an assay for neutralizing antibody activity. It was successfully applied to large-scale testing of live varicella vaccine in humans.

Nucleotide sequence of the two rat cellular rasH genes.

We present the nucleotide sequence of the coding region of the rat c-rasH-1 gene and a partial sequence analysis of the rat c-rasH-2 gene. By comparing these sequences with the Harvey murine sarcoma virus ras gene, we predict that the p21 protein encoded by the Harvey virus differs from the cellular c-rasH-1-encoded p21 at only two amino acids; those at positions 12 and 59. Alterations at each of these positions may play a role in activating the viral p21 protein. The c-rasH-2 gene is likely to be a nonfunctional pseudogene because it lacks introns, cannot be activated to transform NIH 3T3 cells, and differs in sequence from both c-rasH-1 and v-rasH at several base pair positions.

Generation of BALB-MuSV and Ha-MuSV by type C virus transduction of homologous transforming genes from different species

The nature of the cell-derived (bas) sequences of BALB-MuSV, a spontaneous mouse sarcoma virus isolate, was determined. Molecularly cloned bas sequences demonstrated no detectable homology with the onc genes of other mouse transforming viruses, but exhibited a high degree of sequence homology with the ras gene of the rat-derived Harvey murine sarcoma virus (Ha-MuSV) genome. The Ha-MuSV cell-derived sequence (ras) shared a colinear 750 bp region of homology with bas. Moreover, BALB-MuSV transformation was associated with the expression of high levels of a 21,000 dalton protein, immunologically related to the ras gene products, p21. Thus bas and ras represent retroviral transforming gene homologs that were independently transduced by mouse type C viruses from the genomes of different species.

Purification of individual varicella-zoster virus (VZV) glycoproteins gpI, gpII, and gpIII and their use in ELISA for detection of VZV glycoprotein-specific antibodies

We have utilized monoclonal antibodies in immune affinity chromatography to purify each of the 3 major glycoproteins of varicella-zoster virus (VZV), gpI, gpII, and gpIII, in immunologically active form. Upon injection into guinea pigs, each preparation elicited the production of specific antibodies capable of immunoprecipitating the homologous glycoprotein and of neutralizing VZV infectivity in vitro. Also, total glycoproteins from VZV-infected cells have been purified by lectin affinity chromatography. Each of the individual purified glycoproteins, as well as total VZV glycoproteins and appropriate uninfected cell protein controls, have been employed as solid-phase reagents in enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies directed against specific VZV glycoproteins. The specificity of the purified glycoproteins as ELISA reagents was verified by the ability of individual monoclonal antibodies to bind specifically to individual glycoprotein preparations. We have demonstrated the utility of the glycoprotein-specific ELISA by detecting antibodies in sera from post-zoster and post-varicella patients. The assay detects antibodies directed against each of the 3 major glycoproteins and is sensitive enough to detect antibodies in a 1:320 000 dilution of some sera. This assay, as well as the purified individual glycoproteins per se, should prove to be very useful reagents in understanding the role of each of gpI, gpII, and gpIII in immunity to VZV.

Mouse cells contain two distinct ras gene mRNA species that can be translated into a p21 onc protein.

The Kirsten (Ki) and Harvey (Ha) strains of murine sarcoma virus encode a 21,000-dalton protein (p21 ras) which is the product of the transforming gene of these viruses. Normal cells express low levels of p21 ras encoded by cellular genes (Ki-ras and Ha-ras) homologous to the Ki and Ha murine sarcoma virus transformation genes. A bone marrow-derived mouse cell line, 416B, has been shown to express unusually high levels of p21 ras. In this manuscript, we investigated the molecular biology of p21 ras gene expression in 416B and other normal mouse cells. We identified four distinct polyadenylated and polysome-associated RNAs, two related to Ki-ras and two to Ha-ras. The levels in 416B cells of the two Ki-ras RNAs, sized 5.2 and 2.0 kilobases, were both elevated approximately 25-fold over levels found in normal mouse cells; there was no corresponding change in 416B cells in the levels of the two Ha-ras RNAs. We partially purified the two Ki-ras mRNAs and separated them by velocity sedimentation in sucrose density gradients. Both the 5.2- and 2.0-kilobase mRNAs could be translated in vitro into p21 ras. These results show that a cellular onc protein can be translated from two distinct cellular mRNA species.