Biomedical subjects

M M Levine

Publications and source records attributed to M M Levine.

At least 91 records · Page 5Linked to original sources

PubMed1996

Large scale, postlicensure, selective vaccination of Chilean infants with PRP-T conjugate vaccine: practicality and effectiveness in preventing invasive Haemophilus influenzae type b infections.

BACKGROUND: Haemophilus influenzae type b (Hib) conjugate vaccines have demonstrated an impressive impact in diminishing Hib disease in industrialized countries. However, their high cost prompts nonindustrialized countries to corroborate their effectiveness under local conditions before considering their programmatic implementation. Such a postlicensure evaluation of vaccine effectiveness was undertaken in Chile. METHODS: After its licensure in Chile polyribosylribitol phosphate-tetanus toxoid protein conjugate vaccine (PRP-T), combined with diphtheria-tetanus-pertussis vaccine, was introduced into the Expanded Program on Immunization schedules in 36 health centers throughout metropolitan Santiago for 12 months, whereas 35 similar health centers administered only diphtheria-tetanus toxoid-pertussis vaccine. Bacteriologic surveillance data for invasive Hib cases collected over the ensuing 30 months were analyzed. RESULTS: In an intent-to-vaccinate (effectiveness) analysis, PRP-T provided 90.2% protection (95% confidence interval, 74.5 to 100%) against invasive Hib disease (40 vs. 4 cases, P < 0.001). Vaccine effectiveness was 91.3% against meningitis (22 vs. 2 cases) and 80% against pneumonia and empyema (10 vs. 2 cases, P = 0.039). Vaccine efficacy among infants who received all three doses of PRP-T was 91.7% (95% confidence interval, 64.8 to 100%). CONCLUSIONS: Programmatic use of Hib conjugate vaccine administered in combination with diphtheria-tetanus-pertussis vaccine constitutes a highly effective and practical intervention in Chile, a newly industrializing country.

Bacterial Capsules↗

PubMed1996

Factors influencing secondary vibriocidal immune responses: relevance for understanding immunity to cholera.

Although serum vibriocidal activity is used extensively as a marker of immunity to O1 Vibrio cholerae, there are limitations in this assay to detect instances of reexposure. We define the conditions operative in producing secondary vibriocidal responses in North American volunteers primed with either wild-type V. cholerae 1, 4, or 6 months later. Secondary serum vibriocidal responses occurred under two distinct secondary challenge conditions. The first occurred when secondary challenge produced a breakthrough in clinical protection. Following secondary exposure, 14 of 22 (64%) and 1 of 29 (3%) subjects with and without vibrio stool excretion, respectively, had secondary responses (P < 0.001); 5 of 6 (83%) and 10 of 45 (22%) subjects with or without diarrhea, respectively, mounted a secondary response (P = 0.006). The second condition occurred in the presence of full clinical protection but was dependent on the time interval between exposure. No subject (0 to 17) vaccinated with CVD 103-HgR and given homologous wild-type challenge within 4 months mounted a secondary vibriocidal response (P = 0.0009). The majority of the serum vibriocidal activity was of the immunoglobulin M (IgM) isotype, seen in 96 and 73% of subjects following primary and secondary exposure, respectively. Vibriocidal activity in the IgG fraction following primary and secondary exposures occurred with < or = 50% of volunteers; lipopolysaccharide (LPS)-specific IgG1 and IgG3 subclass responses supported the vibriocidal isotype data. However, following primary exposure, IgG4 LPS responses predominated, occurring in 81% of responding volunteers. These data suggest that, under certain conditions of secondary exposure to V. cholerae O1 antigens, when there is sufficient active local immunity present, there is a block of vibrio antigen resampling at the M cell level. We discuss the implications of and possible explanations for these findings.

Adolescent↗

PubMed1996

Further characterization of delta aroA delta virG Shigella flexneri 2a strain CVD 1203 as a mucosal Shigella vaccine and as a live-vector vaccine for delivering antigens of enterotoxigenic Escherichia coli.

The use of attenuated delta aroA delta virG Shigella flexneri 2a strain CVD 1203 as a live vector for enterotoxigenic Escherichia coli (ETEC) antigens is reported. CVD 1203 alone or expressing colonization factor antigen fimbriae and CS3 fibrillae of ETEC was given to guinea pigs and mice, orogastrically (o.g.) or intranasally (i.n.). CVD 1203 given i.n. elicited high titers of antilipopolysaccharide (anti-LPS) immunoglobulin A (IgA) and was protective in guinea pigs against a homologous conjunctival challenge. Whereas a strong IgA response against colonization factor antigen CS3, and Shigella LPS was detected in tears and serum of guinea pigs after o.g. or i.n. immunization, the i.n. route elicited significantly higher antibody titers. A strong serum IgG response was also observed against the ETEC antigens, although no serum anti-LPS IgG response was detected. The immune response in mice followed a pattern similar to that in guinea pigs, and the difference between the responses after o.g. and i.n. administration was even more remarkable.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed1996

Protective immunity to Shiga-like toxin I following oral immunization with Shiga-like toxin I B-subunit-producing Vibrio cholerae CVD 103-HgR.

This study addresses a mechanism for inducing systemic immunity to Shiga-like toxins by oral administration of a Shiga-like toxin I B-subunit-expressing Vibrio cholerae vaccine strain [CVD 103-HgR(pDA60)]. Two sets of three rabbits were given either CVD 103-HgR or CVD 103-HgR(pDA60) orally. All rabbits immunized with CVD 103-HgR(pDA60) developed neutralizing serum antibodies to Shiga-like toxin I. None of the controls developed such antibodies.

Administration, Oral↗

PubMed1996

Expression and immunogenicity of pertussis toxin S1 subunit-tetanus toxin fragment C fusions in Salmonella typhi vaccine strain CVD 908.

Salmonella typhi vaccine strain CVD 908 can deliver heterologous antigens to the host immune system following mucosal immunization. Stable expression of foreign proteins in Salmonella cells often requires antigen-specific engineering strategies. Fusion of antigens to stabilizing proteins has proven to be a successful strategy for rescuing otherwise unstable proteins. We designed plasmids to allow the fusion of antigens to the amino terminus or carboxyl terminus of fragment C of tetanus toxin, separated by a 4-amino-acid hinge region. Towards the ultimate goal of developing a live oral diphtheria-pertussis-tetanus vaccine, we used these plasmids to stably express the S1 subunit of pertussis toxin in CVD 908. Driven by the anaerobically inducible nirB promoter, the S1 subunit alone was expressed poorly in Salmonella cytoplasm. In contrast, hybrid proteins with S1 fused to either the amino or carboxyl terminus of fragment C were expressed at a high level in CVD 908 and were recognized in Western blot (immunoblot) analysis by monoclonal antibodies directed to S1 and to fragment C. Mice were immunized by the oral or intranasal routes with CVD 908 derivatives harboring these recombinant plasmids. All fusion proteins elicited serum antibody responses to fragment C following intranasal immunization, whereas oral inoculation did not. The configuration of antigens constituting the fusion was critical; S1 fused to the amino terminus of fragment C was less effective than S1 fused to the carboxyl terminus in generating anti-fragment C antibodies. CVD 908 expressing truncated S1 fused to the carboxyl terminus of fragment C elicited neutralizing serum pertussis antitoxin following intranasal immunization of mice.

Animals↗

PubMed1996

Safety, immunogenicity, and transmissibility in humans of CVD 1203, a live oral Shigella flexneri 2a vaccine candidate attenuated by deletions in aroA and virG.

We evaluated the safety and immunogenicity of attenuated Shigella flexneri 2a vaccine candidate CVD 1203, which harbors precise deletions in the plasmid gene virG and in the chromosomal gene aroA. CVD 1203 invades epithelial cells but undergoes minimal intracellular proliferation and cell-to-cell spread. Fasting healthy volunteers, aged 18 to 40 years, were randomly allocated (double-blind design) to receive either CVD 1203 vaccine or placebo, along with sodium bicarbonate buffer, on days 0 and 14, as follows. At the time of the first inoculation, 10 subjects received placebo (group 1) and 22 subjects received either 1.5 x 10(8) (group 2; 11 subjects) or 1.5 x 10(9) (group 3; 11 subjects) CFU of CVD 1203. Fourteen days later, subjects from group 1 received 1.2 x 10(6) CFU of CVD 1203 and subjects from groups 2 and 3 received 1.2 x 10(8) vaccine organisms. Clinical tolerance was dose dependent. After a single dose of CVD 1203 at 10(6), 10(8), or 10(9) CFU, self-limited (<48-h duration) objective reactogenicity (fever, diarrhea, or dysentery) developed in 0, 18, and 72% of subjects, respectively, and in no placebo recipients. CVD 1203 induced immunoglobulin G seroconversion to S. flexneri 2a lipopolysaccharide (LPS) in 30, 45, and 36% of subjects from groups 1, 2, and 3, respectively, and stimulated immunoglobulin A-producing anti-LPS antibody-secreting cells in 60, 91, and 100% of subjects, respectively. After vaccination, significant rises in tumor necrosis factor alpha concentration in serum (groups 1, 2, and 3) and stool (group 2) samples were observed. We conclude that engineered deletions in virG and aroA markedly attenuate wild-type S. flexneri but preserve immunogenicity; however, less reactogenic vaccines are needed.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed1996

Engineered deltaguaB-A deltavirG Shigella flexneri 2a strain CVD 1205: construction, safety, immunogenicity, and potential efficacy as a mucosal vaccine.

Shigella flexneri 2a strain CVD 1204, which was constructed by introducing a specific, in-frame deletion mutation in the guaB-A operon, was compared with deltaaroA strain CVD 1201. CVD 1204 was less invasive for HeLa cells than CVD 1201, whereas following invasion, the abilities of the two mutants to proliferate intracellularly were similarly impaired. The reduction in invasiveness was independent of the guanine auxotrophic phenotype and fully recovered when the chromosomal deletion mutation in CVD 1204 was repaired. Following inoculation of the conjunctival sac of guinea pigs (Serény test) at high doses (10(9) CFU per eye), both strains evoked minimal, short- lived conjunctival inflammation, which was significantly milder with strain CVD 1204. Double mutant deltaguaB-A deltavirG (also called icsA) strain CVD 1205 induced, after a single intranasal dose, high mucosal immunoglobulin A antilipopolysaccharide titers, which were significantly boosted further following a second dose of vaccine given 14 days later. Upon Serény test challenge with wild-type S. flexneri 2a, CVD 1205-vaccinated animals were significantly protected against keratoconjunctivitis (zero of eight vaccinees versus five of seven controls, P = 0.03; vaccine efficacy, 100%). CVD 1205 is an attractive candidate for human clinical trials.

Administration, Intranasal↗

PubMed1995

Cytotoxic T lymphocytes after oral immunization with attenuated vaccine strains of Salmonella typhi in humans.

Not only viruses, but certain parasites and bacteria as well, can elicit CTL involved in mediating protection. It has been surmised that CTL able to lyse Salmonella typhi-infected cells are likely to be important in protecting against S. typhi, an intracellular bacterial infection, but heretofore this has not been demonstrated. Consequently, the presence of CTL activity against S. typhi-infected cells was investigated in human volunteers immunized with attenuated vaccine strains of S. typhi. Oral immunization with S. typhi strain CVD 908 elicited circulating CTL effector cells capable of killing S. typhi-infected autologous EBV-transformed cells. CTL activity was observed after 6 to 8 days of in vitro expansion in the presence of S. typhi-infected autologous EBV-transformed cells. Maximum CTL activity was observed 29 days after immunization. Depletion of CD8+ T cells eliminated or markedly reduced the CTL activity, while depletion of CD4+ T cells did not affect CTL responses. CTL activity was blocked by mAbs to human class I MHC Ags, but not by mAbs to class II MHC Ags. This first demonstration that oral immunization of volunteers with attenuated S. typhi elicits CD8+ T cell, MHC class I-restricted, CTL responses raises the possibility that CTL activity might play a significant role in protection during typhoid fever. It also encourages the future use of such attenuated strains as liver vector vaccines to stimulate specific CTL against relevant foreign Ags.

Administration, Oral↗

PubMed1995

Evaluation of the safety, immunogenicity, and efficacy in healthy adults of four doses of live oral hybrid Escherichia coli-Shigella flexneri 2a vaccine strain EcSf2a-2.

In previous trials, live invasive Escherichia coli-Shigella flexneri 2a hybrid vaccine candidate EcSf2a-2, administered to adult volunteers as 3 doses of ca. 2 x 10(9) colony forming units (c.f.u.) spaced over one week, induced fever and/or diarrhea in 11% of subjects and provided only limited protection (36% efficacy) against illness following challenge with virulent S. flexneri 2a. We sought to improve the clinical safety of this vaccine by administering a lower inoculum, and to enhance protective immunity by administering additional booster doses at 2 weeks. Twenty-one healthy adults were immunized with ca. 7 x 10(8) c.f.u. of EcSf2a-2 on days 0, 3, 14, and 17. The vaccine consistently colonized the intestine without causing serious adverse reactions; mild diarrhea developed in one subject and low grade fever in another. Vaccination elicited an antibody secreting cell (ASC) response to lipopolysaccharide (LPS) in all subjects, which was highest on day 7 and notably diminished thereafter on days 10, 16, 21, and 24, suggesting that active mucosal immunity developed rapidly. The magnitude of the response was modest (geometric mean peak = 16 IgA ASC/10(6) peripheral blood mononuclear cells) and an IgG serological response to LPS was detected in only 19% of subjects. Following experimental challenge with virulent S. flexneri 2a administered with bicarbonate buffer, shigellosis (diarrhea, dysentery, or fever) developed in 10 of 16 vaccine recipients (63%) and in 12 of 14 unvaccinated controls (86%), resulting in a vaccine efficacy of 27% (95% confidence limits -197, 82, p = 0.15, 1-tailed).(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗

PubMed1995

Simultaneous expression of CFA/I and CS3 colonization factor antigens of enterotoxigenic Escherichia coli by delta aroC, delta aroD Salmonella typhi vaccine strain CVD 908.

Among the known colonization factors of enterotoxigenic Escherichia coli (ETEC), CFA/I and CS3 (the common antigen in the CFA/II family of fimbrial antigens) are two of the most prevalent fimbrial antigens found in clinical isolates but are never expressed by the same wild-type strain. We manipulated the genetic determinants encoding CS3 and CFA/I fimbriae so that these two important colonization factors are expressed simultaneously in attenuated Salmonella typhi live oral vaccine strain CVD 908, including after growth in liquid medium (CFA/I is poorly expressed by wild-type ETEC in broth culture). The recombinant fimbrial structures produced by CVD 908 are morphologically indistinguishable from the CS3 fibrillae and CFA/I rod-like fimbriae produced by ETEC, and are recognized by monospecific CS3 and CFA/I antibodies. This prototype construct may prove useful in investigating the live vector approach to immunoprophylaxis of ETEC diarrheal disease.

Antigens, Bacterial↗

PubMed1995

Expression of fragment C of tetanus toxin fused to a carboxyl-terminal fragment of diphtheria toxin in Salmonella typhi CVD 908 vaccine strain.

We report the expression of fragment C of tetanus toxin (FC) fused to the eukaryotic cell binding domain (the carboxyl-terminus) of diphtheria toxin (FC-bDt fusion) in attenuated Salmonella typhi live vector vaccine strain CVD 908. The FC-bDt protein fusion was constructed using plasmid pTETnir15 which carries the gene encoding FC under control of the nirB promoter (nirBP). The open reading frame for FC was modified to incorporate an in-frame glycine-proline hinge region and a set of four restriction sites at the 3' end of the FC gene. A 482 bp DNA fragment encoding the eukaryotic cell binding domain of diphtheria toxin was then inserted at the 3' end of the modified FC gene to create an in-frame FC-bDt fusion gene. The resulting plasmid, pOG215, was able to express the FC-bDt fusion protein in both Escherichia coli DH5a and S. typhi CVD 908, as evidenced by Western immunoblots using anti-FC and anti-C-terminal diphtheria toxin monoclonal antibodies. Maximum expression of the FC-bDt fusion protein was achieved by growing CVD 908(pOG215) at the low oxidation-reduction potential of thioglycollate broth, i.e. in conditions that activate nirBP and drive transcription of the FC-bDt fusion gene. Whereas maximum expression of FC alone was also observed using thioglycollate broth, expression of bDt alone was unsuccessful using a variety of growth conditions. FC fusions constitute one strategy to "rescue" expression of proteins which are otherwise difficult to express.

Antigens, Bacterial↗

PubMed1995

A modified Shigella volunteer challenge model in which the inoculum is administered with bicarbonate buffer: clinical experience and implications for Shigella infectivity.

In the absence of a definitive immunologic correlate of protection against shigellosis, promising Shigella vaccine candidates have been selected based on their ability to confer resistance against experimental challenge with wild-type Shigella in healthy adult volunteers. A limitation of this model has been the low and often variable attack rate of illness among controls, necessitating repeated inpatient studies to demonstrate statistically significant results. In this study, the Shigella challenge model was modified by using bicarbonate buffer instead of skimmed milk as the delivery vehicle to enhance survival of the ingested challenge inoculum. To determine the ability of the modified model to detect protective efficacy, 11 veteran volunteers (previously challenged with S. flexneri 2a in bicarbonate buffer) and 12 immunologically naive control subjects were challenged with 1.4 x 10(3) c.f.u. S. flexneri 2a. Shigellosis occurred in 3 veterans and 11 control subjects (27 vs 92%, p = 0.003), yielding a protective efficacy of 70%. Dose response was evaluated in an additional seven naive subjects who were inoculated with a log lower (1.4 x 10(2) c.f.u.) S. flexneri 2a and had a significantly diminished attack rate of shigellosis (317 (43%) vs 11/12 (92%), p = 0.04). These findings indicate that the modified bicarbonate challenge model using an inoculum of 10(3) c.f.u. is a safe, repeatable, and valid method of selecting Shigella vaccines and other immunoprophylactic agents that are likely to confer protection against natural shigellosis.

Adolescent↗

PubMed1995

Infecting dose and severity of typhoid: analysis of volunteer data and examination of the influence of the definition of illness used.

Data from volunteers challenged with Salmonella typhi were reanalysed to explore the relationship between challenge dose and severity of disease. Among 120 ill volunteers who received between 10(5) and 10(9) organisms, dose was weakly correlated with peak temperature (r = 0.22, 95% CI 0.04-0.39), duration of temperature above 103 degrees F (39.4 degrees C: r = 0.13, 95% CI - 0.03 to 0.55) and symptom score (r = 0.27, 95% CI 0.09-0.43). The association with symptom score was lost after adjusting for year, and the findings depended on the definition of illness used: with stricter definitions the associations with temperature were also lost. The study shows the need for caution in interpreting the relationship between dose and severity of disease.

Administration, Oral↗

PubMed1995

Heterogeneity of enteroaggregative Escherichia coli virulence demonstrated in volunteers.

Enteroaggregative Escherichia coli (EAggEC) are diarrheal pathogens defined by aggregative adherence to HEp-2 cells. In an effort to identify pathogenic EAggEC isolates, four groups of 5 volunteers were fed 1 of 4 different EAggEC strains, each at a dose of 10(10) cfu. Strain 042 caused diarrhea in 3 of 5 adults; 3 other EAggEC isolates (17-2, 34b, and JM221) failed to elicit diarrhea. A gene encoding enterotoxin EAST1 was found in strains 042 and 17-2 but not 34b or JM221; a 108-kDa cytotoxin was expressed in all 4 isolates. All 4 isolates showed a modest degree of gentamicin protection in HEp-2 cells. 17-2, 34b, and JM221 expressed the fimbrial antigen AAF/I; 042 did not express this fimbria as determined by immunogold electron microscopy and genetic probe hybridization.

Bacterial Adhesion↗

PubMed1995

Initial clinical studies of CVD 112 Vibrio cholerae O139 live oral vaccine: safety and efficacy against experimental challenge.

Since October 1992, epidemics of cholera associated with Vibrio cholerae O group 139 have occurred in India, Bangladesh, and much of the rest of Asia. A volunteer model was used to determine the safety, immunogenicity, and efficacy of an attenuated delta ctxA delta zot delta ace delta cep V. cholerae O139 vaccine strain, designated CVD 112. Six volunteers received 10(6) cfu and 6 received 10(8) cfu of CVD 112. No subject who received the 10(6) dose had diarrhea or other severe symptoms after vaccination; 3 vaccinees developed mild diarrhea (mean stool volume, 648 mL) after receiving the higher dose. Five weeks after vaccination, 8 vaccinees and 15 unvaccinated control subjects underwent challenge with 10(6) cfu of wild type V. cholerae O139 AI1837. One vaccinee (13%) and 12 control subjects (80%) developed diarrhea after challenge (P = .003). The short-term protective efficacy conferred by vaccine strain CVD 112 was 84% and was remarkably similar to that conferred by primary wild type clinical infection (80%).

Administration, Oral↗

PubMed1995

Prevalence of Shigella enterotoxin 1 among Shigella clinical isolates of diverse serotypes.

Shigella enterotoxin 1 (ShET1) is a novel, iron-dependent, toxin encoded by chromosomal genes (set1). To determine the prevalence of this enterotoxin, 172 Shigella clinical isolates (and 10 enteroinvasive Escherichia coli [EIEC]) from distant areas worldwide, representing all 4 groups and 45 serotypes of Shigella, were screened for set1 by DNA colony hybridization and polymerase chain reaction amplification. set1 was present in all 22 Shigella flexneri 2a strains tested but was rare in isolates of other Shigella serotypes (3.3%, 5/150) and not found in EIEC (0/10). That ShET1 is found almost exclusively in S. flexneri 2a may help explain the epidemiologic predominance of this serotype in the developing world.

Bacterial Toxins↗

PubMed1995

Cholera.

Despite more than a century of study, cholera still presents challenges and surprises to us. Throughout most of the 20th century, cholera was caused by Vibrio cholerae of the O1 serogroup and the disease was largely confined to Asia and Africa. However, the last decade of the 20th century has witnessed two major developments in the history of this disease. In 1991, a massive outbreak of cholera started in South America, the one continent previously untouched by cholera in this century. In 1992, an apparently new pandemic caused by a previously unknown serogroup of V. cholerae (O139) began in India and Bangladesh. The O139 epidemic has been occurring in populations assumed to be largely immune to V. cholerae O1 and has rapidly spread to many countries including the United States. In this review, we discuss all aspects of cholera, including the clinical microbiology, epidemiology, pathogenesis, and clinical features of the disease. Special attention will be paid to the extraordinary advances that have been made in recent years in unravelling the molecular pathogenesis of this infection and in the development of new generations of vaccines to prevent it.

Antibody Formation↗

PubMed1995

The capsule and O antigen in Vibrio cholerae O139 Bengal are associated with a genetic region not present in Vibrio cholerae O1.

Vibrio cholerae O139 Bengal, although closely related to V. cholerae O1 El Tor, produces a polysaccharide capsule and has a distinct O antigen. We have identified a chromosomal region of at least 11 kb, as defined by three TnphoA mutations, that is required for the expression of both polysaccharides. Electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis show that these TnphoA mutants have lost the abilities both to express capsule and to produce lipopolysaccharide beyond the core oligosaccharide. Reactivity with O139 typing serum and resistance to serum are also lost in the mutants. DNA probes for this region do not hybridize with O1 V. cholerae but do react with other vibrios, implying that the region was recently acquired.

Bacterial Capsules↗