Steven S. Wasserman

Role of the eaeA gene in experimental enteropathogenic Escherichia coli infection.

Enteropathogenic Escherichia coli (EPEC) infections are a leading cause of infant diarrhea in developing countries. Recently eaeA, a gene necessary for the characteristic intimate attachment of EPEC to epithelial cells in tissue culture, was described. We conducted a randomized, double-blind study to determine the role of the eaeA gene in human EPEC infection. 11 adult volunteers ingested 2 x 10(10) colony-forming units of O127:H6 EPEC strain E2348/69, and an equal number received the same dose of an isogenic eaeA deletion mutant constructed from E2348/69. Volunteers were monitored for the development of diarrhea, fever, and systemic and gastrointestinal complaints. Diarrhea developed in all 11 volunteers who received E2348/69 and in 4 of 11 who received the mutant (P = 0.002). Fever was more common in recipients of the wild-type strain (P = 0.024). Stool volumes were lower in recipients of the mutant. All volunteers seroconverted to E2348/69 LPS, but the geometric mean peak titers of serum IgG and IgA in recipients of the mutant were lower than those of recipients of the wild-type strain. IgA against LPS was detected in the jejunal fluid of six of six recipients of E2348/69 and 5/6 recipients of the mutant. This study unambiguously assigns a role for eaeA as an EPEC virulence gene, but the residual diarrhea seen in recipients of the mutant indicates that other factors are involved.

Determinants of HIV-1 shedding in the genital tract of women

BACKGROUND Plasma HIV-1 RNA concentration has been the best predictor for risk of heterosexual and perinatal transmission. However, direct contact with HIV-1 present locally in the genital tract might be necessary for transmission. We aimed to assess the relation between HIV-1 shedding (RNA or culturable virus) in female genital secretions and other factors that might affect HIV-1 shedding. METHODS This was a cross-sectional study within the Womens Interagency HIV Study (WIHS), a prospective longitudinal cohort study of HIV-infected women. We enrolled 311 HIV positive women from Jan 30, 1997 to July 1, 1998. We did clinical assessments, cultured HIV-1, and measured RNA in peripheral blood mononuclear cells (PBMC) and genital secretions. We compared the results with univariate and multivariate analyses. Presence of HIV-1 RNA or culturable virus in genital secretions was defined as HIV-1 shedding. FINDINGS HIV-1 RNA was present in genital secretions of 57% (152/268) of women whereas infectious virus was detected only in 6% (17/271). Genital tract HIV-1 shedding was found in 80% (130/163) of women with detectable plasma RNA and 78% (116/148) of women with positive PBMC cultures. 33% (27/83) of women with less than 500 copies/mL plasma RNA and 39% (35/90) of those with negative PBMC cultures also had genital tract shedding. INTERPRETATION Plasma RNA concentration, both qualitatively and quantitatively, was the most important factor in predicting genital HIV-1 shedding, even among women receiving potent antiretroviral therapy. However, HIV-1 shedding did occur in women with less than 500 copies/mL plasma HIV-1 RNA. This finding suggests that a separate reservoir of HIV-1 replication may exist in some women.

Humoral, mucosal, and cellular immune responses to oral Norwalk virus-like particles in volunteers

Norwalk virus-like particles (VLPs), made from recombinant capsid protein, are a promising vaccine. Thirty-six healthy adult volunteers received 250 microg (n = 10), 500 microg (n = 10), or 2000 microg (n = 10) of orally administered VLP or placebo (n = 6). All vaccinees developed significant rises in IgA anti-VLP antibody-secreting cells. Ninety percent who received 250 microg developed rises in serum anti-VLP IgG; neither the rates of seroconversion nor geometric mean titers increased at the higher doses. About 30-40% of volunteers developed mucosal anti-VLP IgA. Lymphoproliferative responses and IFN-gamma production were observed transiently among those who received 250 microg or 500 microg but not 2000 microg of VLP. Studies to increase immunogenicity using a mucosal adjuvant are planned.

Efficacy trial of single-dose live oral cholera vaccine CVD 103-HgR in North Jakarta, Indonesia, a cholera-endemic area

A randomized, double-blind, placebo-controlled efficacy trial of one dose of CVD 103-HgR live oral cholera vaccine was performed in Indonesia from 1993 to 1997. 67,508 persons aged 2-41 years ingested vaccine or placebo and were followed for four years, detecting cholera cases using hospital-based surveillance. A nested reactogenicity study (538 vaccinees, 535 controls) revealed no vaccine-attributable side effects. A nested immunogenicity study (N=657) showed vibriocidal seroresponses in 64-70% of vaccinees vs 1-2% of controls. Cholera incidence was lower than expected. 103 cases of Vibrio cholerae O1 El Tor diarrhea were detected, 93 evaluable for vaccine efficacy (43 vaccine, 50 placebo; efficacy=14%). A suggestion of protection was observed among persons with blood group O [P=0.12]. Only seven cases occurred within six months of vaccination, precluding assessment of short-term efficacy. In Jakarta, single-dose CVD 103-HgR did not confer long-term protection. Short-term protection from a single-dose and long-term protection from two doses have yet to be studied.

Safety and Immunogenicity of Oral Inactivated Whole-Cell Helicobacter pylori Vaccine with Adjuvant among Volunteers with or without Subclinical Infection

ABSTRACT Helicobacter pylori infection of the gastric mucosa can be found in approximately 50% of the worlds population and is associated with a range of pathology, including peptic ulcer, atrophic gastritis, and gastric cancer. To explore immunization as a strategy for preventing and treating H. pylori-associated disease, we assessed the safety and immunogenicity in healthy adults of a formalin-inactivated, oral H. pylori whole-cell (HWC) vaccine, administered with or without mutant Escherichia coli heat-labile toxin (LTR192G) as a mucosal adjuvant. In a dose-response study, 23 subjects with or withoutH. pylori infection were vaccinated with either 2.5 × 106 HWC, 2.5 × 108 HWC, or 2.5 × 1010 HWC, plus 25 μg of LTR192G. Thereafter, a randomized study was conducted in which 18 H. pylori-infected subjects were assigned, in a double-blind fashion, to receive either 2.5 × 1010 HWC plus placebo-adjuvant, placebo-vaccine plus 25 μg of LTR192G, placebo-vaccine plus placebo-adjuvant, or 2.5 × 1010 HWC plus 25 μg of LTR192G. Diarrhea (six subjects), low-grade fever (five subjects), and vomiting (two subjects) were observed, usually after the first dose. Significant rises in geometric mean mucosal (fecal and salivary) anti-HWC immunoglobulin A antibodies occurred amongH. pylori-infected and uninfected subjects following inoculation with 2.5 × 1010 HWC plus 25 μg of LTR192G. Moreover, among H. pylori-negative volunteers, this regimen induced significant lymphoproliferative responses in 5 of 10 subjects and gamma interferon production responses to H. pylori sonicate in 7 of 10 subjects. There was no evidence that vaccination eradicatedH. pylori in infected volunteers. These results suggest that it is possible to stimulate mucosal and systemic immune responses in humans to H. pylori antigens by using an HWC vaccine.

Phase 2 clinical trial of attenuated Salmonella enterica serovar Typhi oral live vector vaccine CVD 908-htrA in U.S. volunteers.

ABSTRACT Salmonella enterica serovar Typhi strain CVD 908-htrA is a live attenuated strain which may be useful as an improved oral typhoid vaccine and as a vector for cloned genes of other pathogens. We conducted a phase 2 trial in which 80 healthy adults received one of two dosage levels of CVD 908-htrA in a double-blind, placebo-controlled, crossover study. There were no differences in the rates of side effects among volunteers who received high-dose vaccine (4.5 × 108 CFU), lower-dose vaccine (5 × 107 CFU), or placebo in the 21 days after vaccination, although recipients of high-dose vaccine (8%) had more frequent diarrhea than placebo recipients (0%) in the first 7 days. Seventy-seven percent and 46% of recipients of high- and lower-dose vaccines, respectively, briefly excreted vaccine organisms in their stools. All blood cultures were negative. Antibody-secreting cells producing antilipopolysaccharide (LPS) immunoglobulin A (IgA) were detected in 100 and 92% of recipients of high- and lower-dose vaccines, respectively. Almost half the volunteers developed serum anti-LPS IgG. Lymphocyte proliferation and gamma interferon production against serovar Typhi antigens occurred in a significant proportion of vaccinees. This phase 2 study supports the further development of CVD 908-htrA as a single-dose vaccine against typhoid fever and as a possible live vector for oral delivery of other vaccine antigens.

Safety and immunogenicity of increasing doses of a Clostridium difficile toxoid vaccine administered to healthy adults.

ABSTRACT Clostridium difficile is a major cause of nosocomial diarrhea in industrialized countries. Although most illnesses respond to available therapy, infection can increase morbidity, prolong hospitalization, and produce life-threatening colitis. Vaccines are being explored as an alternative means for protecting high-risk individuals. We assessed the safety, immunogenicity, and dose response of a parenteral vaccine containing C. difficile toxoids A and B. Thirty healthy adults were assigned to receive four spaced inoculations on days 1, 8, 30, and 60 with one of three doses of vaccine (6.25, 25, or 100 μg). At each dose level, subjects were randomized, in a double-blind fashion, to receive either the soluble toxoids (n = 5) or toxoids adsorbed to alum (n = 5). Subjects were monitored for clinical and immunologic responses to vaccination. Vaccination was generally well tolerated, with occasional, usually mild, systemic reactions (abdominal pain, arthralgia, and diarrhea). The most common local reaction, mild arm pain, was reported by all recipients of the toxoid-alum formulation. Nearly all subjects (≥90%) developed vigorous serum antibody responses to both toxins, as measured by immunoglobulin G (IgG) enzyme-linked immunosorbent assay and neutralization of cytotoxicity, whereas fecal IgA increases occurred in approximately 50%. Statistically significant effects of dose and formulation on immunogenicity were not seen, although antibody levels tended to be higher with the alum-adjuvanted formulations and with increasing doses of soluble toxoid. Serum antibody responses among the toxoid-alum group appeared to plateau at 25 μg. We concluded that theC. difficile toxoid vaccine is safe and immunogenic in healthy volunteers. Further development as a prophylactic vaccine or for producing C. difficile hyperimmune globulin is justified.

Reduction of transmission of shigellosis by control of houseflies (Musca domestica)

The effect of control of houseflies on the incidence of diarrhoea and shigellosis was evaluated in a prospective crossover intervention study at two military field bases several kilometers apart. In early summer, 1988, intensive fly control measures (mainly bait and trap strategy) were introduced on one base, while the other served as a control. After 11 weeks, as new cohorts arrived, the intervention was abruptly discontinued in the first base and instituted in the second for the next 11 weeks. The study was repeated the next summer. Overall, fly counts were 64% lower on the bases exposed to fly control measures (p = 0.024). Concomitantly, clinic visits dropped by 42% (p = 0.146) for diarrhoeal diseases and by 85% for shigellosis (p = 0.015), as did rates of seroconversion, by 76% (p = 0.024) for antibodies to Shigella and by 57% (p = 0.006) for antibodies to enterotoxigenic Escherichia coli. The findings indicate that houseflies, acting as mechanical vectors, transmit Shigella (and possibly enterotoxigenic E coli) diarrhoeal infections.

Safety and immunogenicity of single-dose live oral cholera vaccine CVD 103-HgR in 5-9-year-old Indonesian children.

Oral vaccines offer great promise as public-health measures to prevent disease in less-developed countries. CVD 103-HgR, a genetically engineered, attenuated, Vibrio cholerae O1 strain has proved effective in industrialised countries. We have assessed the safety, immunogenicity, and excretion of this live cholera vaccine in children in north Jakarta, Indonesia. 412 children aged 5-9 years received single doses of 5 x 10(6), 5 x 10(7), 5 x 10(8), 5 x 10(9), or 1 x 10(10) colony forming units (CFU) of CVD 103-HgR or placebo (5 x 10(8) inactivated Escherichia coli K-12) with buffer. All doses were well tolerated. The 5 x 10(8) CFU dose, which is highly immunogenic in subjects in industrialised countries (greater than 90% seroconversion), elicited seroconversions of vibriocidal antibody in only 16% of Indonesian children. By contrast, a single 5 x 10(9) CFU dose of vaccine resulted in high rates (75% and 87%) of seroconversion with two different batches of vaccine. A batch prepared with a centrifugation step gave significantly higher geometric mean titres (16-fold increase over baseline) than did a batch in which there was a filtration step between fermentation and lyophilisation (10-fold increase over baseline). At a 5 x 10(9) CFU dose, CVD 103-HgR is well tolerated and highly immunogenic in Indonesian children and should therefore be further investigated for use as a one-dose live oral cholera vaccine in developing countries.

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.