Immunoprophylaxis of RSV infection: advancing from RSV-IGIV to palivizumab and motavizumab.

Antibodies mediate humoral immune responses and play key roles in the defense of viral infection by the recognition, neutralization, and elimination of viruses from the circulation. For the prevention of respiratory syncytial virus (RSV) infection, the natural immune response to RSV from pooled human plasma has been harvested and successfully developed as a prophylactic polyclonal RSV hyperimmune globulin, RespiGam (RSV-IGIV; MedImmune, Gaithersburg, MD). The success of RSV-IGIV validated the immunoprophylaxis approach for RSV prevention and led to the development of Synagis (palivizumab; MedImmune, Gaithersburg, MD), a humanized monoclonal antibody (mAb) that binds to the RSV F protein. Palivizumab is a potent anti-RSV mAb that is about 50-fold more potent than RSV-IGIV, and since obtaining regulatory approval in 1998 it has been used extensively to help prevent severe RSV disease in high-risk infants and children. However, a very small number of patients receiving the drug do not appear to be adequately protected. To further improve protection against RSV, we have applied a directed evolution approach to enhance the binding of palivizumab to F protein by manipulation of both the on and off rates. These efforts have yielded a more potent second-generation mAb, motavizumab, which is currently under study in phase III clinical trials. Most recently, a third generation mAb, Numax-YTE, has been generated with the intent to extend the serum half-life of the mAb in humans. If successfully developed, this drug may offer the opportunity for less frequent dosing, obviating the need for the monthly treatments that are required with palivizumab. The development of these anti-RSV approaches exemplifies the accelerated pace of drug development made possible with cutting-edge antibody engineering technologies.

Safety and immunogenicity of oral inactivated whole-cell Helicobacter pylori vaccine with adjuvant among volunteers with or without subclinical infection.

Helicobacter pylori infection of the gastric mucosa can be found in approximately 50% of the world's 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 (LT(R192G)) as a mucosal adjuvant. In a dose-response study, 23 subjects with or without H. pylori infection were vaccinated with either 2.5 x 10(6) HWC, 2.5 x 10(8) HWC, or 2.5 x 10(10) HWC, plus 25 microg of LT(R192G). 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 x 10(10) HWC plus placebo-adjuvant, placebo-vaccine plus 25 microg of LT(R192G), placebo-vaccine plus placebo-adjuvant, or 2.5 x 10(10) HWC plus 25 microg of LT(R192G). 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 among H. pylori-infected and uninfected subjects following inoculation with 2.5 x 10(10) HWC plus 25 microg of LT(R192G). 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 eradicated H. 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.

Attenuated Shigella flexneri 2a Delta guaBA strain CVD 1204 expressing enterotoxigenic Escherichia coli (ETEC) CS2 and CS3 fimbriae as a live mucosal vaccine against Shigella and ETEC infection.

To construct a prototype hybrid vaccine against Shigella and enterotoxigenic Escherichia coli (ETEC), the genes encoding the production of ETEC CS2 and CS3 fimbriae were isolated and expressed in attenuated Shigella flexneri 2a guaBA strain CVD 1204. The CS2 cotA to -D genes, isolated from ETEC strain C91F, and the CS3 cstA to -H genes, subcloned from plasmid pCS100, were cloned into ~15-copy-number-stabilized pGA1 behind the osmotically regulated ompC promoter, resulting in high expression of both fimbriae. Under nonselective in vitro growth conditions, pGA1-CS2 and pGA1-CS3 were stable in CVD 1204, exhibiting a plasmid loss of only approximately 1% per duplication. Expression of CS2 and CS3 reduced the invasiveness of Shigella for HeLa cells and slowed the intracellular growth rate. Guinea pigs immunized intranasally with CVD 1204(pGA1-CS2) or CVD 1204(pGA1-CS3), or with a mixture of these strains, developed secretory immunoglobulin A (IgA) in tears and serum IgG antibodies against Shigella lipopolysaccharide, CS2, and CS3 antigens. Moreover, the animals were protected against keratoconjunctivitis following conjunctival challenge with virulent S. flexneri 2a strain 2457T. Animals immunized with Shigella expressing CS2 or CS3 developed serum antibodies that agglutinated Shigella as well as an ETEC strain bearing the homologous fimbriae, whereas animals immunized with combined CVD 1204(pGA1-CS2) and CVD 1204(pGA1-CS3) developed antibodies that agglutinated all three test strains. These observations support the feasibility of a multivalent vaccine against shigellosis and ETEC diarrhea consisting of multiple Shigella live vectors expressing relevant ETEC antigens.

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

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 microg). 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 (> or = 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 microg. We concluded that the C. 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.

Safety and immune responses to attenuated Salmonella enterica serovar typhi oral live vector vaccines expressing tetanus toxin fragment C.

Attenuated Salmonella enterica serovar Typhi vaccine strain CVD 908-htrA was used as a vector to deliver fragment C of tetanus toxin as a single-dose oral tetanus vaccine candidate to elicit protective levels of serum tetanus antitoxin. Twenty-one healthy adult volunteers received doses of 1.6 x 10(7) to 8.2 x 10(9) CFU of one of two strains, CVD 908-htrA(pTETnir15) or CVD 908-htrA(pTETlpp), which contained plasmid-encoded fragment C, with sodium bicarbonate, and the safety and immune responses to serovar Typhi antigens and tetanus toxin were assessed. No volunteer had fever or positive blood cultures after vaccination, although diarrhea occurred in 3 volunteers and vomiting in 2 volunteers within 3 weeks after vaccination. Most volunteers excreted the vaccine strain in the first 72 h after vaccination. Three of nine volunteers who received 10(8) CFU or higher doses of the CVD 908-htrA(pTETlpp) construct developed rises in serum antitoxin antibodies. The serum and cellular immune responses to serovar Typhi antigens were less frequent than those previously observed in volunteers who ingested the parent strain CVD 908-htrA. This study demonstrates that fragment C of tetanus toxin delivered orally to volunteers in an S. Typhi vector can elicit protective levels of serum antitoxin.

Albendazole treatment of children with ascariasis enhances the vibriocidal antibody response to the live attenuated oral cholera vaccine CVD 103-HgR.

Because concurrent infections with geohelminth parasites might impair the immune response to oral vaccines, we studied the vibriocidal antibody response to the oral cholera vaccine CVD 103-HgR in children infected with Ascaris lumbricoides and investigated the effect of albendazole pretreatment on the postvaccination response. Children with ascariasis were randomized to receive either 2 sequential doses of 400 mg of albendazole or placebo. After the second dose, CVD 103-HgR was given, and serum vibriocidal antibody levels were measured before and 10 days after vaccination. Postvaccination rates of seroconversion were greater in the treatment group that received albendazole (P=.06). Significantly greater rates of seroconversion and geometric mean titer were observed in the albendazole group in subjects with non-O ABO blood groups. A significant association was observed between vibriocidal seroconversion rates and treatment group, suggesting that A. lumbricoides infections impair the immune response to oral cholera vaccine, particularly in subjects of non-O blood groups.

Attenuated Shigella flexneri 2a vaccine strain CVD 1204 expressing colonization factor antigen I and mutant heat-labile enterotoxin of enterotoxigenic Escherichia coli.

A multivalent live oral vaccine against both Shigella spp. and enterotoxigenic Escherichia coli (ETEC) is being developed based on the hypothesis that protection can be achieved if attenuated shigellae express ETEC fimbrial colonization factors and genetically detoxified heat-labile toxin from a human ETEC isolate (LTh). Two detoxified derivatives of LTh, LThK63 and LThR72, were engineered by substitution-serine to lysine at residue 63, or lysine to arginine at residue 72. The genes encoding these two derivatives were cloned separately on expression plasmids downstream from the CFA/I operon. Following electroporation into S. flexneri 2a vaccine strain CVD 1204, coexpression of CFA/I and LThK63 or LThR72 was demonstrated by Western blot analysis, GM(1) binding assays, and agglutination with anti-CFA/I antiserum. Hemagglutination and electron microscopy confirmed surface expression of CFA/I. Guinea pigs immunized intranasally on days 0 and 15 with CVD 1204 expressing CFA/I and LThK63 or LThR72 exhibited high titers of both serum immunoglobulin G (IgG) and mucosal secretory IgA anti-CFA/I; 40% of the animals produced antibodies directed against LTh. All immunized guinea pigs also produced mucosal IgA (in tears) and serum IgG anti-S. flexneri 2a O antibodies. Furthermore, all immunized animals were protected from challenge with wild-type S. flexneri 2a. This prototype Shigella-ETEC hybrid vaccine demonstrates the feasibility of expressing multiple ETEC antigens on a single plasmid in an attenuated Shigella vaccine strain and engendering immune responses against both the heterologous antigens and vector strain.

Human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes.

A new approach for delivering vaccine antigens is the use of inexpensive, plentiful, plant-based oral vaccines. Norwalk virus capsid protein (NVCP), assembled into virus-like particles, was used as a test antigen, to determine whether immune responses could be generated in volunteers who ingested transgenic potatoes. Twenty-four healthy adult volunteers received 2 or 3 doses of transgenic potato (n=20) or 3 doses of wild-type potato (n=4). Each dose consisted of 150 g of raw, peeled, diced potato that contained 215-751 microgram of NVCP. Nineteen (95%) of 20 volunteers who ingested transgenic potatoes developed significant increases in the numbers of specific IgA antibody-secreting cells. Four (20%) of 20 volunteers developed specific serum IgG, and 6 (30%) of 20 volunteers developed specific stool IgA. Overall, 19 of 20 volunteers developed an immune response of some kind, although the level of serum antibody increases was modest.

Diminution of the anti-polyribosylribitol phosphate response to a combined diphtheria-tetanus-acellular pertussis/Haemophilus influenzae type b vaccine by concurrent inactivated poliovirus vaccination.

BACKGROUND: Prelicensure studies of Haemophilus influenzae type b vaccines (Hib) and diphtheria-tetanus-acellular pertussis vaccines (DTaP) were evaluated with concurrent oral poliovirus vaccine (OPV). However, inactivated poliovirus vaccine (IPV) is now recommended. A trial was conducted in which infants received a DTaP and Hib vaccine, separately (+) or combined (/), with either all OPV, all IPV or sequential IPV-OPV for the primary series of vaccinations. METHODS: In this protocol 567 infants were equally randomized to receive one of the following: Reference Arm A, DTaP + Hib + OPV; Treatment Arm B, DTaP/Hib + OPV; Treatment Arm C, DTaP/Hib + IPV at 2 and 4 months and OPV at 6 months; or Treatment Arm D, DTaP/Hib + IPV. antibodies against all administered antigens were measured at 7 months of age. Children with an antibody response to Hib (anti-polyribosylribitol phosphate (anti-PRP) <0.15 microg/ml had an antibody titer repeated after the toddler booster immunization. RESULTS: A significant diminution in the anti-PRP response was observed at 7 months of age in children given two or three doses of IPV concurrently with DTaP/Hib, compared with the groups given OPV. The geometric mean concentration of anti-PRP, percentage of children with > or = 0.15 microg/ml and percentage of children with > or = 1.0 microg/ ml, respectively, were: A, 4.4, 98%, 81%; B, 3.2, 94%, 78%; C, 1.3, 86%, 58% and D, 1.2, 84%, 53%. CONCLUSION: In this trial concurrent IPV appeared to interfere with the anti-PRP response to DTaP/Hib vaccine, suggesting that introduction of new vaccines may require evaluation of immune responses to all concurrently administered vaccines.

Role of EspB in experimental human enteropathogenic Escherichia coli infection.

Enteropathogenic Escherichia coli (EPEC), a leading cause of diarrhea among infants in developing countries, induces dramatic alterations in host cell architecture that depend on a type III secretion system. EspB, one of the proteins secreted and translocated to the host cytoplasm via this system, is required for numerous alterations in host cell structure and function. To determine the role of EspB in virulence, we conducted a randomized, double-blind trial comparing the ability of wild-type EPEC and an isogenic DeltaespB mutant strain to cause diarrhea in adult volunteers. Diarrhea developed in 9 of 10 volunteers who ingested the wild-type strain but in only 1 of 10 volunteers who ingested the DeltaespB mutant strain. Marked destruction of the microvillous brush border adjacent to adherent organisms was observed in a jejunal biopsy from a volunteer who ingested the wild-type strain but not from two volunteers who ingested the DeltaespB mutant strain. Humoral and cell-mediated immune responses to EPEC antigens were stronger among recipients of the wild-type strain. In addition, four of the volunteers who ingested the wild-type strain had lymphoproliferative responses to EspB. These results demonstrate that EspB is a critical virulence determinant of EPEC infections and suggest that EspB contributes to an immune response.

Shigella flexneri 2a strain CVD 1207, with specific deletions in virG, sen, set, and guaBA, is highly attenuated in humans.

A phase 1 clinical trial was conducted among 35 healthy adult volunteers to evaluate the safety, immunogenicity, and shedding of different doses of CVD 1207, a live attenuated Shigella flexneri 2a vaccine candidate with specific deletion mutations in virG, sen, set, and guaBA. CVD 1207 retains the ability to invade epithelial cells but cannot effectively spread intercellularly after invasion (DeltavirG), does not produce enterotoxin (Deltasen and Deltaset), and has limited proliferation in vivo (DeltaguaBA). In a consecutive fashion, groups of three to seven subjects ingested a single oral dose of CVD 1207 at an inoculum of either 10(6), 10(7), 10(8), 10(9), or 10(10) CFU. CVD 1207 was remarkably well-tolerated at inocula as high as 10(8) CFU. In comparison, one of 12 subjects who received 10(9) CFU experienced mild diarrhea and another experienced a single episode of emesis. One of five subjects who received 10(10) CFU experienced watery diarrhea and emesis. All subjects who ingested doses of 10(8) to 10(10) CFU excreted the vaccine; in 23 of 25, the duration of excretion was

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

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 x 10(8) CFU), lower-dose vaccine (5 x 10(7) 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.

Production of IFN-gamma and IL-10 to Shigella invasins by mononuclear cells from volunteers orally inoculated with a Shiga toxin-deleted Shigella dysenteriae type 1 strain.

Volunteers were orally administered invasive, non-Shiga toxin-producing Shigella dysenteriae 1 to establish a challenge model to assess vaccine efficacy. In stepwise fashion, four separate groups were given 3 x 10(2), 7 x 10(3), 5 x 10(4), or 7 x 10(5) CFU. Using PBMC, proliferative responses and cytokine production were measured to S. dysenteriae whole-cell preparations and to purified recombinant invasion plasmid Ags (Ipa) C and IpaD. Anti-LPS and anti-Ipa Abs and Ab-secreting cells were also evaluated. Preinoculation PBMC produced considerable quantities of IL-10 and IFN-gamma, probably secreted by monocytes and NK cells, respectively, of the innate immune system. Following inoculation, PBMC from 95 and 87% of volunteers exhibited an increased production of IFN-gamma and IL-10, respectively, in response to Shigella Ags. These increases included responses to IpaC and IpaD among those volunteers receiving the lowest inoculum. No IL-4 or IL-5 responses were detected. Whereas there were no Ab or Ab-secreting cell responses in volunteers receiving the lowest inoculum, other dose groups had moderate to strong anti-LPS and anti-Ipa responses. These results suggest that in humans, type 1 responses play an important role in mucosal and systemic immunity to S. dysentariae 1.

Extensive swelling after booster doses of acellular pertussis-tetanus-diphtheria vaccines.

BACKGROUND: Diphtheria and tetanus toxoid combined with acellular pertussis (DTaP) vaccines are less reactogenic than diphtheria and tetanus toxoid combined with whole cell pertussis (DTwP) vaccines. However, local reactions increase in rate and severity with each successive DTaP dose, and swelling of the entire injected limb has been reported after booster doses. METHODS: We reviewed reports of swelling of the entire thigh or upper arm after the fourth and fifth dose, respectively, of DTaP vaccines administered in the National Institutes of Health multicenter comparative DTaP studies. Relationships were explored among reports of severe swelling, rates of other reactions, quantity of vaccine contents, and prevaccination and postvaccination antibody levels to pertussis toxin, tetanus toxin, and diphtheria toxin. RESULTS: Entire thigh swelling was an unsolicited reaction reported in 20 (2%) of the 1015 children who received 4 consecutive doses of the same DTaP vaccine. The reaction was associated with 9 of the 12 DTaP vaccines evaluated. Although there were no reports of swelling of the entire upper arm in 121 children given a fifth dose of the same DTaP, 4 (2.7%) of 146 recipients of 5 doses of a mixed schedule of DTaP vaccines experienced such swelling. Rates of other reactions were higher in children with entire thigh swelling than in those without. Of the children with entire thigh swelling, 60% had local pain, and 60% had erythema. All swelling subsided spontaneously without sequelae. There was a significant linear association between the rates of entire thigh swelling after dose 4 and diphtheria toxoid content in the DTaP products. Lesser degrees of swelling (>50 mm but less than entire limb) correlated with pertussis toxoid content after dose 4 and aluminum content after dose 5. No relationship was established between levels of serum antibody to diphtheria, tetanus, or pertussis toxin and rates of swelling of the whole thigh. CONCLUSIONS: Booster doses of DTaP vaccines can cause entire limb swelling, which is usually associated with redness and pain. Our data suggest that this extensive swelling reaction may be more common with vaccines containing high diphtheria toxoid content.

Lack of prophylactic efficacy of an enteric-coated bovine hyperimmune milk product against enterotoxigenic Escherichia coli challenge administered during a standard meal.

Orally administered bovine immunoglobulins with specific activity against colonization factors of enterotoxigenic Escherichia coli (ETEC) could provide passive protection against ETEC challenge in volunteers. Twenty healthy adult volunteers ingested either a placebo or a partially enteric-coated preparation of bovine immunoglobulins with activity against the colonization factor antigens CFA/I, CS3, and CS6 and then were challenged with ETEC strain E24377A (CS1+, CS3+) administered with a standard meal. There was no difference in the incidence or severity of diarrhea among the 10 volunteers who received the bovine immunoglobulins and the 10 who received placebo. Either the specificity or titer of anti-colonization factor antibodies or the formulation of antibodies in this product was not adequate to provide passive protection against ETEC challenge.

Validation of a gastrointestinal explant system for measurement of mucosal antibody production.

A gastrointestinal explant culture system was developed and compared to the mononuclear cell extraction and enzyme-linked immunospot assay method for measurement of immunoglobulin A (IgA) and IgG antibody-secreting cells (ASCs) in gastric antral and duodenal biopsies of non-Helicobacter pylori-infected volunteers. IgA and IgG were detected in explant supernatants during 6 to 7 days of culture in all subjects. IgA containing secretory component was also detected throughout the culture period, although peak production occurred only in the first 3 days. During 7 days of culture, the cumulative geometric mean IgA levels produced were 2.2 and 8.02 microg/ml/10 mg of antral and duodenal biopsy tissues, respectively, while the cumulative geometric mean IgG levels were 1.54 and 2.92 microg/ml/10 mg of antral and duodenal biopsy tissues, respectively. Cycloheximide treatment resulted in a >90% reduction in both immunoglobulin classes after 6 days of treatment compared to levels in untreated controls. The detection of IgA and IgG ASCs extracted from biopsies on days 1 and 6 of culture confirmed that the antibody detected was derived from mucosal lamina propria. The IgA and IgG ASC responses were positively correlated with antibody concentrations detected in culture supernatants (r = 0.87 and 0.85, respectively). These results validate the potential usefulness of our gastrointestinal explant system for the evaluation of mucosal effector B-cell function.

Randomized, double-blind, placebo-controlled, multicentered trial of the efficacy of a single dose of live oral cholera vaccine CVD 103-HgR in preventing cholera following challenge with Vibrio cholerae O1 El tor inaba three months after vaccination.

CVD 103-HgR is a live oral cholera vaccine strain constructed by deleting 94% of the gene for the enzymatically active A subunit of cholera toxin from classical Inaba Vibrio cholerae O1 569B; the strain also contains a mercury resistance gene as an identifying marker. This vaccine was well tolerated and immunogenic in double-blind, controlled studies and was protective in open-label studies of volunteers challenged with V. cholerae O1. A randomized, double-blind, placebo-controlled, multicenter study of vaccine efficacy was designed to test longer-term protection of CVD 103-HgR against moderate and severe El Tor cholera in U.S. volunteers. A total of 85 volunteers (50 at the University of Maryland and 35 at Children's Hospital Medical Center/University of Cincinnati) were recruited for vaccination and challenge with wild-type V. cholerae El Tor Inaba. Volunteers were randomized in a double-blind manner to receive, with buffer, a single oral dose of either CVD 103-HgR (2 x 10(8) to 8 x 10(8) CFU) or placebo (killed E. coli K-12). About 3 months after immunization, 51 of these volunteers were orally challenged with 10(5) CFU of virulent V. cholerae O1 El Tor Inaba strain N16961, prepared from a standardized frozen inoculum. Ninety-one percent of the vaccinees had a >/=4-fold rise in serum vibriocidal antibodies after vaccination. After challenge, 9 (39%) of the 23 placebo recipients and 1 (4%) of the 28 vaccinees had moderate or severe diarrhea (>/=3-liter diarrheal stool) (P < 0.01; protective efficacy, 91%). A total of 21 (91%) of 23 placebo recipients and 5 (18%) of 28 vaccinees had any diarrhea (P < 0.001; protective efficacy, 80%). Peak stool V. cholerae excretion among placebo recipients was 1.1 x 10(7) CFU/g and among vaccinees was 4.9 x 10(2) CFU/g (P < 0.001). This vaccine could therefore be a safe and effective tool to prevent cholera in travelers.

Validation and characterization of a human volunteer challenge model for cholera by using frozen bacteria of the new Vibrio cholerae epidemic serotype, O139.

Until recently, all epidemic strains of Vibrio cholerae were of the O1 serotype. Current epidemics have also been caused by a new serotype, Vibrio cholerae O139. Although the pathogenesis and clinical features of O139 cholera are similar to those of O1 cholera, immunity to serotype O1 does not confer immunity to serotype O139. Therefore, prior to beginning vaccine efficacy studies, we sought to validate the use of a large standardized frozen inoculum of virulent V. cholerae O139 4260B for use in a human volunteer challenge model. Healthy volunteers (n = 25) were recruited for an Internal Review Board-approved inpatient dose-escalation challenge. Our goal was to identify a dose at which the cholera attack rate and the geometric mean purge were sufficient for determining vaccine efficacy against moderate and severe disease. At a dose of 10(5) CFU, 8 of 10 volunteers experienced purging and had a positive stool culture for V. cholerae. However, at this dose, the geometric mean stool volume of 2,175 g was insufficient by study criteria. At a dose of 10(6) CFU, 14 of 15 volunteers experienced purging, with a geometric mean stool volume of 5,621 g. Disease severity was significantly greater in volunteers with blood group O than those with non-O blood types (10,353 g versus 3,555 g, P < 0.001). Following challenge, all volunteers demonstrated a significant rise in antitoxin antibodies but the serum vibriocidal titer was attenuated compared to that seen after challenge with an O1 strain. This model provides a reproducible illness of sufficient severity for testing the efficacies of new O139 or combined O1-O139 vaccines.

Effect of small bowel bacterial overgrowth on the immunogenicity of single-dose live oral cholera vaccine CVD 103-HgR.

Several live oral vaccines (polio, bovine rotavirus, CVD 103-HgR cholera) are less immunogenic in developing than in industrialized countries. It was hypothesized that proximal small bowel bacterial overgrowth (common in children in less developed countries but rare in industrialized settings) diminishes the vibriocidal antibody response to CVD 103-HgR. In total, 202 fasting Santiago schoolchildren aged 5-9 years had lactulose breath H2 tests to detect proximal small bowel bacteria 1 day before ingesting CVD 103-HgR. Florid small bowel overgrowth was observed in 10 (5.6%) of 178 analyzable children. In children with florid overgrowth, vibriocidal seroconversion differed little from other children (60% vs. 67%), but the geometric mean titer was lower (160 vs. 368; P=.25). By logistic regression, increased peak breath H2 at small bowel time points was associated with diminished seroconversion (P=.04), as was the interaction of H2 value and weight (children >25 kg had lower seroconversion rates among subjects with heaviest overgrowth).

Palatability, reactogenicity and immunogenicity of engineered live oral cholera vaccine CVD 103-HgR in Chilean infants and toddlers.

BACKGROUND: Live oral cholera vaccine CVD 103-HgR is well-tolerated and immunogenic when administered to adults, school age children and preschool children in a single 5 x 10(9) colony-forming unit dose. Because elicitation of immune responses after administration of a single dose is exceptional for any oral vaccine in any age group, CVD 103-HgR was used as a probe to investigate the clinical acceptability, practicality and immunogenicity of this vaccine in infants and toddlers 3 to 17 months of age. METHODS: The study was undertaken successively in 12- to 17-month-olds (n = 104), 7- to 11-month-olds (n = 106) and 3- to 5-month-olds (n = 102). One-half of the subjects were randomly allocated to receive vaccine and the other one-half to receive placebo, in double blind fashion. After 2 weeks of double blind follow-up, all subjects received a dose of vaccine. Vibriocidal antibody titers were measured on coded sera collected at baseline and 2 weeks after each dosing. The buffered vaccine "cocktail" had a volume of 100 ml; subjects who ingested > or =70 ml were considered fully vaccinated. FINDINGS: Only 37% of subjects overall (25% of 3- to 5-month-olds) ingested > or =70 ml of the cocktail. The vaccine was well-tolerated with no significant differences in the rate or severity of adverse reactions after ingestion of vaccine vs. placebo. Seroconversion after ingestion of a single dose of CVD 103-HgR was similar in fully vaccinated subjects (66%) and in those who ingested a smaller fraction of the vaccine cocktail (63%). Of subjects who ingested two doses, 5 of 118 excreted vaccine organisms on Day 7 after the first dose vs. 0 of 118 after the second dose. INTERPRETATION: Single dose oral CVD 103-HgR is well-tolerated and immunogenic in infants even when a partial dose is ingested. The buffered vaccine cocktail that is readily imbibed by older children is not appealing to young infants, and improved vaccine formulations and delivery vehicles for immunizing infants must be sought.