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.

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.

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.

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

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.

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.

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.

Expanded safety and immunogenicity of a bivalent, oral, attenuated cholera vaccine, CVD 103-HgR plus CVD 111, in United States military personnel stationed in Panama.

To provide optimum protection against classical and El Tor biotypes of Vibrio cholerae O1, a single-dose, oral cholera vaccine was developed by combining two live, attenuated vaccine strains, CVD 103-HgR (classical, Inaba) and CVD 111 (El Tor, Ogawa). The vaccines were formulated in a double-chamber sachet; one chamber contained lyophilized bacteria, and the other contained buffer. A total of 170 partially-immune American soldiers stationed in Panama received one of the following five formulations: (a) CVD 103-HgR at 10(8) CFU plus CVD 111 at 10(7) CFU, (b) CVD 103-HgR at 10(8) CFU plus CVD 111 at 10(6) CFU, (c) CVD 103-HgR alone at 10(8) CFU, (d) CVD 111 alone at 10(7) CFU, or (e) inactivated Escherichia coli placebo. Among those who received CVD 111 at the high or low dose either alone or in combination with CVD 103-HgR, 8 of 103 had diarrhea, defined as three or more liquid stools. None of the 32 volunteers who received CVD 103-HgR alone or the 35 placebo recipients had diarrhea. CVD 111 was detected in the stools of 46% of the 103 volunteers who received it. About 65% of all persons who received CVD 103-HgR either alone or in combination had a fourfold rise in Inaba vibriocidal titers. The postvaccination geometric mean titers were comparable among groups, ranging from 450 to 550. Ogawa vibriocidal titers were about twice as high in persons who received CVD 111 as in those who received CVD 103-HgR alone (600 versus 300). The addition of CVD 111 improved the overall seroconversion rate and doubled the serum Ogawa vibriocidal titers, suggesting that the combination of an El Tor and a classical cholera strain is desirable. While CVD 111 was previously found to be well tolerated in semiimmune Peruvians, the adverse effects observed in this study indicate that this strain requires further attenuation before it can be safely used in nonimmune populations.

Hepatitis B vaccination of premature infants: a reassessment of current recommendations for delayed immunization.

OBJECTIVE: Current American Academy of Pediatrics and United States Public Health Service Immunization Practices Advisory Committee recommendations for hepatitis B immunization in premature infants weighing <2 kg at birth born to hepatitis B surface antigen (HBSAg)-negative mothers are to delay the initiation of vaccination until such infants reach 2 kg or until 2 months of age. This proposal to delay vaccination at birth in these low-risk infants was based on limited studies not conducted in the United States. We sought to reassess current recommendations to delay administration of hepatitis B vaccine in low-risk premature infants by determining the immunogenicity of early hepatitis B vaccination in a US population and identifying variables associated with poor immunogenicity. METHODS: A total of 148 infants <37 weeks' gestation born to mothers negative for HBSAg were recruited at birth and stratified to three birth weight groups: <1000 g, 1000 to 1500 g, and >1500 g. Recombinant hepatitis B vaccine was administered within the first week of life, at 1 to 2 months of age, and at 6 to 7 months of age. Serum obtained at birth and after the second and third doses of vaccine was tested for antibody to HBSAg. Variables associated with poor response were sought prospectively by collecting demographic and clinical data. RESULTS: A total of 118 subjects (83%) completed the study. Postsecond dose sera were available for 117 infants and postthird dose sera were available for 112 infants. The seroprotection rate (attaining >/=10 mIU/mL HBS antibody) after two doses was low (25%) regardless of birth weight; infants weighing <1000 g at birth had the poorest response (11%). The seroprotection response rate after three doses of vaccine increased with birth weight; infants weighing 1500 g at birth (group 3; 84% response rate). The seroprotection response rate of group 3 infants after three doses of vaccine, although low, could not be differentiated from the response rates reported for full-term infants using 95% confidence intervals. Of all infants who did not achieve protective levels of antibody after three doses of vaccine, 96% (26/27) weighed <1700 g at birth. The geometric mean HBS antibody levels in responders were 88 and 386 mIU/mL after two and three doses, respectively. Of 36 children with a birth weight >1500 g, 33 (91%) achieved levels of HBS antibody >100 mIU/mL after three doses of vaccine, compared with 25/35 (71%) of infants with birth weight <1500 g. Using logistic regression analysis, nonresponders were more likely than were responders to have been treated with steroids (26% vs 9%) and to have had a low birth weight (1037 g vs 1455 g). In addition, the seroresponse rate of black infants was more likely than that of white infants to be associated with poor weight gain (falling off 2 percentile ranks in weight) in the first 6 months of life: 22% of black and 60% of white children who failed to gain weight adequately responded to vaccination, compared with 92% of black and 70% of white children who were growing adequately. Of interest, the only infant with a birth weight of >1700 g who did not make protective levels of specific antibody after three doses of vaccine was 2300 g at birth, but had inadequate weight gain in the first 6 months of life. CONCLUSIONS: This study supports current recommendations of the American Academy of Pediatrics and the Centers for Disease Control and Prevention for delaying the initiation of hepatitis B immunization beyond the first week of life for premature infants at low risk for hepatitis B infection, particularly in newborns weighing <1700 g at birth. In addition, we have identified variables other than birth weight that were associated with an inadequate immune response to early hepatitis B vaccination in premature infants, such as poor weight gain in the first 6 months of life

A single dose of live oral cholera vaccine CVD 103-HgR is safe and immunogenic in HIV-infected and HIV-noninfected adults in Mali.

Despite considerable experience with single-dose, live, oral cholera vaccine CVD 103-HgR in Asia, Europe, and the Americas, the vaccine had not been evaluated in sub-Saharan Africa or on individuals infected with human immunodeficiency virus (HIV). We therefore conducted a randomized, placebo-controlled, double-blind, cross-over clinical trial in 38 HIV-seropositive (without clinical acquired immunodeficiency syndrome (AIDS)) and 387 HIV-seronegative adults in Mali to assess its safety and immunogenicity. Adverse reactions (fever, diarrhoea and vomiting) were observed with similar frequency among vaccine and placebo recipients. The vaccine strain was not isolated from the coprocultures of any subject. The baseline geometric mean titre (GMT) of serum vibriocidal antibody was significantly lower in HIV-seropositives (1:23) than in HIV-seronegatives (1:65) (P = 0.002). Significant rises in vibriocidal antibody were observed in 71% of HIV-seronegatives and 58% of HIV-seropositives, and in 40% of HIV-seropositives with CD4+ counts below 500 per microliter. Following immunization, the peak vibriocidal GMT in HIV-seronegatives was 1:584 versus 1:124 in HIV-seropositives (P = 0.0006); in HIV-seropositives with CD4+ counts < 500 per microliter, the peak vibriocidal GMT was 1:40 (P = 0.03 versus other HIV-seropositives). CVD 103-HgR was safe in HIV-infected Malian adults, although serological responses were significantly attenuated among HIV-seropositives (particularly in those with CD4+ counts < 500 per microliter) relative to HIV-seronegatives. These results encourage further evaluations of this single-dose, oral cholera vaccine in high-risk populations such as refugees in sub-Saharan Africa.

PIP: In response to the 1994 cholera outbreak that swept through Rwandan refugee camps near Goma, Zaire, in 1994, the World Health Organization explored the immunogenicity of a new generation of single-dose, live oral cholera vaccines. One such vaccine, CVD 103-HgR, has been evaluated in Asia, Europe, and the Americas, but not in sub-Saharan Africa or in individuals infected with HIV. Therefore, the present study evaluated the safety and immunogenicity of this new vaccine in a randomized, placebo-controlled, double-blind, crossover clinical trial in Mali. Enrolled were 38 HIV-positive individuals without full-blown AIDS and 387 HIV-negative adults. Adverse reactions (fever, diarrhea, and vomiting) occurred with equal frequency in vaccine and placebo recipients. The vaccine strain was not isolated from the coprocultures of any subject. The baseline geometric mean titre (GMT) of serum vibriocidal antibody was significantly lower in HIV-positive subjects (1:23) than HIV-negatives (1:65). Significant rises in vibriocidal antibody were observed in 71% of HIV-seronegatives and 58% of HIV-positives and in 40% of HIV-positives with CD4 counts below 500/mcl. After immunization, the peak vibriocidal GMT in HIV-negative subjects was 1:584 compared with 1:124 in HIV-positive subjects. In HIV-positives with a CD4 count below 500/mcl, the peak vibriocidal GMT was 1:40. Although serologic responses were significantly attenuated among HIV-positive subjects, especially those with CD4 counts below 500/mcl, CVD 103-HgR was safe in HIV-infected Malian adults. Further evaluations of this single-dose oral cholera vaccine are recommended in high-risk populations such as refugees in sub-Saharan Africa.

Economisation of vaccination against Haemophilus influenzae type b: a randomised trial of immunogenicity of fractional-dose and two-dose regimens.

BACKGROUND: The cost of Haemophilus influenzae type b (Hib) conjugate vaccines has limited their use in non-industrialised countries. To identify more economical vaccination schedules, we carried out a randomised trial of the immunogenicity of alternative regimens to the standard three-dose series. METHODS: 627 Chilean infants were randomly allocated to one of four regimens with either Hib polysaccharide-tetanus toxoid conjugate vaccine (PRP-T) or Hib oligosaccharide-diphtheria mutant toxoid conjugate vaccine (PRP-CRM197), for a total of eight groups. All infants receive diphtheria-tetanus-pertussis (DTP) vaccine at ages 2, 4, and 6 months. The regimens included three full doses, three fractional doses consisting of one half or one third of the full dose, and a regimen of two full doses (at age 4 and 6 months). The primary outcome was the proportion of infants with serum anti-polyribosylribitol phosphate (PRP, the type b capsular polysaccharide) concentrations of 0.15 microg/mL or more at age 8 months. FINDINGS: 93% (95% CI 85-98) of infants vaccinated with three full doses of PRP-T or PRP-CRM197 (95% CI 84-98) achieved anti-PRP concentrations of 0.15 microg/mL or more at age 8 months, compared with 91% (83-96) to 100% (95-100) of infants immunised with any fractional-dose regimen. Of the infants vaccinated with two doses of PRP-T or PRP-CRM197, 99% (93-100) and 87% (77-93) developed anti-PRP concentrations of 0.15 microg/mL or more, respectively. INTERPRETATION: 91% (83-96) to 100% (95-100) of infants immunised with one-half or one-third of a full dose of Hib conjugate developed protective antibody concentrations. Carrier priming with DTP may make two-dose schedules an option in some places. These alternative regimens could bring the cost of Hib vaccines within reach of countries that currently cannot afford them.

Validation of a volunteer model of cholera with frozen bacteria as the challenge.

To evaluate a standardized inoculum of Vibrio cholerae for volunteer challenge studies, 40 healthy adult volunteers were challenged at three different institutions with a standard inoculum prepared directly from vials of frozen, virulent, El Tor Inaba V. cholerae N16961, with no further incubation. Groups of 5 volunteers, with each group including 2 volunteers with blood group O, were given a dose of 10(5) CFU, and 34 of the 40 volunteers developed diarrhea (mean incubation time, 28 h). Transient fevers occurred in 15 (37.5%) of the volunteers. V. cholerae was excreted by 36 of 40 volunteers. Five additional volunteers received 10(4) CFU, and four developed diarrhea but with a lower average purging rate than required for the model. Of the 40 volunteers, 37 developed rises in their vibriocidal and antitoxin titers similar to those in previous groups challenged with freshly harvested bacteria. We conclude that challenge with frozen bacteria results in a reproducible illness similar to that induced by freshly harvested bacteria. Use of this model should minimize differences in attack rates or severity when groups are challenged at different times and in different institutions.

Vibriocidal antibody responses in North American volunteers exposed to wild-type or vaccine Vibrio cholerae O139: specificity and relevance to immunity.

The emergence of a new agent of cholera, Vibrio cholerae O139, has prompted a reevaluation of the vibriocidal antibody assay. This assay, primarily directed to lipopolysaccharide, is an important correlate of O1 immunity. V. cholerae O139 strains are encapsulated, rendering them relatively resistant to killing by serum. Recent reports suggest that there is strain-to-strain variability in the sensitivity of the vibriocidal assay to fully encapsulated O139 strains. We have assessed a modified vibriocidal assay for fully encapsulated O139 strain AI-1837 and its unencapsulated mutant 2L in sera from 53 volunteers given wild-type AI-1837 or its attenuated derivative CVD 112 and from 48 controls challenged with V. cholerae O1 or strains of the family Enterobacteriaceae. Vibriocidal responses to the AI-1837 and 2L strains were seen in 67 and 89% of volunteers, respectively, following a single exposure to the wild-type strain. However, >50% of all controls had low-level vibriocidal responses to both strains. These nonspecific responses were transient and of the immunoglobulin G isotype. No binding activity against purified O139 lipopolysaccharide (LPS) by immunoblotting was seen in control sera. In contrast, vibriocidal assay and strain 2L LPS responses by immunoblotting were detectable in 91% of tested volunteers following a single exposure to O139. The presence of vibriocidal antibody to AI-1837 or 2L was not associated with protection in rechallenge studies with O139 strain AI-1837. The vibriocidal assay with unencapsulated strain 2L may be used to detect exposure to O139 strain AI-1837 in controlled research trials. However, its lack of specificity does not make it useful for determining exposure to V. cholerae O139 in the field.

A murine model of intranasal immunization to assess the immunogenicity of attenuated Salmonella typhi live vector vaccines in stimulating serum antibody responses to expressed foreign antigens.

The lack of a practical small animal model to study the immunogenicity of Salmonella typhi-based live vector vaccines expressing foreign antigens has seriously impeded the vaccine development process. For some foreign antigens, stimulation of serum IgG antibody is the desired, protective immune response. We administered to mice, by orogastric or intranasal (i.n.) routes, attenuated delta aroC delta aroD S. typhi CVD 908 carrying a plasmid encoding fragment C (fragC) of tetanus toxin fused to the eukaryotic cell receptor binding domain of diphtheria toxin (fragC-bDt), and monitored serum antibody. While orogastric inoculation of three doses was not immunogenic, i.n. immunization elicited high titers of serum IgG tetanus antitoxin, generating peak ELISA geometric mean titers (GMT) of 27024 and 35658 with 10(8) and 10(9) c.f.u. dosages, respectively; 10(9) c.f.u. i.n. of an delta aroA S. typhimurium live vector stimulated a peak antitoxin GMT of 376 405. Mice immunized with the S. typhi live vector were 100% protected against challenge with 100 50% lethal doses of tetanus toxin that rapidly killed all control mice. Intranasal immunization with two doses of S. typhi expressing unfused fragment C under control of an anaerobically-activated promoter derived from nirB stimulated significantly higher titers of serum neutralizing antitoxin than fused fragC-bDt controlled by the same promoter (GMT 0.10 AU ml-1 vs 0.01 AU ml-1, P = 0.0095). Two i.n. doses of S typhi encoding fragC under control of powerful constitutive promoter 1pp led to significantly higher peak serum neutralizing antitoxin titers than the otherwise identical construct utilizing the nirB promoter (peak GMT 0.72 AU ml-1 vs 0.10 AU ml-1, P = 0.022). The i.n. route of inoculation of mice may constitute a practical breakthrough that could expedite the development of some S. typhi-based live vector vaccines by allowing, for the first time, quantitative measurement of serum antibody responses to candidate constructs following i.n. mucosal immunization.

Safety of live oral Salmonella typhi vaccine strains with deletions in htrA and aroC aroD and immune response in humans.

A single-dose, oral Salmonella typhi vaccine strain has been sought as a carrier or vector of cloned genes encoding protective antigens of other pathogens. Such a hybrid vaccine, administered orally, would stimulate immune responses both at the mucosal surface and in the systemic compartment and would potentially provide protection against multiple pathogens. S. typhi CVD 908 and CVD 906, which harbor deletions in aroC and aroD, were further engineered by deletion in htrA to produce strains CVD 908-htrA and CVD 906-htrA, which are unable to sustain growth and are severely impaired in their ability to survive in host tissues. These strains were fed to humans at doses of 5 x 10(7) to 5 x 10(9) CFU with buffer, and safety and immune responses were assessed. CVD 908-htrA and CVD 906-htrA were well tolerated in volunteers; mild diarrhea in 3 of 36 volunteers and mild fever in 1 volunteer were the only notable adverse responses. The vaccine strains were not detected in blood cultures and only transiently detected in stool. Serum immune responses to S. typhi lipopolysaccharide and H antigens were observed in 75 to 100% of volunteers who received 5 x 10(8) to 5 x 10(9) CFU, and cells secreting S. typhi-specific antibodies were found in all volunteers after ingestion of either strain. Sixty-three percent to 83% of volunteers developed lymphoproliferative responses to S. typhi flagellar and particulate antigens after the higher doses. These studies demonstrate the potential of CVD 908-htrA as a live vector for the delivery of heterologous genes, and a clinical trial of such a construct is planned.