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.     

Randomized, controlled human challenge study of the safety, immunogenicity, and protective efficacy of a single dose of Peru-15, a live attenuated oral cholera vaccine

Peru-15 is a live attenuated oral vaccine derived from a Vibrio cholerae O1 El Tor Inaba strain by a series of deletions and modifications, including deletion of the entire CT genetic element. Peru-15 is also a stable, motility-defective strain and is unable to recombine with homologous DNA. We wished to determine whether a single oral dose of Peru-15 was safe and immunogenic and whether it would provide significant protection against moderate and severe diarrhea in a randomized, double-blind, placebo-controlled human volunteer cholera challenge model. A total of 59 volunteers were randomly allocated to groups to receive either 2 x 10(8) CFU of reconstituted, lyophilized Peru-15 vaccine diluted in CeraVacx buffer or placebo (CeraVacx buffer alone). Approximately 3 months after vaccination, 36 of these volunteers were challenged with approximately 10(5) CFU of virulent V. cholerae O1 El Tor Inaba strain N16961, prepared from a standardized frozen inoculum. Among vaccinees, 98% showed at least a fourfold increase in vibriocidal antibody titers. After challenge, 5 (42%) of the 12 placebo recipients and none (0%) of the 24 vaccinees had moderate or severe diarrhea (> or = 3,000 g of diarrheal stool) (P = 0.002; protective efficacy, 100%; lower one-sided 95% confidence limit, 75%). A total of 7 (58%) of the 12 placebo recipients and 1 (4%) of the 24 vaccinees had any diarrhea (P < 0.001; protective efficacy, 93%; lower one-sided 95% confidence limit, 62%). The total number of diarrheal stools, weight of diarrheal stools, incidence of fever, and peak stool V. cholerae excretion among vaccinees were all significantly lower than in placebo recipients. Peru-15 is a well-tolerated and immunogenic oral cholera vaccine that affords protective efficacy against life-threatening cholera diarrhea in a human volunteer challenge model. This vaccine may therefore be a safe and effective tool to prevent cholera in travelers and is a strong candidate for further evaluation to prevent cholera in an area where cholera is endemic.     

Safety, immunogenicity, and efficacy against cholera challenge in humans of a typhoid-cholera hybrid vaccine derived from Salmonella typhi Ty21a.

A live oral vaccine consisting of attenuated Salmonella typhi Ty21a expressing Vibrio cholerae O1 Inaba lipopolysaccharide (LPS) O antigen was constructed and tested in volunteers for safety, immunogenicity, and efficacy. Fourteen adults ingested three doses of 10(10) viable organisms with buffer. One month later, 8 vaccinees and 13 unimmunized controls were challenged with 10(6) pathogenic V. cholerae O1 E1 T or Inaba organisms. No significant adverse reactions to vaccination were observed. All volunteers had significant rises in serum immunoglobulin G (IgG) antibody to S. typhi LPS. Only 2 (14%) of 14 had significant rises in serum IgA or IgG antibody to Inaba LPS, and 5 (36%) of 14 had fourfold rises in vibriocidal antibody. In the challenge study, diarrhea occurred in 13 of 13 controls and 6 of 8 vaccinees (vaccine efficacy, 25%; P = 0.13). The vaccine significantly reduced the severity of the clinical illness (P less than 0.05) and caused decreased excretion of challenge vibrios (P less than 0.05). Although the typhoid-cholera hybrid vaccine did not provide significant protection overall against experimental cholera, this study demonstrates the importance of antibody to V. cholerae O antigen in ameliorating clinical illness and illustrates the use of an S. typhi carrier vaccine strain expressing a foreign antigen.     

Evaluation in humans of attenuated Vibrio cholerae El Tor Ogawa strain Texas Star-SR as a live oral vaccine

Texas Star-SR, an A- B+ mutant derived by nitrosoguanidine treatment from Vibrio cholerae El Tor Ogawa strain 3083, was fed to 68 volunteers as an oral vaccine in doses of 10(5) to 5 X 10(10) organisms with NaHCO3. Sixteen (24%) vaccinees experienced some loose stools (unrelated to vaccine dose), but in only one did the total stool volume exceed 1.0 liter. The vaccine strain was cultured from duodenal fluid of 35 of 46 (76%) persons who ingested doses of 10(8) organisms or greater. No A+ B+ toxinogenic revertants were found among 456 clinical isolates tested. Sixty-three vaccinees (93%) manifested seroconversions of vibriocidal antibody, whereas only 20 (29%) had significant rises in serum antitoxin titers. Paired intestinal fluids from 41 volunteers showed significant rises of secretory immunoglobulin A against lipopolysaccharide (29%), Ogawa outer membrane preparation (29%), and toxin (12%) antigens. In challenge studies with pathogenic V. cholerae El Tor Ogawa and El Tor Inaba, the attack rate in vaccinees (7 of 25) was significantly lower than in controls (18 of 25) (vaccine efficacy, 61%); furthermore, the diarrheal stool volume in vaccinees was significantly less than that in controls (P less than 0.01). Texas Star-SR served as a prototype to investigate the concept of immunoprophylaxis by means of attenuated strains as oral vaccines. These observations provide an invaluable background for planning future studies with newly developed attenuated strains prepared by recombinant DNA techniques.     

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.     

Construction and characterization of a nonproliferative El Tor cholera vaccine candidate derived from strain 638

In recent clinical assays, our cholera vaccine candidate strain, Vibrio cholerae 638 El Tor Ogawa, was well tolerated and immunogenic in Cuban volunteers. In this work we describe the construction of 638T, a thymidine auxotrophic version of improved environmental biosafety. In so doing, the thyA gene from V. cholerae was cloned, sequenced, mutated in vitro, and used to replace the wild-type allele. Except for its dependence on thymidine for growth in minimal medium, 638T is essentially indistinguishable from 638 in the rate of growth and morphology in complete medium. The two strains showed equivalent phenotypes with regard to motility, expression of the celA marker, colonization capacity in the infant mouse cholera model, and immunogenicity in the adult rabbit cholera model. However, the ability of this new strain to survive environmental starvation was limited with respect to that of 638. Taken together, these results suggest that this live, attenuated, but nonproliferative strain is a new, promising cholera vaccine candidate.     

Enterotoxin-specific immunoglobulin E responses in humans after infection or vaccination with diarrhea-causing enteropathogens

Cholera toxin (CT)-specific antibody responses of the immunoglobulin E (IgE) isotype in the sera of adult patients suffering from infection with either Vibrio cholerae O1, V. cholerae O139, or enterotoxigenic Escherichia coli (ETEC) were analyzed and compared with those in the sera of volunteers immunized with a bivalent B subunit O1/O139 whole-cell cholera vaccine. A significant IgE response to CT was observed in 90% of the patients with V. cholerae O1 infection (18 of 20; P = <0.001) and 95% of the patients with V. cholerae O139 infection (19 of 20; P = <0.001). Similarly, the majority of the patients with ETEC diarrhea (83%; 13 of 15) showed a positive IgE response to CT. Eight of 10 North American volunteers (80%) orally challenged with V. cholerae O1 showed CT-specific IgE responses (P = 0.004). In contrast, Swedish volunteers immunized with the oral cholera vaccine showed no IgE responses to CT (P value not significant). During the study period, total IgE levels in the sera of the diarrheal patients, the North American volunteers, and the Swedish cholera vaccinees alike remained unchanged. However, the total IgE levels in the sera of patients and healthy Bangladeshi controls were on average 89-fold higher than those in the sera of the healthy Swedish volunteers and 34-fold higher than those in the sera of the North American volunteers.     

Preliminary Assessment of the Safety and Immunogenicity of a New CTXΦ-Negative, Hemagglutinin/Protease-Defective El Tor Strain as a Cholera Vaccine Candidate

Vibrio cholerae 638 (El Tor, Ogawa), a new CTXΦ-negative hemagglutinin/protease-defective strain that is a cholera vaccine candidate, was examined for safety and immunogenicity in healthy adult volunteers. In a double-blind placebo-controlled study, no significant adverse reactions were observed in volunteers ingesting strain 638. Four volunteers of 42 who ingested strain 638 and 1 of 14 who received placebo experienced loose stools. The strain strongly colonized the human small bowel, as evidenced by its isolation from the stools of 37 of 42 volunteers. V. cholerae 638, at doses ranging from 4 × 10⁷ to 2 × 10⁹ vibrios, elicited significant serum vibriocidal antibody and anti-Ogawa immunoglobulin A antibody secreting cell responses.     

CpG DNA, liposome and refined antigen oral cholera vaccine

An oral cholera vaccine made up of three Vibrio cholerae antigens, i.e. lipopolysaccharide (LPS), recombinant toxin co-regulated pili (rTcpA) and heat-treated cholera toxin (H-CT) has been developed in six different formulations. Eight-week-old Wistar rats were divided into nine groups and immunized as follows: the first group received the oral vaccine 1 consisting of the three antigens (LPS, rTcpA and H-CT) associated with a liposome (L) and bacterial CpG-DNA (ODN#1826). The rats of groups 2 and 3 received oral vaccines 2 and 3 consisting of the liposome-associated three antigens with and without non-bacterial CpG-DNA (ODN#1982), respectively. Rats of groups 4 received oral vaccine 4 consisting of the three antigens mixed with the ODN#1826, similar to vaccine 1, but without liposome. Rats of groups 5 and 6 received oral vaccines 5 and 6 consisting of the three antigens with and without ODN#1982, respectively, similar to vaccines 2 and 3, but without liposome. Rats of groups 7, 8 and 9 received oral placebos, namely liposomes (L), ODN#1826 (CpG), and vaccine diluent, i.e. 5% NaHCO3 solution, respectively. All vaccines were given in three doses at 14-day intervals. It was found that the combination of liposome and ODN#1826 in vaccine 1 evoked the highest immune response to V. cholerae antigen compared to other vaccine formulations and placebos, as measured by the appearance of antigen-specific antibody-producing cells in the intestinal lamina propria. The immunogenicity according to the magnitude of the immune response was: V1>V2=V3>V4>V5=V6>V7=V8=V9. The results of this study indicate that CpG-DNA and liposome are effective mucosal adjuvants for an oral cholera vaccine prepared from refined V. cholerae antigens and their combination seems to be synergistic. The potential role of liposome as a vaccine delivery vehicle has been confirmed.     

Potential for reacquisition of cholera enterotoxin genes by attenuated Vibrio cholerae vaccine strain CVD 103-HgR.

The potential for reacquisition of ctxA genes by attenuated Vibrio cholerae O1 vaccine strain CVD 103-HgR was examined by performing a series of mating experiments under a variety of in vivo and in vitro conditions. We found no evidence that CVD 103-HgR could reacquire ctxA genes from wild-type V. cholerae O1 strains. However, if the donor V. cholerae O1 strains were genetically manipulated to add genes that allow chromosomal gene transfer, then ctxA sequences could be acquired by CVD 103-HgR. The minimal excretion of CVD 103-HgR by vaccinees and the refractoriness to reacquisition of ctxA sequences suggest that this well-tolerated, highly immunogenic live oral cholera vaccine will have a minimal environmental impact.     

Determinants of immunogenicity and mechanisms of protection by virulent and mutant Vibrio cholerae O1 in rabbits.

The colonizing capacities of 16 Vibrio cholerae strains, including nine genetically and/or phenotypically defined parent-mutant pairs, were determined in unobstructed adult rabbit small bowel. There were marked interstrain differences in colonizing capacity, which was enhanced by bacterial motility and the production of cholera holotoxin but was unrelated to production of cholera toxin B-subunit or hemolysin or to bacterial serotype or biotype. The role of colonizing capacity and other bacterial features in determining the immunizing efficiency of live V. cholerae was studied by determining the efficiency with which graded inocula of each strain immunized against attempted recolonization of the ileum or induction of choleralike diarrhea by the RITARD (removable intestinal tie-adult rabbit diarrhea) challenge technique using standard inocula of virulent V. cholerae. Mucosal colonizing capacity was the only quantitative predictor of bacterial immunizing capacity; none of the other bacterial features cited above influenced bacterial immunogenicity against either type of challenge, except as they affected colonizing capacity. Live V. cholerae immunized much more efficiently than Formalin-killed bacteria; the former caused marked protection after a single inoculum of 10(2) CFU, whereas the latter gave only partial protection after three inoculations of 10(11) killed organisms. Protection induced by live bacteria was due largely to resistance to colonization and included marked inhibition of bacterial growth within the bowel lumen. These findings strongly suggest that an optimally efficient oral cholera vaccine would be composed of avirulent live V. cholerae selected for their capacity to colonize the small-bowel mucosa.     

Vaccination of chickens with strain CVL30, a genetically defined Salmonella enteritidis aroA live oral vaccine candidate.

Newly hatched chicks were vaccinated orally with a genetically defined Salmonella enteritidis aroA candidate, strain CVL30. In chickens immunized with 10(5) or 10(9) CFU and challenged by the intravenous route with 10(8) CFU of S. enteritidis 109 Nalr at 8 weeks old, there were similar reductions in colonization of the spleens, livers, and ceca of vaccinees compared with unvaccinated controls. Two groups of newly hatched female chicks were vaccinated orally with 10(9) CFU of strain CVL30, and one group was revaccinated intramuscularly with 10(9) CFU at 16 weeks old. When challenged intravenously with S. enteritidis 109 Nalr at 23 weeks old, there was a reduction in the colonization of spleens, livers, ovaries, and ceca compared with unvaccinated controls. Inclusion of the intramuscular booster gave increased protection to the ovary, although the vaccine strain was isolated on one occasion from a batch of eggs laid at 20 weeks old. In chickens immunized with 10(9) CFU of strain CVL30 and challenged orally with 10(9) CFU of S. enteritidis 109 Nalr, there was a reduction in intestinal shedding of the challenge strain from vaccines compared with unvaccinated controls. Circulating immunoglobulin G antibodies to lipopolysaccharide (LPS) were detected in unvaccinated controls within 7 to 10 days of oral challenge. In contrast, circulating immunoglobulin G antibodies to LPS in vaccinees were not altered by the oral challenge, which suggested that vaccination reduced or prevented invasion by the challenge strain from the gut or multiplication of the challenge strain in the tissues. Newly hatched chicks were vaccinated orally with ca. 10(9) CFU of strain CVL30, and 1 day later, the vaccines and unvaccinated controls were challenged orally with 10(5) or 10(9) CFU of S. enteritidis 109 Nalr. Colonization of the ceca and invasion from the gut by the S. enteritidis challenge strain was reduced in the vaccines up to 5 days postchallenge compared with controls. In a second trial, vaccinees and controls were challenged orally with 10(7) or 10(9) CFU of S. typhimurium 2391 Nalr. In contrast to the challenge with S. enteritidis, colonization of the ceca and invasion by the S. typhimurium strain were not greatly reduced.     

Construction of a recombinant live oral vaccine from a non-toxigenic strain of Vibrio cholerae O1 serotype inaba biotype E1 Tor and assessment of its reactogenicity and immunogenicity in the rabbit model.

The disease cholera is an important cause of mortality in many developing countries. Though it can be controlled through improved sanitation, this goal is not easily attainable in many countries. Development of an efficacious vaccine offers the best immediate solution. A new oral candidate vaccine has been constructed from a non-toxigenic strain of Vibrio cholerae E1 Tor, Inaba, which is not only devoid of the cholera toxin (CT) virulence cassette but also is completely non-reactogenic in rabbit ileal loop assay. The strain, however, had toxR and tcpA genes. Through a series of manipulations, the ctxB gene of V. cholerae, responsible for the production of the 'B' subunit of the cholera toxin (CTB) was introduced into the cryptic hemolysin locus of the strain. The resulting strain, named vaccine attempt 1.3 (VA1.3), was found to be able to produce copious amounts of CTB. In the RITARD model this strain was found to be non-reactogenic and provided full protection against the challenge doses of both V. cholerae O1, classical and E1 Tor. In the immunized rabbit it invoked significant levels of anti-bacterial and anti-toxin immunity.     

宋内痢疾菌无毒株S7表达霍乱弧菌O抗原及霍乱毒素B亚单位工程菌的构建

以宋内氏痢疾菌无毒株S7作为受体,将含有编码霍乱弧菌O抗原及霍乱毒素B亚单位基因的质粒转移入其中,构建成重组菌株S7COB。质粒电泳图谱显示重组株中存在被转移的外源质粒。重组株的生化特性和毒力表型与受体相同。菌体凝集和全菌体酶联免疫吸附试验(ELISA)证明在重组株的菌体表面同时表达了霍乱弧菌和宋内氏痢疾菌的O抗原。GM1-ELISA表明该重组株能在细胞内表达霍乱毒素B亚单位。以5亿、10亿的重组株免疫LIBP品系小鼠,免疫小鼠对宋内氏毒株S63攻击的保护率为70％～100％,对霍乱弧菌毒株569B攻击的保护率为30％～54％。     

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.     

Protective efficacy in humans of killed whole-vibrio oral cholera vaccine with and without the B subunit of cholera toxin.

Natural protection from cholera is associated with local intestinal antibacterial and antitoxic antibodies, which appear to act synergistically. Although current parenteral cholera vaccines offer insufficient protection, new vaccines administered orally have more promise. Killed Vibrio cholerae, alone or given with the B subunit of cholera toxin, was evaluated in adult volunteers. Vaccinees, who received three doses of either vaccine, and unvaccinated controls ingested 10(6) V. cholerae organisms to determine the protective efficacy of the vaccines. The combination vaccine provided 64% protection, and the whole vibrio vaccine given alone provided 56% protection. In addition, illnesses in vaccines were milder than those in controls, and both vaccines gave complete protection against more severe disease. This substantial level of protection against a dose of V. cholerae that caused cholera in nearly 90% of controls suggests that these vaccines might provide at least as high a level of protection if given to the population of an endemic area. Indeed, a field efficacy trial is underway in Bangladesh, and preliminary data indicate a protective efficacy of 85% for a killed whole vibrio plus B subunit vaccine similar to that tested in volunteers and an efficacy of 58% for the killed whole vibrio vaccine alone. Thus, the studies in human volunteers were successful in predicting the substantial protection afforded by the vaccines in a cholera endemic area.     

Transcutaneous immunization with toxin-coregulated pilin A induces protective immunity against Vibrio cholerae O1 El Tor challenge in mice

Toxin-coregulated pilin A (TcpA) is the main structural subunit of a type IV bundle-forming pilus of Vibrio cholerae, the cause of cholera. Toxin-coregulated pilus is involved in formation of microcolonies of V. cholerae at the intestinal surface, and strains of V. cholerae deficient in TcpA are attenuated and unable to colonize intestinal surfaces. Anti-TcpA immunity is common in humans recovering from cholera in Bangladesh, and immunization against TcpA is protective in murine V. cholerae models. To evaluate whether transcutaneously applied TcpA is immunogenic, we transcutaneously immunized mice with 100 mug of TcpA or TcpA with an immunoadjuvant (cholera toxin [CT], 50 mug) on days 0, 19, and 40. Mice immunized with TcpA alone did not develop anti-TcpA responses. Mice that received transcutaneously applied TcpA and CT developed prominent anti-TcpA immunoglobulin G (IgG) serum responses but minimal anti-TcpA IgA. Transcutaneous immunization with CT induced prominent IgG and IgA anti-CT serum responses. In an infant mouse model, offspring born to dams transcutaneously immunized either with TcpA and CT or with CT alone were challenged with 10(6) CFU (one 50% lethal dose) wild-type V. cholerae O1 El Tor strain N16961. At 48 h, mice born to females transcutaneously immunized with CT alone had 36% +/- 10% (mean +/- standard error of the mean) survival, while mice born to females transcutaneously immunized with TcpA and CT had 69% +/- 6% survival (P < 0.001). Our results suggest that transcutaneous immunization with TcpA and an immunoadjuvant induces protective anti-TcpA immune responses. Anti-TcpA responses may contribute to an optimal cholera vaccine.     

Safety and Immunogenicity of Single-Dose Live Oral Cholera Vaccine Strain CVD 103-HgR,Prepared from New Master and Working Cell Banks

Currently, no cholera vaccine is available for persons traveling from the United States to areas of high cholera transmission and who for reasons of occupation or host factors are at increased risk for development of the disease. A single-dose oral cholera vaccine with a rapid onset of protection would be particularly useful for such travelers and might also be an adjunct control measure for cholera outbreaks. The attenuated Vibrio cholerae O1 vaccine strain CVD 103-HgR harbors a 94% deletion of the cholera toxin A subunit gene (ctxA) and has a mercury resistance gene inserted in the gene encoding hemolysin A. We undertook a phase I randomized placebo-controlled two-site trial to assess the safety and immunogenicity of a preliminary formulation of CVD 103-HgR prepared from new master and working cell banks. Healthy young adults were randomized (5:1 vaccinees to placebo recipients) to receive a single oral dose of ∼4.4 × 10⁸ CFU of vaccine or a placebo. Blood serum vibriocidal and cholera toxin-specific IgG antibodies were measured before and 10, 14, and 28 days following vaccination or placebo. Excretion of the vaccine strain in the stool was assessed during the first week postvaccination. A total of 66 subjects were enrolled, comprising 55 vaccinees and 11 placebo recipients. The vaccine was well tolerated. The overall vibriocidal and anti-cholera toxin seroconversion rates were 89% and 57%, respectively. CVD 103-HgR is undergoing renewed manufacture for licensure in the United States under the auspices of PaxVax. Our data mimic those from previous commercial formulations that elicited vibriocidal antibody seroconversion (a correlate of protection) in ∼90% of vaccinees. (This study has been registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT01585181","term_id":"NCT01585181"}}NCT01585181.)     

Phase I evaluation of delta virG Shigella sonnei live, attenuated, oral vaccine strain WRSS1 in healthy adults

We conducted a phase I trial with healthy adults to evaluate WRSS1, a live, oral Delta virG Shigella sonnei vaccine candidate. In a double-blind, randomized, dose-escalating fashion, inpatient volunteers received a single dose of either placebo (n = 7) or vaccine (n = 27) at 3 x 10(3) CFU (group 1), 3 x 10(4) CFU (group 2), 3 x 10(5) CFU (group 3), or 3 x 10(6) CFU (group 4). The vaccine was generally well tolerated, although a low-grade fever or mild diarrhea occurred in six (22%) of the vaccine recipients. WRSS1 was recovered from the stools of 50 to 100% of the vaccinees in each group. The geometric mean peak anti-lipopolysaccharide responses in groups 1 to 4, respectively, were 99, 39, 278, and 233 for immunoglobulin (IgA) antibody-secreting cell counts; 401, 201, 533, and 284 for serum reciprocal IgG titers; and 25, 3, 489, and 1,092 for fecal IgA reciprocal titers. Postvaccination increases in gamma interferon production in response to Shigella antigens occurred in some volunteers. We conclude that WRSS1 vaccine is remarkably immunogenic in doses ranging from 10(3) to 10(6) CFU but elicits clinical reactions that must be assessed in further volunteer trials.     

A clinical trial with Alice/R-75 strain,live attenuated serum inhibitor-resistant intranasal bivalent influenza A/B vaccine

A clinical trial was conducted with Alice/R-75 strain live attenuated intranasal influenza A/B vaccine. With double blind control 88 adult volunteers were administered 2 doses of Alice/R-75 vaccine, 93 volunteers received one dose of Alice/R-75 vaccine and one dose placebo solution and 94 subjects were administered 2 doses of placebo solution. Twenty-three other subjects received Alice strain monovalent influenza A vaccine. For comparison, data from 21 subjects who received monovalent intranasal R-75 strain influenza B in two doses is included. The vaccine was generally well tolerated. Four-fold serum hemagglutination-inhibiting (HAI) antibody titer rises to A/England/42/72 occurred in 39% of the monovalent Alice strain vaccinees; in contrast 18% of those given 2 doses of bivalent Alice/R-75 vaccine and 11% of those given 1 dose of bivalent vaccine had similar four-fold HAI antibody titer rises. HAI antibody titer rises to influenza B/Hong Kong/72 occurred in 38% of R-75 strain monovalent vaccinees, 14% of Alice/R-75 2-dose vaccinees and 11% of Alice/R-75 one dose vaccinees. An epidemic of influenza at the onset of the study made evaluation of the efficacy of the vaccine impossible.This study was supported by a grant from Smith, Kline and French Laboratories, Philadelphia, Pennsylvania