Immunogenicity of two formulations of oral cholera vaccine comprised of killed whole vibrios and the B subunit of cholera toxin.

Two formulations of oral cholera vaccine were evaluated for safety and immunogenicity in adult volunteers in Thailand, an area with sporadic cholera outbreaks. One formulation consisted of 2 x 10(11) killed vibrios and 5 mg of cholera toxin B subunit, as was previously evaluated in North American volunteers, and the other consisted of 1 x 10(11) killed vibrios and 1 mg of B subunit, as was recently evaluated in a field trial in Bangladesh. Three doses of each formulation were given with citrate-bicarbonate buffer. Neither formulation had adverse effects. The formulations stimulated similar serum immunoglobulin G (IgG) and IgA responses to Vibrio cholerae lipopolysaccharide and cholera toxin and intestinal secretory IgA responses to lipopolysaccharide and toxin after three doses. The formulation containing twice the quantity of killed vibrios stimulated better vibriocidal responses, especially to Ogawa serotype. A formulation of oral vaccine containing more killed vibrios than were included in the vaccine studied in the Bangladesh field trial may provide greater protection against cholera.     

The major subunit of the toxin-coregulated pilus TcpA induces mucosal and systemic immunoglobulin A immune responses in patients with cholera caused by Vibrio cholerae O1 and O139

Diarrhea caused by Vibrio cholerae is known to give long-lasting protection against subsequent life-threatening illness. The serum vibriocidal antibody response has been well studied and has been shown to correlate with protection. However, this systemic antibody response may be a surrogate marker for mucosal immune responses to key colonization factors of this organism, such as the toxin-coregulated pilus (TCP) and other factors. Information regarding immune responses to TCP, particularly mucosal immune responses, is lacking, particularly for patients infected with the El Tor biotype of V. cholerae O1 or V. cholerae O139 since highly purified TcpA from these strains has not been available previously for use in immune assays. We studied the immune responses to El Tor TcpA in cholera patients in Bangladesh. Patients had substantial and significant increases in TcpA-specific antibody-secreting cells in the circulation on day 7 after the onset of illness, as well as similar mucosal responses as determined by an alternate technique, the assay for antibody in lymphocyte supernatant. Significant increases in antibodies to TcpA were also seen in sera and feces of patients on days 7 and 21 after the onset of infection. Overall, 93% of the patients showed a TcpA-specific response in at least one of the specimens compared with the results obtained on day 2 and with healthy controls. These results demonstrate that TcpA is immunogenic following natural V. cholerae infection and suggest that immune responses to this antigen should be evaluated for potential protection against subsequent life-threatening illness.     

Synthetic fragments of Vibrio cholerae O1 Inaba O-specific polysaccharide bound to a protein carrier are immunogenic in mice but do not induce protective antibodies

Development of Vibrio cholerae lipopolysaccharide (LPS) as a cholera vaccine immunogen is justified by the correlation of vibriocidal anti-LPS response with immunity. Two V. cholerae O1 LPS serotypes, Inaba and Ogawa, are associated with endemic and pandemic cholera. Both serotypes induce protective antibody following infection or vaccination. Structurally, the LPSs that define the serotypes are identical except for the terminal perosamine moiety, which has a methoxyl group at position 2 in Ogawa but a hydroxyl group in Inaba. The terminal sugar of the Ogawa LPS is a protective B-cell epitope. We chemically synthesized the terminal hexasaccharides of V. cholerae serotype Ogawa, which comprises in part the O-specific polysaccharide component of the native LPS, and coupled the oligosaccharide at different molar ratios to bovine serum albumin (BSA). Our initial studies with Ogawa immunogens showed that the conjugates induced protective antibody. We hypothesized that antibodies specific for the terminal sugar of Inaba LPS would also be protective. Neoglycoconjugates were prepared from synthetic Inaba oligosaccharides (disaccharide, tetrasaccharide, and hexasaccharide) and BSA at different levels of substitution. BALB/c mice responded to the Inaba carbohydrate (CHO)-BSA conjugates with levels of serum antibodies of comparable magnitude to those of mice immunized with Ogawa CHO-BSA conjugates, but the Inaba-specific antibodies (immunoglobulin M [IgM] and IgG1) were neither vibriocidal nor protective in the infant mouse cholera model. We hypothesize that the anti-Inaba antibodies induced by the Inaba CHO-BSA conjugates have enough affinity to be screened via enzyme-linked immunosorbent assay but not enough to be protective in vivo.     

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.     

Analysis of expression of toxin-coregulated pili in classical and El Tor Vibrio cholerae O1 in vitro and in vivo.

The expression of toxin-coregulated pili (TCP) and their structural subunit TcpA was compared in 20 strains of Vibrio cholerae of the classical and El Tor biotypes. Bacteria were isolated from the intestines of rabbits with experimental cholera and compared with the same strains grown under optimal TCP expression conditions in vitro. Immunoblotting revealed that TcpA production was induced in both biotypes after vibrios entered the intestinal milieu; TcpA-negative inocula gave rise to TcpA-positive vibrios after multiplication in the gut. The levels of TcpA expressed during growth in the intestine were, for most strains, comparable to those attained under optimal growth conditions in vitro. Of 11 classical strains tested, 10 expressed TCP antigen on the bacterial surface at levels comparable to or exceeding those seen after growth in vitro as determined by an inhibition enzyme-linked immunosorbent assay. In contrast, only one of the nine El Tor strains studied produced detectable amounts of TCP surface antigen in vivo and no fimbriae or surface antigen reacting with anti-TCP serum was found on El Tor vibrios from human cholera stools. Distinct TCP fimbriae were observed by immunoelectron microscopy on classical-biotype vibrios grown either in rabbit intestines or in vitro but were not detected on El Tor vibrios. The results show that TCP is expressed on V. cholerae O1 of the classical biotype but not on V. cholerae O1 of the El Tor biotype in the intestines of rabbits with experimental cholera infection.     

Vibrio cholerae O139 conjugate vaccines: synthesis and immunogenicity of V. cholerae O139 capsular polysaccharide conjugates with recombinant diphtheria toxin mutant in mice

Epidemiologic and experimental data provide evidence that a critical level of serum immunoglobulin G (IgG) antibodies to the surface polysaccharide of Vibrio cholerae O1 (lipopolysaccharide) and of Vibrio cholerae O139 (capsular polysaccharide [CPS]) is associated with immunity to the homologous pathogen. The immunogenicity of polysaccharides, especially in infants, may be enhanced by their covalent attachment to proteins (conjugates). Two synthetic schemes, involving 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as activating agents, were adapted to prepare four conjugates of V. cholerae O139 CPS with the recombinant diphtheria toxin mutant, CRMH21G. Adipic acid dihydrazide was used as a linker. When injected subcutaneously into young outbred mice by a clinically relevant dose and schedule, these conjugates elicited serum CPS antibodies of the IgG and IgM classes with vibriocidal activity to strains of capsulated V. cholerae O139. Treatment of these sera with 2-mercaptoethanol (2-ME) reduced, but did not eliminate, their vibriocidal activity. These results indicate that the conjugates elicited IgG with vibriocidal activity. Conjugates also elicited high levels of serum diphtheria toxin IgG. Convalescent sera from 20 cholera patients infected with V. cholerae O139 had vibriocidal titers ranging from 100 to 3,200: absorption with the CPS reduced the vibriocidal titer of all sera to < or =50. Treatment with 2-ME reduced the titers of 17 of 20 patients to < or =50. These data show that, like infection with V. cholerae O1, infection with V. cholerae O139 induces vibriocidal antibodies specific to the surface polysaccharide of this bacterium (CPS) that are mostly of IgM class. Based on these data, clinical trials with the V. cholerae O139 CPS conjugates with recombinant diphtheria toxin are planned.     

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.     

A Vibrio cholerae classical TcpA amino acid sequence induces protective antibody that binds an area hypothesized to be important for toxin-coregulated pilus structure

Vibrio cholerae is a gram-negative bacterium that has been associated with cholera pandemics since the early 1800s. Whole-cell, killed, and live-attenuated oral cholera vaccines are in use. We and others have focused on the development of a subunit cholera vaccine that features standardized epitopes from various V. cholerae macromolecules that are known to induce protective antibody responses. TcpA protein is assembled into toxin-coregulated pilus (TCP), a type IVb pilus required for V. cholerae colonization, and thus is a strong candidate for a cholera subunit vaccine. Polypeptides (24 to 26 amino acids) in TcpA that can induce protective antibody responses have been reported, but further characterization of their amino acid targets relative to tertiary or quaternary TCP structures has not been done. We report a refinement of the TcpA sequences that can induce protective antibody. One sequence, TcpA 15 (residues 170 to 183), induces antibodies that bind linear TcpA in a Western blot as well as weakly bind soluble TcpA in solution. These antibodies bind assembled pili at high density and provide 80 to 100% protection in the infant mouse protection assay. This is in sharp contrast to other anti-TcpA peptide sera (TcpA 11, TcpA 13, and TcpA 17) that bind very strongly in Western blot and solution assays yet do not provide protection or effectively bind TCP, as evidenced by immunoelectron microscopy. The sequences of TcpA 15 that induce protective antibody were localized on a model of assembled TCP. These sequences are centered on a site that is predicted to be important for TCP structure.     

Construction and evaluation of a safe, live, oral Vibrio cholerae vaccine candidate, IEM108

IEM101, a Vibrio cholerae O1 El Tor Ogawa strain naturally deficient in CTXPhi, was previously selected as a live cholera vaccine candidate. To make a better and safer vaccine that can induce protective immunity against both the bacteria and cholera toxin (CT), a new vaccine candidate, IEM108, was constructed by introducing a ctxB gene and an El Tor-derived rstR gene into IEM101. The ctxB gene codes for the protective antigen CTB subunit, and the rstR gene mediates phage immunity. The stable expression of the two genes was managed by a chromosome-plasmid lethal balanced system based on the housekeeping gene thyA. Immunization studies indicate that IEM108 generates good immune responses against both the bacteria and CT. After a single-dose intraintestinal vaccination with 10(9) CFU of IEM108, both anti-CTB immunoglobulin G and vibriocidal antibodies were detected in the immunized-rabbit sera. However, only vibriocidal antibodies are detected in rabbits immunized with IEM101. In addition, IEM108 but not IEM101 conferred full protection against the challenges of four wild-type toxigenic strains of V. cholerae O1 and 4 micro g of CT protein in a rabbit model. By introducing the rstR gene, the frequency of conjugative transfer of a recombinant El Tor-derived RS2 suicidal plasmid to IEM108 was decreased 100-fold compared to that for IEM101. This indicated that the El Tor-derived rstR cloned in IEM108 was fully functional and could effectively inhibit the El Tor-derived CTXPhi from infecting IEM108. Our results demonstrate that IEM108 is an efficient and safe live oral cholera vaccine candidate that induces antibacterial and antitoxic immunity and CTXPhi phage immunity.     

B-cell responses to lipopolysaccharide epitopes: Who sees what first - does it matter?

PROBLEM: Cholera is the paradigm for waterborne bacterial diseases. For over a 100 years, scientists have tried to develop a universally effective vaccine for cholera. We are hampered in our efforts because we do not know the details of the basic immune response to Vibrio cholerae antigens. What are the most proactive antigens? What special needs for immunization are engendered by previous exposure to cholera or the age of the individual? How long does immunity last, and is this immunity a classic immunologic memory or re-exposure and continual boosting? METHOD OF STUDY: Immunization with synthetic derivatives of the carbohydrate moieties of V. cholerae lipopolysaccharide (LPS) coupled to different carrier proteins (neoglycoconjugates, NGC) has allowed dissection of the response to the disaccharide array of perosamine that represent either the Inaba or the Ogawa serotype. Studying serum anti-LPS endpoint titers and the serum vibriocidal response to NGC provides insight into the importance of LPS serotype-specific B-cell epitopes and how antibody response are influenced by the form of the LPS immunogen. RESULTS: We found that murine serum antibody responses to V. cholerae LPS are dynamic. The magnitude of serum anti-LPS antibody titers and the capacity to induced vibriocidal antibodies (immunoglobulin M) are influenced by the initial immunizing serotype of LPS, the structure of the LPS immunogen (native LPS versus NGC), and the order of serotype immunization in a prime boost immunization strategy. The dynamic of the immune response to LPS immunogens is typified by the fact that the host species can affect the immunization response. We found mice do not make vibriocidal antibody to Inaba NGC but rabbits do. This is in contrast to the Ogawa NGC that induced vibriocidal antibody in mice. CONCLUSION: The results suggest that the host's B-cell repertoire can influence the immunization efficacy; therefore, the development of the new generation of NGC V. cholerae vaccines should focus on human volunteers and their ability to mount protective responses.     

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.     

Anti-class II monoclonal antibody-targeted Vibrio cholerae TcpA pilin: modulation of serologic response, epitope specificity, and isotype.

Toxin-coregulated pilus (TCP) is a colonization factor required for cholera infection. It is not a strong immunogen when delivered in the context of whole cells, yet pilus subunits or TcpA derivative synthetic peptides induce protective responses. We examined the efficacy of immunizing mice with TCP conjugated to anti-class II monoclonal antibodies (MAb) with or without the addition of cholera toxin (CT) or anti-CD40 MAb to determine if the serologic response to TcpA could be manipulated. Anti-class II MAb-targeted TCP influenced the anti-TCP peptide serologic response with respect to titer and isotype. Responses to TcpA peptide 4 were induced with class II MAb-targeted TCP and not with nontargeted TCP. Class II MAb-targeting TcpA reduced the response to peptide 6 compared to the nontargeted TCP response. Class II MAb-targeted TcpA, if delivered with CT, enhanced the serologic response to TcpA peptides. The effectiveness of the combination of targeted TCP and CT was reduced if anti-CD40 MAb were included in the primary immunization. These data establish the need to understand the role of TCP presentation in the generation of B-cell epitopes in order to optimize TcpA-based cholera vaccines.     

Mucosal and Systemic Immune Responses in Humans after Primary and Booster Immunizations with Orally Administered Invasive and Noninvasive Live Attenuated Bacteria

The mucosal and systemic immune responses after primary and booster immunizations with two attenuated live oral vaccine strains derived from a noninvasive (Vibrio cholerae) and an invasive (Salmonella typhi) enteric pathogen were comparatively evaluated. Vaccination with S. typhi Ty21a elicited antibody-secreting cell (ASC) responses specific for S. typhi O9, 12 lipopolysaccharide (LPS), as well as significant increases in levels of immunoglobulin G (IgG) and IgA antibodies to the same antigen in serum. A strong systemic CD4(+) T-helper type 1 cell-mediated immune (CMI) response was also induced. In contrast to results with Ty21a, no evidence of a CMI response was obtained after primary immunization with V. cholerae CVD 103-HgR in spite of the good immunogenicity of the vaccine. Volunteers who received a single dose of CVD 103-HgR primarily developed an IgM ASC response against whole vaccine cells and purified V. cholerae Inaba LPS, and seroconversion of serum vibriocidal antibodies occurred in four of five subjects. Serum IgG anti-cholera toxin antibody titers were of lower magnitude. For both live vaccines, the volunteers still presented significant local immunity 14 months after primary immunization, as revealed by the elevated baseline antibody titers at the time of the booster immunization and the lower ASC, serum IgG, and vibriocidal antibody responses after the booster immunization. These results suggest that local immunity may interfere with colonization of the gut by both vaccine strains at least up to 14 months after basis immunization. Interestingly, despite a low secondary ASC response, Ty21a was able to boost both humoral (anti-LPS systemic IgG and IgA) and CMI responses. Evidence of a CMI response was also observed for one of three volunteers given a cholera vaccine booster dose. The direct comparison of results with two attenuated live oral vaccine strains in human volunteers clearly showed that the capacity of the vaccine strain to colonize specific body compartments conditions the pattern of vaccine-induced immune responses.     

Live Oral Cholera Vaccine: Evaluation of the Clinical Effectiveness of Two Strains in Humans

El Tor Ogawa C14-S5 and EW-6, two live vaccine candidate strains, were given to volunteers in varying doses with and without bicarbonate. Vibrios were found in the stool of one of 32 men given the vaccine strain, and only three men developed a significant titer rise (fourfold or greater) at 2 weeks of vibriocidal or antitoxic antibody. Five men who had previously received 10⁹ organisms of the C14-S5 strain were challenged subsequently with virulent Ogawa 395 Vibrio cholerae. The rate of clinical infection in these men was no different than in unvaccinated controls. It was demonstrated that the live oral cholera vaccines did not remain viable in the intestine long enough to act antigenically.     

Immune response genes modulate serologic responses to Vibrio cholerae TcpA pilin peptides.

Cholera is an enteric disease caused by Vibrio cholerae. Toxin-coregulated pilus (TCP), a type 4 pilus expressed by V. cholerae, is a cholera virulence factor that is required for host colonization. The TCP polymer is composed of subunits of TcpA pilin. Antibodies directed against TcpA are protective in animal models of cholera. While natural or recombinant forms of TcpA are difficult to purify to homogeneity, it is anticipated that synthesized TcpA peptides might serve as immunogens in a subunit vaccine. We wanted to assess the potential for effects of the immune response (Ir) gene that could complicate a peptide-based vaccine. Using a panel of mice congenic at the H-2 locus we tested the immunogenicity of TcpA peptide sequences (peptides 4 to 6) found in the carboxyl termini of both the classical (Cl) and El Tor (ET) biotypes of TCP. Cl peptides have been shown to be immunogenic in CD-1 mice. Our data clearly establish that there are effects of the Ir gene associated with both biotypes of TcpA. These effects are dynamic and dependent on the biotype of TcpA and the haplotypes of the host. In addition to the effects of the classic class II Ir gene, class I (D, L) or nonclassical class I (Qa-2) may also affect immune responses to TcpA peptides. To overcome the effects of the class II Ir gene, multiple TcpA peptides similar to peptides 4, 5, and 6 could be used in a subunit vaccine formulation. Identification of the most protective B-cell epitopes of TcpA within a particular peptide and conjugation to a universal carrier may be the most effective method to eliminate the effects of the class II and class I Ir genes.     

Phase 1 Evaluation of Vibrio cholerae O1, Serotype Inaba, Polysaccharide-Cholera Toxin Conjugates in Adult Volunteers

Conjugate vaccines were prepared by binding hydrazine-treated lipopolysaccharide (DeALPS) from Vibrio cholerae O1, serotype Inaba, to cholera toxin (CT) variants CT-1 and CT-2. Volunteers (n = 75) were injected with either 25 μg of DeALPS, alone or as a conjugate, or the licensed cellular vaccine containing 4 × 10⁹ organisms each of serotypes Inaba and Ogawa per ml. No serious adverse reactions were observed. DeALPS alone did not elicit serum LPS or vibriocidal antibodies in mice and only low levels of immunoglobulin M (IgM) anti-LPS in the volunteers. Recipients of the cellular vaccine had the highest IgM anti-LPS levels, but the difference was not statistically significant from that elicited by the conjugates. The conjugates elicited the highest levels of IgG anti-LPS (DeALPS-CT-2 > DeALPS-CT-1 > cellular vaccine). Both conjugates and the cellular vaccine elicited vibriocidal antibodies: after 8 months, recipients of cellular vaccine had the highest geometric mean titer (1,249), followed by DeALPS-CT-2 (588) and DeALPS-CT-1 (330). The correlation coefficient between IgG anti-LPS and 2-mercaptoethanol (2-ME)-resistant vibriocidal antibodies was 0.81 (P = 0.0004). Convalescent sera from cholera patients had a mean vibriocidal titer of 2,525 that was removed by treatment with 2-ME. The vibriocidal activities of sera from all vaccine groups and from the patients were absorbed (>75%) by LPS but not by either CT-1 or CT-2. Conjugate-induced IgG vibriocidal antibodies persisted longer than those elicited by the whole-cell vaccine. Both conjugates, but not the cellular vaccine, elicited IgG anti-CT.     

Comparison of memory B cell, antibody-secreting cell, and plasma antibody responses in young children, older children, and adults with infection caused by Vibrio cholerae O1 El Tor Ogawa in Bangladesh

Children bear a large component of the global burden of cholera. Despite this, little is known about immune responses to cholera in children, especially those under 5 years of age. Cholera vaccine studies have demonstrated lower long-term protective efficacy in young children than in older children and adults. Memory B cell (MBC) responses may correlate with duration of protection following infection and vaccination. Here we report a comparison of immune responses in young children (3 to 5 years of age; n = 17), older children (6 to 17 years of age; n = 17), and adults (18 to 60 years of age; n = 68) hospitalized with cholera in Dhaka, Bangladesh. We found that young children had lower baseline vibriocidal antibody titers and higher fold increases in titer between day 2 and day 7 than adults. Young children had higher baseline IgG plasma antibody levels to Vibrio cholerae antigens, although the magnitudes of responses at days 7 and 30 were similar across age groups. As a surrogate marker for mucosal immune responses, we assessed day 7 antibody-secreting cell (ASC) responses. These were comparable across age groups, although there was a trend for older age groups to have higher levels of lipopolysaccharide-specific IgA ASC responses. All age groups developed comparable MBC responses to V. cholerae lipopolysaccharide and cholera toxin B subunit at day 30. These findings suggest that young children are able to mount robust vibriocidal, plasma antibody, ASC, and MBC responses against V. cholerae O1, suggesting that under an optimal vaccination strategy, young children could achieve protective efficacy comparable to that induced in adults.     

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 of cholera vaccines formulated with toxin-coregulated pilin peptide plus polymer adjuvant in mice.

Cholera is an acute diarrheal disease that is caused by the gram-negative bacterium Vibrio cholerae. The low efficacy of currently available killed-whole-cell vaccines and the reactinogenicity coupled with potential reversion of live vaccines have thus far precluded widespread vaccination for the control of cholera. Recent studies on the molecular nature of the virulence components that contribute to V. cholerae pathogenesis have provided insights into possible approaches for the development of a defined subunit cholera vaccine. Genetic analysis has demonstrated that the toxin-coregulated pilus (TCP) is the major factor that contributes to colonization of the human intestine by V. cholerae. In addition, polyclonal and several monoclonal antibodies directed against TCP have been shown to provide passive immunity to disease in the infant mouse cholera model. In the present study, synthetic peptides corresponding to portions of the C-terminal disulfide region of TcpA pilin were formulated with polymer adjuvants currently in clinical trials and used to actively immunize adult female CD-1 mice. The experimental vaccine formulations elicited high levels of antigen-specific immunoglobulin G (IgG), including a broad spectrum of subclasses (IgG1, IgG2a, IgG2b, and IgG3), and lower levels of IgA. Infant mice born to the immunized mothers showed 100% protection against a 50% lethal dose (1 LD(50)) challenge and 50% protection against a 10-LD(50) challenge with virulent strain O395. These results indicate that specific regions of TcpA, including those delineated by the peptides used in this study, have the potential to be incorporated into an effective defined subunit vaccine for cholera.     

Microtiter enzyme-linked immunosorbent assay for immunoglobulin g cholera antitoxin in humans: sensitivity and specificity

Serum samples were obtained from 92 informed, community volunteers before and 10, 21, and 28 days after they ingested 10(3) to 10(6)Vibrio cholerae of Inaba or Ogawa serotype and classical or El Tor biotype as part of a cholera vaccine development program. Pre- and postchallenge sera were examined for neutralizing antibody to cholera toxin by the rabbit skin permeability factor and adrenal cell techniques. Immunoglobulin G-binding antibodies to cholera toxin were quantitated by enzyme-linked immunosorbent assay (ELISA) in serum diluted 1:200. The results obtained in these cholera volunteers were compared with a negative control population comprising 30 people who ingested enteropathogenic Escherichia coli or E. coli which produced heat-stable but not heat-labile enterotoxin. Although all three antitoxin assays correlated closely with each other in both groups of volunteers, ELISA was more sensitive than either neutralization assay in detecting both subclinical and overt cholera infections. Seroconversion was demonstrated by ELISA in 58 of 66 (88%) volunteers who excreted V. cholerae, including 50 of 54 (93%) with clinical cholera, compared with 47 of 66 (71%) and 52 of 66 (79%) by the rabbit skin permeability factor and adrenal cell techniques, respectively. Although ELISA does not measure the toxin-neutralizing activity of antibodies directly, it provides a practical alternative to the rabbit skin permeability factor and adrenal cell assays.