INTRODUCTION

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Infection by Vibrio cholerae remains a major cause of morbidity and mortality throughout the world (2). Adequate supplies of uncontaminated food and water coupled with proper sanitary measures, if supported by the appropriate infrastructure, would control cholera, but, unfortunately, this is still not feasible for many affected countries. An alternate means to control cholera is an effective economical cholera vaccine that would mitigate the spread of cholera and stabilize areas of endemicity.

Currently, the following two types of cholera vaccines are approved and in general use for humans: (i) a killed-whole-cell formulation representing both biotypes and serotypes representative of serogroup O1 (35) that is combined with the purified cholera toxin (CT-B) subunit and (ii) a genetically engineered, live-attenuated V. cholerae vaccine (e.g., CVD 103-HgR, O1, classical biotype, Inaba serotype) (17). Multiple doses of the killed-whole-cell vaccine afforded 50% protection in field trials. The most important target population for a cholera vaccine, young children, was even less well protected by this vaccine (less than 25%) (6). A single dose of the live-attenuated vaccine used in clinical trials of North American adults provided >90% protection against the virulent homologous strain and 65 to 80% protection against virulent El Tor biotype, Inaba serotype strains (18, 37, 38). Administration of the CVD 103-HgR vaccine in a large-scale field trial in an area of endemicity of Indonesia did not show any correlation between vaccination and increased protection (12). Another new oral vaccine strain CVD 111, which is a live-attenuated El Tor biotype, Ogawa serotype strain, provided 80% protection in adult volunteers (36). This vaccine is being evaluated together with CVD 103-HgR to determine if the combination can provide further protection against both biotypes in a single dose (40).

Despite the potential of live vaccine strains, two problems are related to their use. First, the live-attenuated strains cause side effects such as mild diarrhea, abdominal cramps, and low fever. Second, because these live strains are attenuated by deleting the ctx genes that are carried on a bacteriophage, there is concern that infection of vaccine strains by ctx-carrying phage may lead to reversion to virulence if these vaccines are used in regions where cholera is endemic (15).

An alternative approach to cholera vaccine design is the development of a subunit vaccine. Research during the last decade has provided new insights into the molecular mechanisms of V. cholerae pathogenesis. Prominent among these is the identification and characterization of the major colonization factor, toxin-coregulated pilus (TCP) (13, 16, 28, 31, 33, 34, 39, 41). TCP and its antigens are obvious targets for testing for inclusion in a subunit cholera vaccine. Of note, there is very little TCP detectable in the commercially available killed-whole-cell cholera vaccines (31), perhaps due to certain culture conditions that need to be optimized for TCP expression.

TCP is composed of a homopolymer of TcpA pilin, which is a 20.5-kDa pilin subunit (41). Rabbit polyclonal antibodies directed against TCP provide 100% protection against a challenge with 100 times the 50% lethal dose (100 LD₅₀) in the infant mouse cholera model (31). Various levels of protection in the infant mouse cholera model were achieved by passive administration of monoclonal antibodies (MAbs) raised against TCP. All the MAbs recognized TcpA, but the most protective MAbs mapped to the C-terminal region of the pilin (32, 33). Synthetic peptides TcpA4, TcpA5, and TcpA6 represent contiguous overlapping peptides corresponding to the carboxyl disulfide bond region of the TcpA pilin. Peptides 5 and 6 were recognized by protective MAbs. In other studies, rabbit antibodies raised against TcpA peptides 4 and 6 were found to be the most protective while antibodies to peptide 5 afforded some protection (34).

One of the problems of utilizing a peptide-based antigen is that, because of their small size, peptides are not likely to elicit a robust stimulation of the immune system. The inclusion of the appropriate adjuvant in vaccine formulations can overcome this problem (9). Polydi(carboxylatophenoxy) phosphazene (PCPP) (Avant Immunotherapeutics, Inc., Needham, Mass.) is a water-based ionically cross-linkable polymer adjuvant that has been used in human clinical trials. PCPP promotes sustained antigen release while retaining antigenic integrity (25, 26). Another promising adjuvant for human use is the nonionic block copolymer mixture of polyoxyethylene (POE) and polyoxypropylene (POP) (Vaxcel, Inc., Norcross, Ga.). By varying both the molecular weight and the proportions of hydrophilic and hydrophobic components of the POP and POE molecules, the formulations can be designed to achieve differential levels and specificities of adjuvant activity (21, 44). One of these copolymer mixtures, termed CRL-1005, has been used to augment the immune response of mice and rhesus monkeys to a commercially available human influenza vaccine (42, 43).

To date none of these adjuvants have been tested for their efficacy in enhancing immune responses directed against small synthetic peptides. In the present study, TcpA peptides 4 and 6 were formulated with either PCPP or CRL-1005 and used to immunize adult CD-1 mice. The immune responses to peptides alone and to the peptide-adjuvant mixture were assessed. The efficacy of each combination was then determined by challenging infant mice born to vaccinated and nonvaccinated adults.

	MATERIALS AND METHODS

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Peptide antigens and challenge strain. Peptides corresponding to portions of TcpA from classical biotype strain O395 were synthesized on an Applied Biosystems model 430A synthesizer using the N-hydroxybenzotriazole esters of tert-butoxycarbonyl amino acids in an N-methylpyrrolidone solvent coupling system as described by the manufacturer. Peptides 4 and 6 correspond to amino acid residues 145 to 168 and 174 to 199, respectively, of TcpA pilin (33). PCPP adjuvant was kindly provided by Avant Immunotherapeutics, Inc. The POE and POP copolymers (CRL-1005) were kindly provided by Vaxcel, Inc. V. cholerae O395 (classical, Ogawa) was used for challenge studies after growth in Luria-Bertani broth with a starting pH of 6.5 at 30°C for 18 h with aeration.

Experimental vaccination protocol. Female CD-1 mice, 6 to 8 weeks of age, were immunized in groups of five for each experiment. For all experimental and control formulations, mice were immunized three times subcutaneously at 4-week intervals. Experimental groups received formulations containing 100 µg of TcpA peptide/dose emulsified in adjuvant. The negative-control groups received phosphate-buffered saline (PBS) or adjuvant alone. For PCPP formulations, aqueous suspensions of TcpA peptides were freshly mixed with 100 µg of adjuvant/dose. The vaccine formulations containing TcpA peptide and the Vaxcel copolymer adjuvant were made by emulsifying 1 volume of aqueous antigen with 1 volume of oil phase copolymer adjuvant. Four weeks after each immunization, sera were collected for antibody analysis. Four to 6 weeks after the third immunization, female CD-1 mice were boosted intraperitoneally and mated with male CD-1 mice. Newborn suckling pups (4 to 5 days old) from the immunized or control mothers were used for challenge studies, with each challenge group containing 8 to 12 neonatal mice.

Measurement and characterization of immune responses. Murine antibody responses were measured by enzyme-linked immunosorbent assay (ELISA). TcpA peptide antigens were diluted in PBS to a concentration of 5 µg/ml and passively adsorbed to the wells of Immulon-2 assay plates (Dynex Technologies, Inc., Chantilly, Va.) by incubation of 100 µl/well for 18 h at 4°C. Sera from individual mice were diluted in PBS containing 4% (wt/vol) bovine serum albumin using a log₁₀ titration scheme. For immunoglobulin G (IgG) subclass analysis, sera were serially diluted fivefold starting with a 1:10 dilution. Antibody binding to the test antigen was detected using horseradish peroxidase (HRP)-conjugated goat anti-mouse Igs (IgG and IgA; Sigma, St. Louis, Mo.) and HRP-conjugated goat anti-IgG subclasses (IgG1, IgG2a, IgG2b, and IgG3), obtained from Southern Biotechnology Associates, Inc. (Birmingham, Ala.). HRP activity was measured using the colorimetric substrate 3',3',5',5'-tetramethylbenzidine (Sigma), and the absorbance was read at 450 nm by a 96-well microplate reader. Serum samples from individual mice were tested in triplicate to determine the means and standard deviations (SD). Endpoint titers were defined as the highest dilution of serum that yielded an absorbance value of at least twice that of the background. The levels of mucosal secretory IgA were approximated by measuring intestinal samples using the method described by Dickinson and Clements (10). The small intestine from duodenum to ileal-cecal junction was excised and homogenized with PBS. Samples were centrifuged, and the supernatants were used in ELISA.

Determination of immunization efficacy using the infant mouse model. Neonatal mice (4 to 5 days old) were removed from mothers and orally challenged with 1 LD₅₀ (5 × 10⁵ CFU) or 10 LD₅₀ (5 × 10⁶ CFU) of virulent V. cholerae O395 (41). Animals were monitored for 48 h. After 48 h, any surviving animals were euthanized and sera were collected for antibody analysis.

Statistical analyses. Antibody titers were scored as endpoint titers for a particular serum sample for individual mice in all groups. The log₁₀ titers were used to generate means and SD that were analyzed by analysis of variance (ANOVA) and Tukey's posttest analysis using Prizm software (Graph Pad Software, San Diego, Calif.). The survival curves were also generated using Prizm software. Survival fractions were calculated using the Kaplan-Meier method. To compare survival curves, the log rank test was used to determine if there was a linear trend for the survival curves. Statistical comparisons were based on the null hypothesis that the treatment did not change the slope of the curves. P values of >0.05 are not considered significant.

	RESULTS

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Enhanced immunogenicity of TcpA synthetic peptides in the presence of copolymer adjuvants. Previously, rabbit polyclonal antibodies were raised against individual TCP peptides TcpA4 and TcpA6 coupled to keyhole limpet hemocyanin and emulsified in Freund's complete adjuvant (31, 32). Anti-TcpA4 sera (titer of 1:3,200) were shown to be very effective at providing passive immunity against cholera infection in the infant mouse cholera model, while the sera specific for TcpA6 (1:3,200) were somewhat less effective.

View larger version (45K): [in this window] [in a new window]   FIG. 1.   Measurement of mouse serum antibodies to TcpA4 and TcpA6 peptides by ELISA. Female CD-1 mice were immunized with TcpA peptides alone and TcpA peptides formulated with PCPP or CRL-1005 copolymer adjuvants. All mice were immunized three times at 4-week intervals. Serum samples were taken 4 weeks after each immunization. The data shown represent the log10 means of the endpoint titers. The data were evaluated by ANOVA and Tukey multiple means comparison test. Those means which are significantly different within a group comparison (e.g., for the tertiary-serum comparison, TcpA4 alone [*] versus TcpA4 plus PCPP [**] or TcpA4 plus CRL-1005 [***]) have different numbers of asterisks above the bar. Immunization of mice with PBS or either adjuvant alone did not induce anti-TcpA peptide titers (data not shown).

Characterization of the Ig profile induced by vaccination with the peptides alone and in combination with the polymer adjuvants. To characterize the nature of the immune responses induced by the peptide-adjuvant formulations, the anti-TcpA peptide titers of IgG subclasses and of IgA (serum and secretory IgA [sIgA]) were determined from mice after the third immunization with the TcpA4 and TcpA6 peptides alone or peptides in combination with the PCPP or CRL-1005 adjuvant (Table 1). This analysis revealed that the vaccine formulations induced a broad spectrum of IgG subclasses (IgG1, IgG2a, IgG2b, and IgG3), with IgG1 and IgG2a being predominant.

Induction of protective host immunity by TcpA peptide vaccine formulations. Since the acquisition of passive immunity in young children from mother's milk is one of the goals of cholera vaccine development, we used a modification of the infant mouse protection assay based on the types of methods used in rotavirus vaccine development, whereby mothers are immunized and protective antibodies are acquired by placental transfer or in milk (5, 7, 23, 24, 29). The use of this method not only assesses the ability of the immunization regimen to select a VDJ, VJ solution for the specific antibodies but also assesses the efficacy of inducing a functional antibody that is delivered via natural means, which is not the case in the typical infant mouse cholera protection assay. In this study, after the third immunization, females were mated and pups were born on average 35 days postimmunization. Newborn CD-1 suckling pups were challenged with either 1 LD₅₀ (5 × 10⁵ CFU) or 10 LD₅₀ (5 × 10⁶ CFU) of virulent V. cholerae O395, and the survival rate was monitored for 48 h (Fig. 2). Statistical comparisons of the survival curves for selected treatment groups are shown in Table 2. For vaccine formulations that included TcpA4, the results with the polymer adjuvants were nearly identical. All the neonatal mice from mothers immunized with TcpA4 plus polymer adjuvant survived the 1-LD₅₀ challenge (Fig. 2A). This difference was statistically significant (P = 0.0017 for PBS versus TcpA4 plus PCPP; P = 0.0017 for PBS versus TcpA4 plus CRL-1005) in comparison with that for the neonatal mice born to nonimmunized CD-1 mothers given PBS (Table 2). The difference in protection afforded by the adjuvants was not significant. Neonatal mice from mothers immunized with TcpA4 peptide alone were partially protected (60%) from 1 LD₅₀, and the time until death was increased, but the protection was not significantly different (Fig. 2A and Table 2). The greater protection (1 LD₅₀) for neonatal mice born to mothers immunized with TcpA4 plus adjuvant than for those immunized with TcpA4 alone was statistically significant (P = 0.0297 for TcpA4 versus TcpA4 plus PCPP; P = 0.0297 for TcpA4 versus TcpA4 plus CRL-1005).

View larger version (24K): [in this window] [in a new window]   FIG. 2.   Protection from O395 challenge of neonatal CD-1 mice born to mothers immunized with TcpA4, TcpA4 plus PCPP, TcpA4 plus CRL-1005, TcpA6, TcpA6 plus PCPP, or TcpA6 plus CRL-1005. Five-day-old neonatal mice born to mothers immunized with TcpA4, TcpA4 plus PCPP, or TcpA4 plus CRL-1005 adjuvant (10 to 14 neonatal mice from each experimental group for TcpA4) were challenged with 1 LD50 (A) or 10 LD50 (B) of virulent V. cholerae O395. For TcpA6-immunized groups, eight neonatal mice were challenged with 1 LD50 (C) or 10 LD50 (D) of virulent V. cholerae O395. The survival rates were monitored every 2 h for 48 h. The data shown represent the combined results of two independent experiments.

Titer, antibody source, and LD₅₀ influence survival. We correlated the serologic titer of the IgG1 responses to peptides alone or preparations of peptides with the two adjuvants to the percent protection provided in the modified infant mouse challenge assay (Fig. 3). We chose to focus on IgG1 as it is representative of the isotypes (IgG1 and IgA) that completely correlate with protection. We developed a model that relates the mean log₁₀ titer of the mice for a particular treatment group/LD₅₀ of bacteria given plotted against the percent protection. Retrospective data (Table 3) for percent protection following in vitro mixing of antibody and bacteria were also analyzed and compared to the trend of the line for antibody mixed with bacteria in vivo. There is a direct correlation of anti-TcpA IgG1 titers and protection. The slopes are significantly different from each other (in vivo, 31.47; in vitro, 12.92) and indicate that, if bacteria are mixed with antibodies in vitro, the protection does not fall off as quickly as it does if they are mixed in vivo. For example, if the ratio of the log₁₀ titer is 2 to 1, for the in vivo mixture the protection decreases from 50.4 to 19.0%. The decrease for an in vitro mixture is a change of only about 13% (94.3 to 81.4%).

View larger version (14K): [in this window] [in a new window]   FIG. 3.   Correlation of the percent protection in the neonatal mouse assay with anti-TcpA titer/LD50. Data from this study and from others presented in Table 2 were used to generate curves that show the relationship between anti-TcpA titer and LD50 challenge dose and the percent protection provided by an antibody mixed with bacteria in vitro or obtained passively in vivo. The in vitro studies used 500 LD50, while the in vivo curve based on data from this study combined data for pups challenged with either 1 LD50 or 10 LD50. The curves were generated using the Prizm program, whereby the percent protection was plotted against log10 transformed titer/LD50. The equations for each curve are representative of the standard equation for a line, y = (slope ± SDx + y intercept ± SD).

	DISCUSSION

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In this report, we show that TcpA peptide antigens formulated with newly developed adjuvants intended for use in humans can be used to immunize adult female CD-1 mice, which can then provide passive immunity to their neonatal offspring. Vaccine formulations containing TcpA4 or TcpA6 peptides emulsified in PCPP or CRL-1005 adjuvants augmented antibody responses beyond those achieved for either peptide alone, which in turn corresponded to greater protection against V. cholerae challenge. The amount of peptide used in these studies, 100 µg, is on the high end of the immunogenic dose in mice. Smaller amounts of TcpA peptides (e.g., 25 µg) are immunogenic even if given in only two immunizations (W. F. Wade, personal observation). However, the dose and immunization schedule and route of administration of TcpA peptides in humans remain to be determined, and the amount of antigen will likely be different depending on the route and method chosen to deliver the peptides.

The finding that the vaccine formulations containing the TcpA4 peptide were more protective than those containing TcpA6 is consistent with previous passive immunization studies (34). Importantly, female mice immunized with TcpA peptides in adjuvant were able to transfer an antibody that was protective to suckling pups challenged with virulent V. cholerae O1. It is not known if the protective antibody in our present report was present in milk, transferred through the placenta, or both (1, 4). In humans, IgG has been shown to bind to placental Fc receptors, which facilitate its transport into the fetal circulation (14). Alternatively, there is IgG among other isotypes present in human breast milk. The extension of the adjuvants described in this study and TcpA peptides into humans could result in protective immunity due to IgG acquired by several means. The identity of the protective isotype that is most effective for protection against cholera is an open question. Historically, sIgA, which is usually present in mucosal secretions, has been considered the likely protective isotype against cholera. In support of the potential role of other isotypes, recent evidence indicates that IgG as well as IgM can enter the gut and that the entry of IgG may be enhanced in the local area where there is infection (3). The selective IgA deficiency is one of the most common immunodeficiencies, with 0.5% of the population having no or very low sIgA. There are no published reports of increased incidence of cholera or increased severity of disease in individuals of that phenotype. These patients clearly respond to vaccination with cholera vaccines with increased IgG (22). More data need to be acquired based on the responses of humans to determine the contribution of single or multiple isotypes to affording protection from cholera.

The formulations of TcpA peptides with PCPP or copolymer CRL-1005 adjuvants induced a broad spectrum of IgG subclasses. The highest titers were predominantly for the IgG1 and IgG2a subtypes, suggesting activation of both Th1 and Th2 type T helper cells. This is consistent with the PCPP-formulated influenza vaccine, which induced all IgG subclasses (14, 26). Copolymer CRL-1005 also activates both Th1 and Th2 type responses (43). While the TcpA-specific IgG induced by the vaccine formulations could contribute to the protection observed for the infant mice in the present study, a more accepted mechanism of protection involves mucosal IgA. Studies have shown that both Th1 and Th2 type cytokines can stimulate a mucosal immune response (8, 11, 19, 20).

Although the anti-TcpA peptide IgA titers induced by these experimental vaccine formulations were not as high as the IgG titers, they may contribute to the immune protection seen in the infant mouse challenge. Clearly there were significant increases in sIgA that paralleled the IgG1 increase. Thus, this adjuvant-peptide combination could be an important vaccine component that would generate a protective antibody titer in both the human intestine and breast milk. Since the protective antigens of cholera have not been conclusively defined, it is difficult to assess why people are protected after surviving infection or being immunized with the current whole-cell vaccines. Clearly, antilipopolysaccharide (anti-LPS) titers increase and the specific anti-LPS antibodies in the IgG and IgA compartments as well as in the IgM compartment can be measured. Work in our laboratory suggests that certain somatic mutations need to be acquired by the antibodies for anti-Ogawa immunity directed at the terminal LPS sugar (A. Chernyak et al., submitted for publication). However, since TcpA is not a strong immunogen in natural infections, it is not known how anti-TcpA antibodies mature upon repeated exposure to V. cholerae. The elements of what make a protective anticholera antiserum are usually associated with anti-LPS antibodies rather than anti-TcpA antibodies. This, however, does not preclude TcpA from being used as an immunogen with the types of adjuvants we described here. We expect that vaccination with TcpA would result in TcpA serologic responses that would also mature with the affinity and specificity that would be protective for subsequent exposure to V. cholerae.

The means by which an anti-TcpA antibody is combined with or is available along with the infectious inoculum is an important issue to understand. The standard infant mouse model mixes the antibody and bacteria in vitro, which is a very efficient means of linking the antibody to target epitopes. In the modification of the infant challenge assay that we report here, the antibody has to transfer to the infant mouse naturally and find the target epitope in the context of the gut milieu. Thus, the amounts of antibodies (titer) needed for protection for the two assays are likely to be different. A similar model to evaluate the efficacy of vaccine preparation has been utilized in studies of rotavirus. Rotavirus infection of infants causes disease by replication of the virus in mature epithelial cells of the small intestine. Like V. cholerae, rotavirus will not colonize the intestines in adult animals (27). Most animal studies with respect to rotavirus vaccine development have been performed by immunizing adult female mice and determining the efficacy by challenge of infant mice (5, 7, 23, 24, 28). To our knowledge this is the first study to show the efficacy of evaluating a maternally transferred mouse antibody for its role in protection against cholera. This is particularly relevant as infants are often cited as the population most at risk and are the targeted group for improved vaccines. Our new method of challenge will allow us to assess vaccine formulations in a setting that has direct relevance to human health applications.

Our results show that TcpA peptides can be combined with either PCPP or copolymer adjuvant CRL-1005 for use in inducing protective immunity to cholera infection. The V. cholerae TcpA antigen formulated with polymer adjuvants could have utility for human use. A subunit or peptide vaccine would be safer and less reactinogenic than a live cholera vaccine. Subunit vaccines can also be rigorously standardized during the manufacturing process, eliminating lot-to-lot variance that is problematic with attenuated vaccines. TCP is an obvious choice for this type of vaccine as it is a member of the type 4 pili, which contribute to the colonization capability of many species of gram-negative bacteria, such as Neisseria gonorrhoeae, Pseudomonas aeruginosa, and pathogenic strains of Escherichia coli (30).

Clearly, a data set that indicates that whole V. cholerae does not have to be used to induce antibodies that can protect against cholera is developing. The challenge is to find the right formulation (antigens and adjuvants) and the most effective method to deliver the vaccines that promote the concentration, affinity, and fine specificity of the protective antibodies that protect adults, children, and infants.