Antibody-Secreting Cell Responses after Vibrio cholerae O1 Infection and Oral Cholera Vaccination in Adults in Bangladesh

Infection with Vibrio cholerae and oral cholera vaccines (OCVs) induce transient circulating plasmablast responses that peak within approximately 7 days after infection or vaccination. We previously demonstrated that plasmablast responses strongly correlate with subsequent levels of V. cholerae-specific duodenal antibodies up to 6 months after V. cholerae infection. Hence, plasmablast responses provide an early window into the immunologic memory at the mucosal surface. In this study, we characterized plasmablast responses following V. cholerae infection using a flow cytometrically defined population and compared V. cholerae-specific responses in adult patients with V. cholerae O1 infection and vaccinees who received the OCV Dukoral (Crucell Vaccines Canada). Among flow cytometrically sorted populations of gut-homing plasmablasts, almost 50% of the cells recognized either cholera toxin B subunit (CtxB) or V. cholerae O1 lipopolysaccharide (LPS). Using a traditional enzyme-linked immunosorbent spot assay (ELISPOT), we found that infection with V. cholerae O1 and OCVs induce similar responses to the protein antigen CtxB, but responses to LPS were diminished after OCV compared to those after natural V. cholerae infection. A second dose of OCV on day 14 failed to boost circulating V. cholerae-specific plasmablast responses in Bangladeshi adults. Our results differ from those in studies from areas where cholera is not endemic, in which a second vaccination on day 14 significantly boosts plasmablast responses. Given these results, it is likely that the optimal boosting strategies for OCVs differ significantly between areas where V. cholerae infection is endemic and those where it is not.     

Cholera Caused by Vibrio cholerae O1 Induces T-Cell Responses in the Circulation

Considerable effort is being made to understand the acute and memory antibody responses in natural cholera infection, while rather less is known about the roles of cellular immune responses involving T and B lymphocytes. We studied responses in adult patients hospitalized with cholera caused by Vibrio cholerae O1. Peripheral blood mononuclear cells from patients (n = 15) were analyzed by flow cytometry after stimulation with V. cholerae O1 membrane protein (MP) or toxin-coregulated pilus antigen (TcpA). The gamma interferon (IFN-γ) and interleukin-13 (IL-13) responses in stimulated-lymphocyte supernatants were studied. The responses were compared with those of healthy controls (n = 10). Patients responded with increased frequencies of gut-homing CD4⁺ T cells (CD4⁺ β7⁺), gut-homing CD8⁺ T cells (CD8⁺ β7⁺), and gut-homing B cells (CD19⁺ β7⁺) at the early and/or late convalescent stages compared to the acute stage. After stimulation with MP or TcpA, proliferation of CD4⁺ and CD8⁺ T cells was increased at the acute stage and/or early convalescent stage compared to healthy controls. Increased IL-13 and IFN-γ responses were observed after antigenic stimulation at the acute and convalescent stages compared to healthy controls. Thus, increases in the levels of gut-homing T and B cells, as well as involvement of CD8 and CD4 Th1-mediated (IFN-γ) and CD4 Th2-mediated (IL-13) cytokine responses, take place in acute dehydrating disease caused by V. cholerae O1. Further studies are needed to determine if such responses are also stimulated after immunization with oral cholera vaccines and if these responses play a role in protection following exposure to cholera.Vibrio cholerae O1 is a common causative agent of acute watery diarrhea in children and adults in the developing world (1, 3, 10, 19). After colonizing the proximal small intestine, this bacterium produces cholera toxin, which induces a profuse secretory diarrhea. Cholera remains a key public health problem that results in epidemics in resource-poor settings.It is believed that the immune response to cholera is initiated by antigen presentation in the Peyer''s patches of the gastrointestinal mucosa, followed by migration of the stimulated antigen-specific B cells to regional lymph nodes and differentiation of these cells into specific antibody-secreting cells (28). Stimulation of the common mucosal immune system leads to production of both local and systemic antibodies (2, 15, 27) to virulence antigens of V. cholerae (25, 28).Natural cholera infection is believed to give rise to long-term protection against subsequent disease. Robust systemic and mucosal antibodies are produced to the V. cholerae lipopolysaccharide, to cholera toxin, and to colonization factors, including the major subunit of the toxin-coregulated pilus, TcpA (2, 24, 25, 28). We have recently shown that there is induction of memory B-cell responses following infection, which may play a role in longer-lasting protection (14). In addition, recent evidence suggests that an innate component of the immune system may also play a role in the host response to cholera (9, 22, 26). Studies with experimental animals have shown that the mucosal immune response to cholera toxin is T cell dependent and that CD4 T helper cells have an important role (7, 12, 13). However, not much is known about the role of the adaptive cellular immune responses in patients with cholera. The aim of the present study was to decipher the role of T- and B-cell-mediated immune responses in natural cholera infection in adults hospitalized with dehydrating illness, who were followed from the acute stage to convalescence.     

Comparison of Immune Responses to the O-Specific Polysaccharide and Lipopolysaccharide of Vibrio cholerae O1 in Bangladeshi Adult Patients with Cholera

Immunity against Vibrio cholerae O1 is serogroup specific, and serogrouping is defined by the O-specific polysaccharide (OSP) part of lipopolysaccharide (LPS). Despite this, human immune responses to V. cholerae OSP have not previously been characterized. We assessed immune responses against V. cholerae OSP in adults with cholera caused by V. cholerae O1 El Tor serotype Inaba or Ogawa in Dhaka, Bangladesh, using O1 OSP-core–bovine serum albumin (OSPc:BSA) conjugates; responses targeted OSP in these conjugates. Responses of Inaba-infected patients to Inaba OSP and LPS increased significantly in IgG, IgM, and IgA isotypes from the acute to convalescent phases of illness, and the responses correlated well between OSP and LPS (R = 0.86, 0.73, and 0.91, respectively; P < 0.01). Plasma IgG, IgM, and IgA responses to Ogawa OSP and LPS in Ogawa-infected patients also correlated well with each other (R = 0.60, 0.60, and 0.92, respectively; P < 0.01). Plasma IgM responses to Inaba OSP and Ogawa OSP correlated with the respective serogroup-specific vibriocidal antibodies (R = 0.80 and 0.66, respectively; P < 0.001). Addition of either OSPc:BSA or LPS, but not BSA, to vibriocidal assays inhibited vibriocidal responses in a comparable and concentration-dependent manner. Mucosal IgA immune responses to OSP and LPS were also similar. Our study is the first to characterize anti-OSP immune responses in patients with cholera and suggests that responses targeting V. cholerae LPS, including vibriocidal responses that correlate with protection against cholera, predominantly target OSP. Induction of anti-OSP responses may be associated with protection against cholera, and our results may support the development of a vaccine targeting V. cholerae OSP.     

O-Specific Polysaccharide-Specific Memory B Cell Responses in Young Children,Older Children,and Adults Infected with Vibrio cholerae O1 Ogawa in Bangladesh

Cholera caused by Vibrio cholerae O1 confers at least 3 to 10 years of protection against subsequent disease regardless of age, despite a relatively rapid fall in antibody levels in peripheral blood, suggesting that memory B cell responses may play an important role in protection. The V. cholerae O1-specific polysaccharide (OSP) component of lipopolysaccharide (LPS) is responsible for serogroup specificity, and it is unclear if young children are capable of developing memory B cell responses against OSP, a T cell-independent antigen, following cholera. To address this, we assessed OSP-specific memory B cell responses in young children (2 to 5 years, n = 11), older children (6 to 17 years, n = 21), and adults (18 to 55 years, n = 28) with cholera caused by V. cholerae O1 in Dhaka, Bangladesh. We also assessed memory B cell responses against LPS and vibriocidal responses, and plasma antibody responses against OSP, LPS, and cholera toxin B subunit (CtxB; a T cell-dependent antigen) on days 2 and 7, as well as days 30, 90, and 180 after convalescence. In all age cohorts, vibriocidal responses and plasma OSP, LPS, and CtxB-specific responses peaked on day 7 and fell toward baseline over the follow-up period. In comparison, we were able to detect OSP memory B cell responses in all age cohorts of patients with detectable responses over baseline for 90 to 180 days. Our results suggest that OSP-specific memory B cell responses can occur following cholera, even in the youngest children, and may explain in part the age-independent induction of long-term immunity following naturally acquired disease.     

Memory B Cell Responses to Vibrio cholerae O1 Lipopolysaccharide Are Associated with Protection against Infection from Household Contacts of Patients with Cholera in Bangladesh

Vibrio cholerae O1 causes cholera, a dehydrating diarrheal disease. We have previously shown that V. cholerae-specific memory B cell responses develop after cholera infection, and we hypothesize that these mediate long-term protective immunity against cholera. We prospectively followed household contacts of cholera patients to determine whether the presence of circulating V. cholerae O1 antigen-specific memory B cells on enrollment was associated with protection against V. cholerae infection over a 30-day period. Two hundred thirty-six household contacts of 122 index patients with cholera were enrolled. The presence of lipopolysaccharide (LPS)-specific IgG memory B cells in peripheral blood on study entry was associated with a 68% decrease in the risk of infection in household contacts (P = 0.032). No protection was associated with cholera toxin B subunit (CtxB)-specific memory B cells or IgA memory B cells specific to LPS. These results suggest that LPS-specific IgG memory B cells may be important in protection against infection with V. cholerae O1.     

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.     

Vaccination with a DNA Vaccine Coding for Perforin-Like Protein 1 and MIC6 Induces Significant Protective Immunity against Toxoplasma gondii

Host cell invasion by Toxoplasma gondii is tightly related to microneme protein 6 (MIC6) and T. gondii perforin-like protein 1 (TgPLP1). In this study, we constructed a DNA vaccine expressing a TgPLP1/MIC6 fusion protein using the pIRESneo vector, and we evaluated the immune response induced by this vaccine in Kunming mice. Levels of IgG antibody, gamma interferon (IFN-γ), interleukin 2 (IL-2), IL-12, IL-4, and IL-10 were examined. Five mice were chosen randomly from every group (vaccinated groups or the nonvaccinated control group) and were challenged intragastrically with 80 cysts of T. gondii strain PRU (genotype II) in order to observe mortality daily. To analyze protection against a less-virulent challenge, eight mice of each group were orally infected with 20 cysts of strain PRU at the 14th day after the last immunization. The brain parasite load was evaluated 6 weeks after infection. The results demonstrated that immunization with pIRESneo/MIC6/PLP1 resulted in the lowest brain cyst count and prolonged the survival time of immunized mice. The levels of Toxoplasma-specific IgG, IFN-γ, IL-2, and IL-12 increased significantly, and the numbers of cysts in brains decreased more obviously, in the group immunized with plasmid pIRESneo/MIC6/PLP1 than in the other groups (P < 0.05). Compared with pIRESneo/MIC6/PLP1, coimmunization with pIRESneo/MIC6/PLP1 and adjuvant murine IL-18 promoted cellular and humoral immune responses but did not contribute significantly to cyst reduction (65.43% versus 61.60%) or the survival of immunized mice (45.0 ± 2.9 days versus 42.8 ± 2.9 days) (P > 0.05). Furthermore, the study also showed that the immune efficacy induced by pIRESneo/MIC6/PLP1 was better than that induced by pVAX/PLP1 or pVAX/MIC6 alone.     

The capsule and O antigen in Vibrio cholerae O139 Bengal are associated with a genetic region not present in Vibrio cholerae O1.

Vibrio cholerae O139 Bengal, although closely related to V. cholerae O1 El Tor, produces a polysaccharide capsule and has a distinct O antigen. We have identified a chromosomal region of at least 11 kb, as defined by three TnphoA mutations, that is required for the expression of both polysaccharides. Electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis show that these TnphoA mutants have lost the abilities both to express capsule and to produce lipopolysaccharide beyond the core oligosaccharide. Reactivity with O139 typing serum and resistance to serum are also lost in the mutants. DNA probes for this region do not hybridize with O1 V. cholerae but do react with other vibrios, implying that the region was recently acquired.     

Development of Immunoglobulin M Memory to Both a T-Cell-Independent and a T-Cell-Dependent Antigen following Infection with Vibrio cholerae O1 in Bangladesh

Vibrio cholerae O1 can cause severe watery diarrhea that can be life-threatening without treatment. Infection results in long-lasting protection against subsequent disease. Development of memory B cells of the immunoglobulin G (IgG) and IgA isotypes to V. cholerae O1 antigens, including serotype-specific lipopolysaccharide (LPS) and the B subunit of cholera toxin (CTB), after cholera infection has been demonstrated. Memory B cells of the IgM isotype may play a role in long-term protection, particularly against T-cell-independent antigens, but IgM memory has not been studied in V. cholerae O1 infection. Therefore, we assayed acute- and convalescent-phase blood samples from cholera patients for the presence of memory B cells that produce cholera antigen-specific IgM antibody upon polyclonal stimulation in in vitro culture. We also examined the development of serological and antibody-secreting cell responses following infection. Subjects developed significant IgM memory responses by day 30 after infection, both to the T-cell-independent antigen LPS and to the T-cell-dependent antigen CTB. No significant corresponding elevations in plasma IgM antibodies or circulating IgM antibody-secreting cells to CTB were detected. In 17 subjects followed to day 90 after infection, significant persistence of elevated IgM memory responses was not observed. The IgM memory response to CTB was negatively correlated with the IgG plasma antibody response to CTB, and there was a trend toward negative correlation between the IgM memory and IgA plasma antibody responses to LPS. We did not observe an association between the IgM memory response to LPS and the vibriocidal titer.Vibrio cholerae continues to be a significant global health burden as a cause of severe secretory diarrhea, resulting in an estimated three to five million annual cases, with more than 100,000 deaths from rapid dehydration (47); cholera has recently become endemic in new regions (44, 45). V. cholerae is a noninvasive pathogen that colonizes the mucosal surface of the small intestine. Strains can be distinguished serologically by the O antigen of the lipopolysaccharide (LPS); V. cholerae O1 is the most common cause of cholera in South Asia as well as globally. The O1 serogroup has two major biotypes, El Tor and classical, and two major serotypes, Inaba and Ogawa (35). Natural infection with V. cholerae O1 El Tor induces protective immunity that lasts for at least 3 to 10 years in both areas where cholera is not endemic and areas where it is endemic (21). It remains unknown, however, what aspects of the adaptive immune response to cholera confer this long-term protection.V. cholerae-infected patients mount immunologic responses to both protein and polysaccharide antigens, including rises in both serum immunoglobulin G (IgG) and IgA antibodies (14). A number of these serological responses have been shown to correlate with protection against reinfection; these include the complement-dependent serum vibriocidal antibody (14) and IgA (but not IgG) responses to LPS, cholera toxin B subunit (CTB), and toxin coregulated pilus A (TcpA) (17). These serological responses, however, are short-lived (4, 32), and the association of the vibriocidal titer with protection is not absolute (36), suggesting that these responses may reflect protection from more recent exposure but that other immunologic mechanisms mediate longer-term protection. In addition to serological responses, development of mucosal immune responses to intestinal antigens can be detected in the blood, when B cells activated by antigen in the gut-associated lymphoid tissues circulate transiently in the blood as antibody-secreting cells (ASCs), before homing back to intestinal mucosal surfaces (11, 26). Circulation of ASCs specific to both LPS and CTB is seen after cholera infection, peaking around the seventh day after infection and declining by day 11 (32).Responses of the IgM isotype to cholera antigens have been less thoroughly investigated than the IgG and IgA responses. However, IgM defenses may be an important component of the overall immunologic response to cholera, since vibriocidal antibodies are principally of the IgM isotype (22) and IgM levels of pooled convalescent-phase serum samples correspond closely with vibriocidal activity (24), which in turn correlates with immunity (14). The pentameric structure of IgM facilitates strong cross-linking of antigens and activation of complement in the defense against other gram-negative enteric bacteria (2).We have recently shown development of memory B cells of both the IgG and IgA isotypes to LPS, CTB, and TcpA; these cells persisted in the circulation beyond 1 year for the protein antigens CTB and TcpA, but were not measurably above baseline levels by 9 to 12 months after infection for the polysaccharide-containing antigen LPS (16, 18). These circulating memory B cells can be detected by ex vivo polyclonal stimulation of peripheral blood mononuclear cells (PBMCs); stimulated memory B cells mature into ASCs detectable by enzyme-linked immunospot (ELISPOT) assay. Alternatively, memory B-cell responses can be detected by measuring antigen-specific antibodies secreted by maturing ASCs during the ex vivo stimulation of PBMCs in the memory B-cell assay (18).Memory B cells relevant for cholera immunity may include IgM⁺ as well as switched-memory (IgA⁺ and IgG⁺) populations. The majority of circulating IgM⁺ cells are naïve B cells, but some IgM⁺ cells bear the memory cell marker CD27⁺, and recent evidence suggests that these IgM⁺CD27⁺ cells are true memory B cells whose immunoglobulin variable region genes have undergone somatic hypermutation in response to antigen in early-stage germinal centers (39). IgM⁺ memory cells can undergo isotype switching to produce IgG, IgA, or IgE antibody, but they also have a role in producing rapid, high-affinity IgM antibody responses to acute infection (19, 37, 46). In this study, we have measured the development of memory B-cell responses of the IgA, IgG, and IgM isotypes to both a protein (CTB) and a nonprotein (LPS) antigen, and we compared these memory responses with other immunologic responses in patients after V. cholerae infection in Bangladesh.     

Molecular Evidence of Cholera Outbreak Caused by a Toxigenic Vibrio cholerae O1 El Tor Variant Strain in Kelantan,Malaysia

A total of 20 Vibrio cholerae isolates were recovered for investigation from a cholera outbreak in Kelantan, Malaysia, that occurred between November and December 2009. All isolates were biochemically characterized as V. cholerae serogroup O1 Ogawa of the El Tor biotype. They were found to be resistant to multiple antibiotics, including tetracycline, erythromycin, sulfamethoxazole-trimethoprim, streptomycin, penicillin G, and polymyxin B, with 35% of the isolates being resistant to ampicillin. All isolates were sensitive to ciprofloxacin, norfloxacin, chloramphenicol, gentamicin, and kanamycin. Multiplex PCR analysis confirmed the biochemical identification and revealed the presence of virulence genes, viz., ace, zot, and ctxA, in all of the isolates. Interestingly, the sequencing of the ctxB gene showed that the outbreak strain harbored the classical cholera toxin gene and therefore belongs to the newly assigned El Tor variant biotype. Clonal analysis by pulsed-field gel electrophoresis demonstrated that a single clone of a V. cholerae strain was responsible for this outbreak. Thus, we present the first molecular evidence that the toxigenic V. cholerae O1 El Tor variant has invaded Malaysia, highlighting the need for continuous monitoring to facilitate early interventions against any potential epidemic by this biotype.Vibrio cholerae strains belonging to the O1 and O139 serogroups are agents of endemic and pandemic cholera, a potentially life-threatening diarrheal disease. As a water- and food-borne disease, cholera infection is linked to poverty and poor sanitation in many developing countries (13). From an epidemiological point of view, cholera tends to occur in explosive outbreaks throughout several regions simultaneously; likewise, extensive pandemics have followed a progressive pattern, affecting many countries across the continents and extending over many years (17). Cholera pandemics long have been believed to be exclusively associated with the toxigenic V. cholerae O1 serogroup, which consists of two biotypes: classical and El Tor. The classical biotype was responsible for the world''s first six pandemics, but the seventh pandemic was caused by the O1 El Tor biotype and was exceedingly more extensive in geographic spread and duration (13).The first non-O1 strain recognized as having caused an explosive cholera epidemic was discovered in 1992, but it did not belong to any of the 138 serogroups previously described. Thus, the new epidemic strain was designated serogroup O139 and subsequently has been linked to extensive outbreaks in various regions of Bangladesh and India (14). The discovery of its ability to cause large outbreaks and rapid spread to neighboring countries suggested the possibility that the new serogroup will facilitate the eighth pandemic of cholera if outbreaks continue to occur and more countries are affected (42). The strains of V. cholerae O1 El Tor in the seventh pandemic experienced an initial displacement by O139 strains in the Indian subcontinent (1) but recovered their prominence within a year and established a coexistence with O139 strains, causing subsequent cholera outbreaks throughout India and Bangladesh (12, 27).In Malaysia, cholera caused by V. cholerae O1 El Tor is endemic and often has been associated with sporadic outbreaks (24, 33, 46). El Tor is one of the most established biotypes within the V. cholerae O1 serogroup and is differentiated from the classical biotype based on a number of phenotypic traits, such as polymyxin B susceptibility, chicken cell agglutination, Voges-proskauer (VP) test positivity, and phage susceptibility (17). In addition, comparative genomic analysis has revealed the presence of genes that are unique to the El Tor biotype (11). Some of these genes, including the major toxin-coregulated pilus (TCP) tcpA (19) and rtxC of the repeat in toxin (RTX) cluster, have since been used as genetic markers for the identification of El Tor strains (8). The classical and El Tor biotypes also differ in the infection pattern of disease they elicit; the El Tor strains have a higher ratio of asymptomatic carriers (37), survive better in the environment and human host, and are more efficiently transmitted from host to host (http://www.ncbi.nlm.nih.gov/bookshelf/br.fegi?book=mmed&part=A1358).The public health significance of a V. cholerae isolate is routinely assessed by two critical properties: the production of cholera toxin (CT) and the possession of either the O1 or O139 antigen, which acts as a marker of epidemic potential (17). So far, agents of endemic and pandemic cholera have been represented exclusively by CT-producing V. cholerae strains. CT has been shown to be the key virulence factor responsible for the manifestation of massive, dehydrating diarrhea (21). Although CTs from classical and El Tor strains are structurally and functionally similar, differences in immunological and genetic attributes have enabled several methods to differentiate between the two biotypes. Specifically, the molecular techniques include the direct sequencing of the ctxB gene (30), a ganglioside GM₁ enzyme-linked immunosorbent assay (GM₁-ELISA) with specific monoclonal antibody to classical or El Tor CT (29), and a mismatch amplification mutation assay (MAMA) PCR to detect polymorphism at nucleotide position 203 in the ctxB genes (26).In the study presented here, we performed the genetic analysis and molecular typing of V. cholerae O1 isolated from patients during a cholera outbreak in Kelantan, Malaysia, that occurred between November and December 2009. The existence of an outbreak was established based on the region''s previous incidence rate of cholera, which was reported to be 0.0 per 100,000 for four consecutive years (2005 to 2008) (http://statistics.gov.my). The duration of the outbreak was only 21 days, and 33 cholera cases were confirmed. Among those 33, 21 victims were local Kelantanese and the remaining 12 were migrant Thai workers. The patient demographic data revealed that six different districts were involved, and the patients ranged widely in age. Isolates recovered from symptomatic patients were characterized for their antimicrobial susceptibility patterns, the presence of various toxigenic and pathogenic genes, the production of cholera toxin, the nucleotide sequence of cholera toxin B subunits, and the demonstration of the clonal relationships between these isolates. The ultimate findings from this research have provided important insights into the causative agent of the recent cholera outbreak in peninsular Malaysia.(This study was presented in part at the 2nd National Conference on Environment and Health, Kota Bharu, Kelantan, Malaysia, 17 to 18 March 2010.)     

Oral Immunization with Attenuated Salmonella enterica Serovar Typhimurium Encoding Cryptosporidium parvum Cp23 and Cp40 Antigens Induces a Specific Immune Response in Mice

Attenuated Salmonella enterica serovar Typhimurium vaccine strain SL3261 was used as an antigen delivery system for the oral immunization of mice against two Cryptosporidium parvum antigens, Cp23 and Cp40. Each antigen was subcloned into the pTECH1 vector system, which allows them to be expressed as fusion proteins with highly immunogenic fragment C of tetanus toxin under the control of the anaerobically inducible nirB promoter. The recombinant vector was introduced into Salmonella Typhimurium vaccine strain SL3261, and the stable soluble expression of the chimeric protein was evaluated and confirmed by Western blotting with polyclonal C. parvum antisera. Mice were inoculated orally with a single dose of SL3261/pTECH-Cp23 or Cp40, respectively, and plasmid stability was demonstrated both in vitro and in vivo. Specific serum immunoglobulin G (IgG) antibodies against the Cp23 or Cp40 antigen were detected by enzyme-linked immunosorbent assay 35 days after immunization. Also, serum IgA and mucosal (feces) IgA antibodies were detected in 30% of the mice immunized with Cp23. In addition, prime-boosting with Cp23 and Cp40 DNA vaccine vectors followed by Salmonella immunization significantly increased antibody responses to both antigens. Our data show that a single oral inoculation with recombinant S. Typhimurium SL3261 can induce specific antibody responses to the Cp23 or Cp40 antigen from C. parvum in mice, suggesting that recombinant Salmonella is a feasible delivery system for a vaccine against C. parvum infection.Cryptosporidium parvum is an obligate intracellular parasite that infects intestinal epithelial cells and has been identified as being a significant cause of diarrheal disease in a variety of mammalian species including rodents, livestock, and humans (24). Infection is usually self-limiting in immunocompetent individuals but can be severe and even life-threatening for those that have compromised immune systems, such as human immunodeficiency virus-infected individuals, transplant recipients, children, and the elderly (30). The incidence of cryptosporidiosis has been reported to be in the range of 1 to 10% (34) but has been reported to be as high as 30% in children in India and Saudi Arabia (1, 10, 14). In light of the fact that chemotherapeutic agents for the treatment of infections of immunodeficient individuals are limited and not always efficacious, the development of a vaccine that is capable of inducing at least partial protection would be beneficial to specific high-risk populations. Data from human volunteer studies have suggested that at least partial immunity develops, as subsequent exposures with the parasite resulted in less-severe clinical signs (26).Since all life cycle stages occur in the host epithelium, the mucosal immune response is paramount to providing resistance and protection. The use of live oral Salmonella vaccines has been successful at delivering heterologous antigens and at generating a mucosal immune response against a number of organisms including intestinal parasitic species such as Toxoplasma gondii and Eimeria tenella (18, 29). Advantages of attenuated Salmonella vaccines include the fact that they induce both cell-mediated and humoral responses, elicit a systemic and local response, are easy to administer, and are affordable (13). To date, reports of the use of attenuated Salmonella as a vaccine vector in C. parvum are not available. Through this study, we assessed the use of an attenuated Salmonella strain carrying specific C. parvum antigens as a vaccine vector and the potential that it offers against C. parvum infection.In this study, we compared the abilities of attenuated strains of Salmonella to express the immunodominant antigens Cp23 and Cp40. These surface antigens of C. parvum are considered to be immunodominant since they are recognized by serum antibodies of humans and many other animals (25, 31, 36). Moreover, the level of oocyst secretion was reduced following the administration of colostrum directed against the Cp23 antigen (26). T-cell responses to Cp23 from infected mice (3), calves (36), and human peripheral blood mononuclear cells (33) with C. parvum infection have been reported, indicating its role in the immune response to C. parvum. Recombinant Cp40 antigen was previously shown to generate a T-cell proliferation response in mice (31). Also, monoclonal antibodies against Cp40 antigen have been shown to neutralize C. parvum infection and inhibit attachment in vitro (4). We also report the safety and plasmid stability of the Salmonella vaccine vector in mice as well as the ability to induce an antibody response against the expressed antigens.     

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.     

Protective Effect of Vaccination with a Combination of Recombinant Surface Antigen 1 and Interleukin-12 against Toxoplasmosis in Mice

We studied the immune response induced in mice by recombinant Toxoplasma gondii surface antigen 1 (rSAG1) protein, alone or combined with interleukin-12 (IL-12) as an adjuvant, and the protective effect against toxoplasmosis. Immunization with rSAG1 alone induced a specific humoral type 2 immunity and did not protect the animals from infection. In contrast, immunization with rSAG1 plus IL-12 redirected humoral and cellular immunity toward a type 1 pattern and reduced the brain parasite load by 40%.     

IC31®, a Two-Component Novel Adjuvant Mixed with a Conjugate Vaccine Enhances Protective Immunity against Pneumococcal Disease in Neonatal Mice

IC31^® is a novel adjuvant which combines the immunostimulatory effects of an 11-mer antibacterial peptide (KLKL₅KLK) and a synthetic oligodeoxynucleotide (ODN1a) which is a Toll-like receptor 9 agonist without containing cytosine phosphate guanine (CpG) motifs. The effects of IC31^® on neonatal immune response to vaccination have not been reported. Neonatal mice were immunized once or twice with a Streptococcus pneumoniae serotype 1 polysaccharide conjugate containing Tetanus Toxoid (Pnc1-TT) carrier protein, with or without IC31^® or CpG-ODN. IC31^® significantly enhanced IgG1, IgG2a and IgG2b antibodies (Ab) to the serotype 1 polysaccharide. One dose of Pnc1-TT and low dose IC31^® elicited high Ab levels that protected the neonatal mice completely from bacteraemia and significantly reduced lung infection following i.n. challenge with serotype 1 pneumococcal strain. One-sixth of an adult murine dose of IC31^® was sufficient and optimal for induction of protective immunity in neonatal mice. Two doses of Pnc1-TT with or without adjuvants protected the neonatal mice completely, but more rapid Ab response was observed when IC31^® was given with the Pnc1-TT. IC31^® is a promising new adjuvant for neonatal vaccinations, rapidly enhancing protective humoral responses when combined with Pnc1-TT.     

Antibody Responses to Bordetella pertussis Fim2 or Fim3 following Immunization with a Whole-Cell,Two-Component,or Five-Component Acellular Pertussis Vaccine and following Pertussis Disease in Children in Sweden in 1997 and 2007

Bordetella pertussis fimbriae (Fim2 and Fim3) are components of a five-component acellular pertussis vaccine (diphtheria–tetanus–acellular pertussis vaccine [DTaP5]), and antibody responses to fimbriae have been associated with protection. We analyzed the IgG responses to individual Fim2 and Fim3 in sera remaining from a Swedish placebo-controlled efficacy trial that compared a whole-cell vaccine (diphtheria-tetanus-whole-cell pertussis vaccine [DTwP]), a two-component acellular pertussis vaccine (DTaP2), and DTaP5. One month following three doses of the Fim-containing vaccines (DTwP or DTaP5), anti-Fim2 geometric mean IgG concentrations were higher than those for anti-Fim3, with a greater anti-Fim2/anti-Fim3 IgG ratio elicited by DTaP5. We also determined the responses in vaccinated children following an episode of pertussis. Those who received DTaP5 showed a large rise in anti-Fim2 IgG, reflecting the predominant Fim2 serotype at the time. In contrast, those who received DTwP showed an equal rise in anti-Fim2 and anti-Fim3 IgG concentrations, indicating that DTwP may provide a more efficient priming effect for a Fim3 response following contact with B. pertussis. Anti-Fim2 and anti-Fim3 IgG concentrations were also determined in samples from two seroprevalence studies conducted in Sweden in 1997, when no pertussis vaccine was used and Fim2 isolates predominated, and in 2007, when either DTaP2 or DTaP3 without fimbriae was used and Fim3 isolates predominated. Very similar distributions of anti-Fim2 and anti-Fim3 IgG concentrations were obtained in 1997 and 2007, except that anti-Fim3 concentrations in 1997 were lower. This observation, together with the numbers of individuals with both anti-Fim2 and anti-Fim3 IgG concentrations, strongly suggests that B. pertussis expresses both Fim2 and Fim3 during infection.     

Persistence of cholera in the United States: isolation of Vibrio cholerae O1 from a patient with diarrhea in Maryland.

A case of cholera was identified in Baltimore County, Md., in October 1984. The Vibrio cholerae O1 isolate from the patient was hemolytic, biotype El Tor, serotype Inaba, and was toxigenic by the Y-1 adrenal cell assay; on Southern blot analysis, the strain had a unique HindIII restriction site in the cholera toxin gene identical to that of other U.S. V. cholerae O1 isolates. Two days before he became ill, the patient had eaten meat from crabs harvested along the Texas coast.     

Induction of Protective T Cells against Listeria monocytogenes in Mice by Immunization with a Listeriolysin O-Negative Avirulent Strain of Bacteria and Liposome-Encapsulated Listeriolysin O

Only listeriolysin O (LLO)-producing strains of Listeria monocytogenes generate protective immunity in mice. Based on the findings that endogenous gamma interferon (IFN-γ) production was induced only by such strains and that purified LLO could induce IFN-γ from NK cells, we have postulated that LLO may play a pivotal role in the induction of Th1-type protective T cells, which are highly dependent on IFN-γ. In this study, mice were immunized with L. monocytogenes ATCC 15313, an LLO-nonproducing avirulent strain, along with LLO encapsulated in liposome (LLO-liposome). LLO-liposome was highly potent in the induction of various cytokines, including IFN-γ. Immunization of mice with either LLO-liposome or the viable strain ATCC 15313 alone did not induce protection against challenge infection. In contrast, the combination of LLO-nonproducing bacteria plus LLO-liposome induced a significant level of protective immunity mediated mainly by Th1-type cells capable of producing a large amount of IFN-γ in an antigen-specific manner. The protection afforded by the combination was not dependent on LLO-specific cytotoxic T cells. These results support the idea that the inability of an LLO-nonproducing avirulent strain or killed bacteria to induce the generation of protective T cells is due not to the lack of a central T-cell epitope(s) but to the lack of ability to induce the production of endogenous cytokine during the early stage of immunization; the results also suggest that an appropriate use of LLO at least in an animal model may be effective in the induction of antigen-specific Th1-dependent protective immunity to various kinds of intracellular parasitic bacteria.     

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.     

Adequate Expression of Protective Immunity in the Absence of Granuloma Formation in Mycobacterium tuberculosis-Infected Mice with a Disruption in the Intracellular Adhesion Molecule 1 Gene

It remains unknown whether the expression of cell-mediated protective immunity and the capacity to mount a delayed-type hypersensitivity (DTH) reaction in tuberculosis infection represent two manifestations of a basic response or are dissociable events. In this study, we present data in favor of the latter hypothesis, by showing that tuberculosis infection in the lungs of mice possessing only a truncated form of intracellular adhesion molecule 1 due to gene disruption was still adequately controlled by the expression of protective immunity in the absence of any sustained influx of macrophages and the lack of formation of appreciable granulomas. These animals also had no detectable DTH response to mycobacterial proteins in the footpad assay, indicating that the accumulation of blood-borne macrophages at sites of mycobacterial infection or antigen deposition is not essential to control of the infection. These data support the hypothesis that the DTH component of the cellular response is not protective but contributes by walling off the sites of infection to prevent dissemination and reactivation disease.     

A Francisella tularensis Live Vaccine Strain (LVS) Mutant with a Deletion in capB,Encoding a Putative Capsular Biosynthesis Protein,Is Significantly More Attenuated than LVS yet Induces Potent Protective Immunity in Mice against F. tularensis Challenge

Francisella tularensis, the causative agent of tularemia, is in the top category (category A) of potential agents of bioterrorism. The F. tularensis live vaccine strain (LVS) is the only vaccine currently available to protect against tularemia; however, this unlicensed vaccine is relatively toxic and provides incomplete protection against aerosolized F. tularensis, the most dangerous mode of transmission. Hence, a safer and more potent vaccine is needed. As a first step toward addressing this need, we have constructed and characterized an attenuated version of LVS, LVS ΔcapB, both as a safer vaccine and as a vector for the expression of recombinant F. tularensis proteins. LVS ΔcapB, with a targeted deletion in a putative capsule synthesis gene (capB), is antibiotic resistance marker free. LVS ΔcapB retains the immunoprotective O antigen, is serum resistant, and is outgrown by parental LVS in human macrophage-like THP-1 cells in a competition assay. LVS ΔcapB is significantly attenuated in mice; the 50% lethal dose (LD₅₀) intranasally (i.n.) is >10,000-fold that of LVS. Providing CapB in trans to LVS ΔcapB partially restores its virulence in mice. Mice immunized with LVS ΔcapB i.n. or intradermally (i.d.) developed humoral and cellular immune responses comparable to those of mice immunized with LVS, and when challenged 4 or 8 weeks later with a lethal dose of LVS i.n., they were 100% protected from illness and death and had significantly lower levels (3 to 5 logs) of LVS in the lung, liver, and spleen than sham-immunized mice. Most importantly, mice immunized with LVS ΔcapB i.n. or i.d. and then challenged 6 weeks later by aerosol with 10× the LD₅₀ of the highly virulent type A F. tularensis strain SchuS4 were significantly protected (100% survival after i.n. immunization). These results show that LVS ΔcapB is significantly safer than LVS and yet provides potent protective immunity against virulent F. tularensis SchuS4 challenge.Francisella tularensis is a Gram-negative coccobacillus that causes tularemia, a zoonotic disease spread among small animals such as rabbits and rodents by blood-sucking insects. Humans typically acquire tularemia by handling infected animals or from the bite of infected insects. There are four subspecies of F. tularensis: F. tularensis subsp. tularensis, holarctica, mediasiatica, and novicida (41); of these, F. tularensis subsp. tularensis, found in North America and also known as type A, causes the most severe disease. Following cutaneous exposure, tularemia typically presents as an ulceronodular disease with painful, ulcerated skin lesions and swollen lymph nodes. Following inhalation exposure, tularemia presents with acute flu-like symptoms followed by pleuropneumonic and typhoidal illness. The pneumonic form of tularemia has a high fatality rate (11).Because of its high pathogenicity in humans, especially after respiratory exposure, its low infectious dose, and the relative ease with which it can be cultured and disseminated, F. tularensis is classified as a category A agent of bioterrorism, i.e., among bioterrorist agents thought to pose the greatest risk to the public. Indeed, F. tularensis was previously developed as a bioweapon and stockpiled by Japan during World War II (16) and by the United States and the Soviet Union during the Cold War (1, 6). Although tularemia can be treated with available antibiotics, F. tularensis can be genetically engineered to be antibiotic resistant (30). Moreover, pneumonic tularemia frequently requires hospitalization and intensive care, and even when an infected individual is treated with antibiotics to which the organism is sensitive, the disease may resolve slowly (12); even a moderately sized outbreak could rapidly overwhelm medical facilities (11). Hence, relying on antibiotics to protect against a bioterrorist attack with F. tularensis is not a practical public health approach. A safe and potent vaccine, on the other hand, would appear to offer a much more reliable approach.An unlicensed vaccine known as the live vaccine strain (LVS), an attenuated mutant of F. tularensis subsp. holarctica, was developed in the mid-1900s and is the only vaccine currently available in the United States. The underlying mechanism of attenuation is not fully characterized genetically, although recently, the reintroduction of deleted genes pilA and FTT0918 was shown to restore virulence to the level of virulent type B strains (35). The LVS vaccine has several drawbacks. The vaccine, which retains considerable virulence in animals, shows significant toxicity in humans after both intradermal (i.d.) and aerosol administration (19, 37). Moreover, it provides incomplete protection to humans challenged with type A F. tularensis by aerosol, the route of transmission of greatest concern in a bioterrorist attack (19, 29, 37).In a search for a vaccine that is safer and more potent than LVS, we sought to rationally attenuate LVS and to use the attenuated LVS as both a vaccine and a vector to overexpress immunogenic F. tularensis proteins. We hypothesized that we would render LVS safer by further attenuating it and that we would render it more potent by overexpressing key immunoprotective antigens. This overall strategy mirrors that used successfully to develop the first vaccine against tuberculosis that is more potent than the current Mycobacterium bovis BCG vaccine, rBCG30, a recombinant BCG vaccine overexpressing the Mycobacterium tuberculosis 30-kDa major secretory protein, and to develop the first vaccine both safer and more potent than BCG, rBCG(mbtB)30, an attenuated version of rBCG30 that is engineered and propagated such that it can multiply only a few times in the host (20, 21, 45).In attenuating LVS, we sought a mutation that would greatly reduce virulence but have a minimal impact on immunogenicity and protective efficacy. Transposon mutagenesis studies of F. tularensis subsp. novicida and holarctica (LVS) have shown that mutants with transposon insertions in genes (FTT0806, FTT0805, and FTT0798) encoding proteins putatively involved in capsular biosynthesis, on the basis of partial amino acid sequence homology with capsular biosynthesis proteins of Bacillus anthracis, are highly attenuated (∼100- to 1,000-fold) in mice (43, 47). Consequently, we decided to evaluate the vaccine potential of an LVS mutant with a deletion in one of these genes.In this study, we describe the construction of an antibiotic resistance marker-free FTL_1416/FTT0805 (capB) deletion mutant of F. tularensis LVS (LVS ΔcapB) and show that LVS ΔcapB is resistant to serum killing, outgrown by its parental LVS in human macrophage-like THP-1 cells, and highly attenuated in mice. We demonstrate further that this vaccine, after both i.d. and intranasal (i.n.) administration, induces potent cellular and humoral immune responses and significant protective immunity against respiratory challenge with virulent F. tularensis.