Immune Responses to the O-Specific Polysaccharide Antigen in Children Who Received a Killed Oral Cholera Vaccine Compared to Responses following Natural Cholera Infection in Bangladesh

Current oral cholera vaccines induce lower levels of protective efficacy and shorter durations of protection in young children than in adults. Immunity against cholera is serogroup specific, and immune responses to Vibrio cholerae lipopolysaccharide (LPS), the antigen that mediates serogroup-specific responses, are associated with protection against disease. Despite this, responses against V. cholerae O-specific polysaccharide (OSP), a key component of the LPS responsible for specificity, have not been characterized in children. Here, we report a comparison of polysaccharide antibody responses in children from a region in Bangladesh where cholera is endemic, including infants (6 to 23 months, n = 15), young children (24 to 59 months, n = 14), and older children (5 to 15 years, n = 23) who received two doses of a killed oral cholera vaccine 14 days apart. We found that infants and young children receiving the vaccine did not mount an IgG, IgA, or IgM antibody response to V. cholerae OSP or LPS, whereas older children showed significant responses. In comparison to the vaccinees, young children with wild-type V. cholerae O1 Ogawa infection did mount significant antibody responses against OSP and LPS. We also demonstrated that OSP responses correlated with age in vaccinees, but not in cholera patients, reflecting the ability of even young children with wild-type cholera to develop OSP responses. These differences might contribute to the lower efficacy of protection rendered by vaccination than by wild-type disease in young children and suggest that efforts to improve lipopolysaccharide-specific responses might be critical for achieving optimal cholera vaccine efficacy in this younger age group.     

Antibody Avidity in Humoral Immune Responses in Bangladeshi Children and Adults following Administration of an Oral Killed Cholera Vaccine

Antibody avidity for antigens following disease or vaccination increases with affinity maturation and somatic hypermutation. In this study, we followed children and adults in Bangladesh for 1 year following oral cholera vaccination and measured the avidity of antibodies to the T cell-dependent antigen cholera toxin B subunit (CTB) and the T cell-independent antigen lipopolysaccharide (LPS) in comparison with responses in other immunological measurements. Children produced CTB-specific IgG and IgA antibodies of high avidity following vaccination, which persisted for several months; the magnitudes of responses were comparable to those seen in adult vaccinees. The avidity of LPS-specific IgG and IgA antibodies in vaccinees increased significantly shortly after the second dose of vaccine but waned rapidly to baseline levels thereafter. CTB-specific memory B cells were present for only a short time following vaccination, and we did not find significant memory B cell responses to LPS in any age group. For older children, there was a significant correlation between CTB-specific memory T cell responses after the second dose of vaccine and CTB-specific IgG antibody avidity indices over the subsequent year. These findings suggest that vaccination induces a longer-lasting increase in the avidity of antibodies to a T cell-dependent antigen than is measured by a memory B cell response to that antigen and that early memory T cell responses correlate well with the subsequent development of higher-avidity antibodies.     

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.     

Kinetics of Local and Systemic Immune Responses to an Oral Cholera Vaccine Given Alone or Together with Acetylcysteine

The possibility that a mucolytic drug, i.e., acetylcysteine, given orally may enhance the gut mucosal or systemic immune response to an oral B-subunit–whole-cell (B-WC) cholera vaccine was evaluated for 40 adult Swedish volunteers, and the kinetics of the immune responses were monitored for responding volunteers. Two doses of vaccine induced similar frequencies of immunoglobulin A (IgA) and IgG antitoxin responses (80 to 90%) and vibriocidal titer increases (60 to 65%) in serum irrespective of whether the vaccine was given alone or together with 2 g of acetylcysteine. In feces the frequencies of IgA antitoxin (67%) and antibacterial (33 to 40%) antibody responses were also comparable in the two immunization groups. Six months after vaccination, IgA and IgG antitoxin as well as vibriocidal antibody titer increases in serum could still be detected in approximately 80% of initially responding vaccinees. Significantly elevated fecal antitoxin and antibacterial IgA antibody levels were found in, respectively, 50 and 43% of those volunteers who initially had responded to the vaccine. Determination of IgA antibodies in feces does not seem to offer any advantages compared to determination in serum for assessment of immune responses after immunization with inactivated cholera vaccine.     

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.     

Intestinal Immune Responses to an Inactivated Oral Enterotoxigenic Escherichia coli Vaccine and Associated Immunoglobulin A Responses in Blood

An inactivated oral enterotoxigenic Escherichia coli (ETEC) vaccine against ETEC diarrhea was given to 25 adult Swedish volunteers. The vaccine consisted of formalin-killed E. coli bacteria expressing the most common colonization factor antigens (CFAs), i.e., CFA/I, -II, and -IV, and recombinantly produced cholera B subunit (CTB). Immunoglobulin A (IgA) antibody responses in intestinal lavage fluid to CTB and CFAs were determined and compared with corresponding responses in stool extracts and serum as well as with IgA antibody-secreting cell (ASC) responses in peripheral blood. Two doses of vaccine induced significant IgA responses to the different CFAs in lavage fluid in 61 to 87% of the vaccinees and in stool in 38 to 81% of them. The most frequent responses were seen against CFA/I. The magnitudes of the antibody responses against CTB and CFA/I in stool correlated significantly (CTB, P < 0.01; CFA/I, P < 0.05) with those in intestinal lavage. Intestinal lavage responses against CFAs were best reflected by the ASC responses, with the sensitivity of the ASC assay being 80 to 85%, followed by stool (sensitivity of 50 to 88%) and serum antibody (sensitivity of 7 to 65%) analyses. CTB-specific immune responses were seen in >90% of the vaccinees in all assays.     

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.     

Ex Vivo Triggering of T-Cell-Mediated Immune Responses by Autologous Tumor Cell Vaccine in Oral Cancer Patients

We investigated immunomodulatory activity of autologous tumor cell vaccine from oral cancer patients ex vivo by lymphoproliferation assay and two color flow cytometry. Vaccine treatment lead to 10-fold higher proliferation of lymphocytes compared with the untreated controls. A significant increase in CD69⁺ and HLA-DR⁺ T-cells was observed in vaccine pulsed cultures compared with untreated (p<0.0001) controls. The frequency of IFN-γ and IL-2 expressing CD4⁺/CD8⁺T-cell subsets was significantly higher with a concomitant reduction in IL-4 and IL-10 expression in the vaccine-treated group (p<0.0001) compared with the untreated controls. Vaccine treatment further increased T-cell receptor Vβ3, Vβ5, and Vβ8 usage. It seems that the autologous tumor cell vaccine triggers T‐cell responses ex vivo, hence it may have a protective role in oral cancer patients.     

Triggering of T Cell-Mediated Immune Responses by Allogenic Tumor Cell Vaccine in Patients with Oral Cancer

We investigated the immunomodulatory activity of allogenic whole tumor cell vaccine in oral cancer patients in vitro by two-color flow cytometry. Vaccine treatment significantly increased the expression of CD69 and HLA-DR in CD3⁺ T-cell subsets. The frequency of Interferon-gamma and Interleukin (IL)-2 expressing CD4⁺/CD8⁺ T-cell subsets was significantly higher with a concomitant reduction in IL-4 and IL-10 expressing T-cells in the vaccine treated group as compared with the untreated controls. Vaccine treatment significantly increased T-cell receptor (TCR), Vβ3, Vβ5 and Vβ8 usage. The results indicate that the allogenic whole tumor cell vaccine is able to trigger T-cell mediated immunity in patients with intraoral squamous cell carcinoma.     

Immune Responses in Ileostomy Fluid and Serum after Oral Cholera Vaccination of Patients Colectomized because of Ulcerative Colitis

The capacity of an oral inactivated B-subunit–whole-cell cholera vaccine to induce immune responses in patients colectomized due to ulcerative colitis was studied. Two doses of vaccine induced significant mucosal immunoglobulin A (IgA) antibody responses in ileostomy fluid against cholera toxin in 14 of 15 (93%) patients and against whole vibrios in 9 of 15 (60%) cases. The serological responses were lower (but not significantly) than those observed in healthy Swedish volunteers. Increased IgA antitoxin levels were found in ileostomy fluid as late as 2 years after vaccination.     

Dose-Dependent Circulating Immunoglobulin A Antibody-Secreting Cell and Serum Antibody Responses in Swedish Volunteers to an Oral Inactivated Enterotoxigenic Escherichia coli Vaccine

The immunogenicity of different preparations of an oral inactivated enterotoxigenic Escherichia coli (ETEC) vaccine was evaluated in Swedish volunteers previously unexposed to ETEC infection. The vaccine preparations consisted of recombinant cholera toxin B subunit (CTB) and various amounts of formalin-killed whole bacteria expressing the most prevalent colonization factor antigens (CFAs). Significant immunoglobulin A (IgA) antibody-secreting cell (ASC) responses against CTB and the various CFA components were seen in a majority of volunteers after two doses of ETEC vaccine independent of the vaccine lot given. The IgA ASC responses against CTB were significantly higher after the second than after the first immunization, whereas the CFA-specific IgA ASC responses were almost comparable after the first and second doses of ETEC vaccine. Two immunizations with one-third of a full dose of CFA-ETEC bacteria induced lower frequencies of IgA ASC responses against all the different CFAs than two full vaccine doses, i.e., 63 versus 80% for CFA/I, 56 versus 70% for CS1, 31 versus 65% for CS2, and 56 versus 75% for CS4. The proportion of vaccinees responding with rises in the titer of serum IgA antibody against the various CFA antigens was also lower after immunization with the reduced dose of CFA-ETEC bacteria. These findings suggest that measurements of circulating IgA ASCs can be used not only for qualitative but also for quantitative assessments of the immunogenicity of individual fimbrial antigens in various preparations of ETEC vaccine.     

Safety and Immunogenicity Assessment of an Oral Cholera Vaccine through Phase I Clinical Trial in Korea

The safety, tolerability and immunogenicity of an oral cholera vaccine (OCV) was assessed in adult Korean male through an open-label, non-comparative clinical study. Two doses of vaccine with an interval of 2 weeks were given to 20 healthy subjects. A total of 7 adverse events occurred in 6 subjects. However, no clinically significant change was observed in electrocardiograms, vital signs, physical examinations, and clinical laboratory tests. The immunogenicity of OCV was evaluated by serum vibriocidal assay where anti-Vibrio cholerae O1 and O139 antibodies were measured at day 0, 14, and 28 of vaccine administration. The antibody titers ranged from < 2.5-5,120 for V. cholerae O1 Inaba, < 2.5-10,240 for V. cholerae O1 Ogawa and < 2.5-480 for V. cholerae O139. In addition, the fold increase in antibody titers ranged from 1-4,096 for O1 Inaba, 1-8,192 for O1 Ogawa, and 1-384 for O139. The seroconversion rate was 95% and 45% for O1 and O139 antibodies, respectively. Our study clearly shows that administration of two doses of OCV at a 2 week-interval increases an appropriate level of antibody titer in the serum of healthy Korean adult males (Clinical Trial Number, NCT01707537).

Graphical Abstract

     

Enhancement of Serum and Mucosal Immune Responses to a Haemophilus influenzae Type B Vaccine by Intranasal Delivery

Intranasal (i.n.) vaccination is potentially the most direct method for conveying upper respiratory and mucosal immunity to respiratory pathogens. However, for unclear reasons, vaccines introduced into the nasal sinuses often have lower efficacy than vaccines administered by the more frequently used parenteral routes. We examined i.n. vaccination in a mouse immune-response model with a commonly used Haemophilus influenzae type B vaccine (Hibv) composed of the polyribosylribitol phosphate (PRP) capsule antigen conjugated to tetanus toxoid. Intranasal vaccination with Hibv using a Toll-like receptor 4 (TLR4) agonist as an adjuvant significantly increased the levels of IgA specific for the PRP capsule antigen in blood serum, saliva, and mucosal secretion specimens. In contrast, control mice vaccinated transdermally (t.d.) with Hibv did not produce significant levels of PRP-specific IgA in the blood serum and saliva, and anti-PRP IgG was increased only in serum. The i.n. and t.d. vaccinations resulted in equivalent bactericidal antibody responses in blood serum, suggesting that vaccine-derived IgG is protective against infection. Elevated levels of IgG specific for the tetanus toxoid carrier protein were measured in nasal sinuses and vaginal secretions in mice vaccinated by either the t.d. or i.n. route. Tissue culture studies confirmed that the nasopharynx-associated lymphoid tissue (NALT) was at least one of the sources of PRP-specific IgA and carrier-specific IgG within the nasal sinuses. We conclude that i.n. vaccination aided by a TLR4 agonist results in robust immune responses to both the carrier protein and bacterial polysaccharide components of the Hibv.     

Kinetics of Memory B Cell and Plasma Cell Responses in the Mice Immunized with Plague Vaccines

In our previous studies, we found that plague vaccines can induce long‐term antibody response, but no significant antibody boost was observed when the immunized mice were challenged with virulent Yersinia pestis. However, a booster vaccination of subunit vaccine on week 3 after primary immunization elicited a significantly higher antibody titre than a single dose, whereas no significant antibody titre difference was observed between a single dose and two doses of EV76 vaccination. To address these issues, in this study, we first investigated the kinetics of memory B cells and plasma cells in the mice immunized with EV76 or F1 protein by flow cytometry and then determined antibody titre in five groups of mice immunized with various vaccination strategy. The results showed that memory B cells dropped to a low level at day 56 after primary immunization. In contrast, plasma cells were maintained for more than 98 days. The group with primary immunization of EV76 and booster of F1 antigen developed a higher antibody titre than the group with immunization of F1 antigen and booster of EV76. This result supports a hypothesis that an excess of antigens can neutralize pre‐existing antibodies, and then the redundant antigen induces antibody boost. Taken together, a boost of antibody titre after revaccination may be dependent on the existence of memory B cells and an excess of antigen vaccination. In addition, this study showed an ideal immunization strategy that involves first immunization with a live attenuated vaccine, such as EV76, and then with a subunit vaccine.     

Kinetics of Local and Systemic Immune Responses after Vaginal Immunization with Recombinant Cholera Toxin B Subunit in Humans

Vaginal vaccination seems to be the best strategy for inducing specific immunoglobulin A (IgA) and IgG antibody responses in the female genital tract. The relative efficiencies of one, two, and three vaginal doses of recombinant cholera toxin B subunit (CTB) in generating mucosal and systemic immune responses in healthy women were evaluated, and the kinetics of the immune responses were monitored for responding volunteers for up to 12 months after the last vaccination. A single dose of CTB failed to generate CTB-specific IgA antibody responses in cervical secretions. Two vaccinations induced significant increases in IgA antitoxin titers in seven of nine volunteers, and four volunteers also developed IgG antitoxin responses. The magnitudes of the responses were 20-fold for IgA antitoxin and 7.1-fold for IgG antitoxin. A third vaccination did not significantly increase the antitoxin responses, although the frequency of IgG responses was slightly higher than that after the second vaccination. In serum, CTB-specific antibodies were observed already after a single vaccination. However, two vaccinations were required to induce marked IgA as well as IgG antitoxin titer increases in the majority of volunteers. The postvaccination levels of antitoxin antibodies in serum were comparable after two and three vaccinations. At 12 months after vaccination, significantly elevated IgA and IgG antitoxin levels in cervical secretions could still be detected in approximately half of the volunteers who had initially responded to the vaccine. Antitoxin titer increases in serum were found in most of the vaccinees at follow-up.     

Immune Activation and Viral Replication after Vaccination with an Influenza A H1N1 2009 Vaccine in HIV-Infected Children Receiving Antiretroviral Therapy

Immunization with a pandemic influenza A H1N1 2009 was recommended for HIV-infected patients. However, there is limited information concerning the impact of immunization with this vaccine on immune activation and HIV viral replication. In this study, 45 HIV-infected children and adolescents receiving antiretroviral therapy were immunized with a 2-dose series of nonadjuvated monovalent influenza A H1N1 2009 vaccine upon enrollment and approximately 1 month later. Immunogenicity was determined by haemagglutination inhibition assay. The level of immune activation was determined by identification of CD38 and HLA-DR on CD8+ T cells. Patients were divided into 2 groups which include patients who had an undetectable HIV viral load (HIV detectable group) and patients who show virological failure (HIV nondetectable group). The results showed seroconversion rate of 55.2% in HIV nondetectable group, whereas 31.3% was found in HIV detectable group. Both groups of patients showed no major increase in immune activation after immunization. Interestingly, a decrease in the frequency of CD8+ T cells that coexpressed CD38 and HLA-DR was observed after immunization in both groups of patients. We suggested that immunization with influenza A H1N1 2009 vaccine can induce immune response to the pandemic virus without major impact on HIV viral replication and immune activation.     

Synergistic and Additive Effects of Cimetidine and Levamisole on Cellular Immune Responses to Hepatitis B virus DNA Vaccine in Mice

We and others have previously shown that both cimetidine (CIM) and levamisole (LMS) enhance humoral and cellular responses to DNA vaccines via different mechanisms. In this study, we investigated the synergistic and additive effects of CIM and LMS on the potency of antigen‐specific immunities generated by a DNA vaccine encoding the hepatitis B surface antigen (HBsAg, pVax‐S2). Compared with CIM or LMS alone, the combination of CIM and LMS elicited a robust HBsAg‐specific cellular response that was characterized by higher IgG2a, but did not further increase HBsAg‐specific antibody IgG and IgG1 production. Consistent with these results, the combination of CIM and LMS produced the highest level of IL‐2 and IFN‐γ in antigen‐specific CD4⁺ T cells, whereas the combination of CIM and LMS did not further increase IL‐4 production. Significantly, a robust HBsAg‐specific cytotoxic response was also observed in the animals immunized with pVax‐S2 in the presence of the combination of CIM and LMS. Further mechanistic studies demonstrated that the combination of CIM and LMS promoted dendritic cell (DC) activation and blocked anti‐inflammatory cytokine IL‐10 and TGF‐β production in CD4⁺CD25⁺ T cells. These findings suggest that CIM and LMS have the synergistic and additive ability to enhance cellular response to hepatitis B virus DNA vaccine, which may be mediated by DC activation and inhibition of anti‐inflammatory cytokine expression. Thus, the combination of cimetidine and levamisole may be useful as an effective adjuvant in DNA vaccinations for chronic hepatitis B virus infection.