Cecil Czerkinsky

Cholera toxin and cholera B subunit as oral-mucosal adjuvant and antigen vector systems.

Cholera toxin (CT) and the analogous heat-labile enterotoxin (LT) from Escherichia coli have several immunomodulating effects which alone or in combination might explain their strong adjuvant action in stimulating mucosal IgA and other immune responses to admixed unrelated antigens after oral immunization. These effects include, depending on animal species and experimental systems, enhanced antigen presentation by a variety of cell types; promotion of isotype differentiation in B cells leading to increased IgA formation; and complex stimulatory as well as inhibitory effects on T-cell proliferation and lymphokine production. This adjuvant activity appears to be closely linked to the ADP-ribosylating action of CT and LT associated with enhanced cyclic AMP formation in the affected cells, and thus it may prove difficult to eliminate the enterotoxic activity without loss of adjuvanticity. However, through a separate mechanism, as an antigen-carrier system providing specific binding to epithelium including the M cells of intestinal Peyers patches, both CT and its non-toxic binding subunit moiety (CTB) have been shown to markedly enhance the mucosal immune response to various foreign antigens or epitopes covalently linked to these molecules. This gives promise for the future use of CTB or related non-toxic binding derivatives as vehicles to facilitate induction of mucosal immune responses to a broad range of antigens for human vaccination purposes.

Mucosal immunisation and adjuvants: a brief overview of recent advances and challenges.

Mucosal immunisation may be used both to prevent mucosal infections through the activation of anti-microbial immunity and to treat selected autoimmune, allergic or infectious-immunopathological disorders through the induction of antigen-specific tolerance. The development of mucosal vaccines, whether for prevention of infectious diseases or for immunotherapy, requires antigen delivery and adjuvant systems that can efficiently help to present vaccine or immunotherapy antigens to the mucosal immune system. Promising advances have recently been made in the design of more efficient mucosal adjuvants based on detoxified bacterial toxin derivatives or CpG motif-containing DNA, and perhaps even more striking progress has been done in the use of virus-like particles as mucosal delivery systems for vaccines and of cholera toxin B subunit as antigen vector for immunotherapeutic tolerance induction. However, it is a memento that two recently developed mucosal vaccines for human use against rotavirus diarrhoea and influenza were withdrawn after a short period in the market because of adverse reactions among the vaccinees, thus emphasising the difficult and challenging task also for mucosal immunisation of combining vaccine and adjuvant efficacy with safety and acceptability.

Sublingual Immunization with Nonreplicating Antigens Induces Antibody-Forming Cells and Cytotoxic T Cells in the Female Genital Tract Mucosa and Protects against Genital Papillomavirus Infection

We have recently reported that the sublingual (s.l.) mucosa is an efficient site for inducing systemic and mucosal immune responses. In this study, the potential of s.l. immunization to induce remote Ab responses and CD8+ cytotoxic responses in the female genital tract was examined in mice by using a nonreplicating Ag, OVA, and cholera toxin (CT) as an adjuvant. Sublingual administration of OVA and CT induced Ag-specific IgA and IgG Abs in blood and in cervicovaginal secretions. These responses were associated with large numbers of IgA Ab-secreting cells (ASCs) in the genital mucosa. Genital ASC responses were similar in magnitude and isotype distribution after s.l., intranasal, or vaginal immunization and were superior to those seen after intragastric immunization. Genital, but not blood or spleen, IgA ASC responses were inhibited by treatment with anti-CCL28 Abs, suggesting that the chemokine CCL28 plays a major role in the migration of IgA ASC progenitors to the reproductive tract mucosa. Furthermore, s.l. immunization with OVA induced OVA-specific effector CD8+ cytolytic T cells in the genital mucosa, and these responses required coadministration of the CT adjuvant. Furthermore, s.l. administration of human papillomavirus virus-like particles with or without the CT adjuvant conferred protection against genital challenge with human papillomavirus pseudovirions. Taken together, these findings underscore the potential of s.l. immunization as an efficient vaccination strategy for inducing genital immune responses and should impact on the development of vaccines against sexually transmitted diseases.

CCR7-CCL19/CCL21-Regulated Dendritic Cells Are Responsible for Effectiveness of Sublingual Vaccination

Our previous studies demonstrated the potential of the sublingual (s.l.) route for delivering vaccines capable of inducing mucosal as well as systemic immune responses. Those findings prompted us to attempt to identify possible inductive mechanism of s.l. vaccination for immune responses. Within 2 h after s.l. administration with cholera toxin (CT), significantly higher numbers of MHC class II+ cells accumulated in the s.l. mucosa. Of note, there were brisk expression levels of both CCL19 and CCL21 in cervical lymph nodes (CLN) 24 h after s.l. vaccination with CT. In reconstitution experiments using OVA-specific CD4+ or CD8+ T cells, s.l. vaccination elicited strong Ag-specific T cell proliferation mainly in CLN. Interestingly, Ag-specific T cell proliferation completely disappeared in CD11c-depleted and CCR7−/− mice but not in Langerin-depleted, macrophage-depleted, and CCR6−/− mice. Similar to CD4+ T cell responses, induction of Ag-specific IgG (systemic) and IgA (mucosal) Ab responses were significantly reduced in CD11c-depleted and CCR7−/− mice after s.l. vaccination with OVA plus CT. Although CD8α− dendritic cells ferried Ag from the s.l. mucosa, both migratory CD8α− and resident CD8α+ dendritic cells were essential to prime CD4+ T cells in the CLN. On the basis of these findings, we believe that CCR7 expressed CD8α−CD11c+ cells ferry Ag in the s.l. mucosa, migrate into the CLN, and share the Ag with resident CD8α+CD11c+ cells for the initiation of Ag-specific T and B cell responses following s.l. challenge. We propose that the s.l. mucosa is one of the effective mucosal inductive sites regulated by the CCR7-CCL19/CCL21 pathway.

Local and Systemic Immune Responses to Rectal Administration of Recombinant Cholera Toxin B Subunit in Humans

ABSTRACT The induction of immune responses to rectally administered recombinant cholera toxin B subunit (CTB) in humans was studied. Three immunizations induced high levels of CTB-specific antibody-secreting cells, particular of the immunoglobulin A isotype, in both rectum and peripheral blood. Antitoxin antibody responses in rectal secretions and serum were also found.

Sublingual Immunization Protects against Helicobacter pylori Infection and Induces T and B Cell Responses in the Stomach

ABSTRACT Sublingual (SL) immunization has been described as an effective novel way to induce mucosal immune responses in the respiratory and genital tracts. We examined the potential of SL immunization against Helicobacter pylori to stimulate immune responses in the gastrointestinal mucosa and protect against H. pylori infection. Mice received two SL immunizations with H. pylori lysate antigens and cholera toxin as an adjuvant, and after challenge with live H. pylori bacteria, their immune responses and protection were evaluated, as were immune responses prior to challenge. SL immunization induced enhanced proliferative responses to H. pylori antigens in cervicomandibular lymph nodes and provided at least the same level of immune responses and protection as corresponding intragastric immunization. Protection in SL-immunized mice was associated with strong H. pylori-specific serum IgG and IgA antibody responses in the stomach and intestine, with strong proliferation and gamma interferon (IFN-γ) and interleukin-17 (IL-17) production by spleen and mesenteric lymph node T cells stimulated with H. pylori antigens in vitro, and with increased IFN-γ and IL-17 gene expression in the stomach compared to levels in infected unimmunized mice. Immunohistochemical studies showed enhanced infiltration of CD4+ T cells and CD19+ B cells into the H. pylori-infected stomach mucosa of SL-immunized but not unimmunized H. pylori-infected mice, which coincided with increased expression of the mucosal addressin cell adhesion molecule (MAdCAM-1) and T and B cell-attracting chemokines CXCL10 and CCL28. We conclude that, in mice, SL immunization can effectively induce protection against H. pylori infection in association with strong T and B cell infiltration into the stomach.

Immunoglobulin secretion by the normal and the infected newborn infant

The development of quantitative single-cell immunoassays has provided a novel opportunity to demonstrate the isotype-specific immunoglobulin responses in normal and infected neonates. The reverse enzyme-linked immunospot assay was used to determine the number of immunoglobulin-secreting cells (IgSCs) in peripheral blood. Baseline numbers of IgSCs were established in 69 uninfected term and preterm infants within 5 days of birth; values above the 99th percentile were considered elevated. The IgSCs were also measured in 266 infants with proved or suspected infections or congenital anomalies. A subset of newborn infants was retested weekly. Few IgSCs (mostly IgMSCs) were detected within 5 days of birth in uninfected neonates, but by 1 month 77% had increased numbers of IgSCs, primarily IgASCs. Sixty-three (24%) of 266 study infants had increased IgSCs on initial sampling (predominantly IgMSCs); these included infants as immature as 25 to 27 weeks of gestational age; elevations in IgSCs were most frequent in infants with intrauterine infections. Increased numbers of IgSCs were uncommon in infants with early-onset sepsis in the first 5 days but were frequent by the second week, consistent with acquisition of infection near the time of delivery. We conclude that the presence of elevated numbers of IgSCs soon after birth may be a useful surrogate marker of untreated intrauterine infection. The development of predominantly IgASCs in the first month of life suggests postnatal exposure to common mucosal antigens.

Enzyme-linked immunospot assays for direct ex vivo measurement of vaccine-induced human humoral immune responses in blood

The enzyme-linked immunospot (ELISPOT) assay was originally developed to enumerate antigen-specific antibody-secreting cells (ASCs), and has subsequently been adapted for various applications, including the detection cytokine-secreting cells. Owing to its exceptionally high sensitivity, the ELISPOT has proven to be especially useful for detecting discrete populations of active cells (e.g., antigen-specific cells). Because of its versatility, the ELISPOT assay is used for a wide range of applications, including clonal analyses of immune responses after vaccination or after immunotherapy. Here we describe standard protocols for the detection of human ASCs specific to virtually any vaccine antigen after enrichment of circulating plasmablasts. In addition, a protocol is described for the measurement of mucosal ASC responses after prior immunomagnetic enrichment of mucosally derived blood lymphocytes. The protocols described allow rapid (∼6–8 h) detection of specific ASCs in small (1–2 ml) samples of blood and can be performed in resource-poor settings.

Current status and future prospects for a vaccine against schistosomiasis

Schistosomiasis remains an intractable problem in many parts of the world. Whereas the schistosome parasites cause little in the way of disease, their eggs become trapped in tissues of the host and elicit powerful and potentially damaging immune responses that are responsible for the pathology. Despite nearly four decades of effort there is still no effective vaccine against schistosomiasis, although a single vaccine candidate is undergoing clinical trials at present. Animal models have revealed much about disease progression and pathology. However, problems remain in identifying appropriate protective antigens to elicit immune responses that will attack the parasite but will not cross-react with egg antigens and thus increase the chance of developing severe chronic disease in individuals that have already been infected. This review summarizes the life-cycle of the parasite, current knowledge of pathogenesis and acquired immunity based on animal studies and observations in humans and the status of efforts in the vaccine development field.

Virus-specific antibody production and polyclonal B-cell activation in the intestinal mucosa of HIV-infected individuals

ObjectiveTo examine possible changes in mucosal B-cell activation status. DesignTo examine the frequency and isotype distribution of total and HIV-specific antibody-secreting cells (ASC) in the intestinal mucosa of HIV-infected individuals. MethodsMucosal lymphocytes were obtained by enzymatic treatment of duodenal pinch biopsies and the numbers of ASC were assayed with the enzyme-linked immunospot technique. ResultsHigh numbers of HIV-specific ASC were found in the intestine of all HIV-infected individuals despite low levels of HIV-specific blood ASC. All HIV-infected individuals had large numbers of intestinal immunoglobulin (Ig) A-ASC against the HIV envelope glycoprotein gp160. Eight out of nine patients also had HIV gp160-specific intestinal IgG-ASC. These HIV-specific ASC were detected irrespective of disease stage, route of infection, or levels of circulating CD4+ T cells. HIV-specific ASC were found in peripheral blood from patients with CD4+ T cells ≥100×106/l blood, but in none of three patients with low CD4+ T-cell counts. The frequencies of virus-specific ASC in the blood were on average 100-fold lower than that observed within the intestinal mucosa. Mucosal polyclonal B-cell activation was evident in HIV-infected individuals, as documented by significantly elevated numbers of Ig-secreting cells (ISC) in all three major Ig classes; on average, seven-, five- and 20-fold numbers of IgA, IgG and IgM-ISC compared with healthy controls. Furthermore, substantial numbers of ASC reacting with unrelated antigens such as dog albumin and keyhole limpet haemocyanin were detected in HIV-infected patients. Interestingly, patients with CD4+T cells < 100×106/l blood displayed large numbers of HIV-specific intestinal ASC even though total numbers of ISC, including ASC reactive to unrelated antigens, were decreased. ConclusionsThe large numbers of virus-specific ASC found in the intestine of HIV-infected individuals may be a consequence of local replication of HIV-1 resulting in a continuous antigen stimulation. The persistence of strong intestinal anti-HIV responses even at late stages of disease suggest that the mucosal B-cell responses are functionally intact throughout the disease. Furthermore, these results suggest that there is no correlation between HIV-specific ASC numbers and polyclonal B-cell activation. These observations indicate that intestinal B-cell activation is profoundly disregulated in HIV-infected individuals.