Marianne Lindblad

Oral tolerization with peptide 336–351 linked to cholera toxin B subunit in preventing relapses of uveitis in Behcet's disease

Behcets disease (BD) specific peptide (p336–351) was identified within the human 60 kD heat shock protein (HSP60). Oral p336–351 induced uveitis in rats which was prevented by oral tolerization with the peptide linked to recombinant cholera toxin B subunit (CTB). This strategy was adopted in a phase I/II clinical trial by oral administration of p336–351‐CTB, 3 times weekly, followed by gradual withdrawal of all immunosuppressive drugs used to control the disease in 8 patients with BD. The patients were monitored by clinical and ophthalmological examination, as well as extensive immunological investigations. Oral administration of p336–351‐CTB had no adverse effect and withdrawal of the immunosuppressive drugs showed no relapse of uveitis in 5 of 8 patients or 5 of 6 selected patients who were free of disease activity prior to initiating the tolerization regimen. After tolerization was discontinued, 3 of 5 patients remained free of relapsing uveitis for 10–18 months after cessation of all treatment. Control of uveitis and extra‐ocular manifestations of BD was associated with a lack of peptide‐specific CD4+ T cell proliferation, a decrease in expression of TH1 type cells (CCR5, CXCR3), IFN‐γ and TNF‐α production, CCR7+ T cells and costimulatory molecules (CD40 and CD28), as compared with an increase in these parameters in patients in whom uveitis had relapsed. The efficacy of oral peptide‐CTB tolerization will need to be confirmed in a phase III trial, but this novel strategy in humans might be applicable generally to autoimmune diseases in which specific antigens have been identified.

Systemic and Mucosal Immune Responses in Mice after Mucosal Immunization with Group B Streptococcus Type III Capsular Polysaccharide-Cholera Toxin B Subunit Conjugate Vaccine

ABSTRACT Group B streptococci (GBS) colonize the female genital and rectal tracts and can cause invasive infection in susceptible newborns. An optimally effective GBS vaccine should induce mucosal and systemic immunity. In this study, we investigate the local and systemic immune responses to GBS type III capsular polysaccharide (CPS) after mucosal vaccination of mice via intranasal, peroral, rectal, and vaginal routes, with GBS type III CPS conjugated with recombinant cholera toxin B subunit (GBS III CPS-rCTB). Cholera toxin (CT) was added as an adjuvant. Immunoglobulin G (IgG) and IgA antibodies to the CPS were tested in serum, lungs, and intestinal, rectal, and vaginal extracts by enzyme-linked immunosorbent assay. The conjugated CPS administered by intranasal, peroral, rectal, and vaginal routes was much more effective at inducing both mucosal and systemic antibody responses to GBS III CPS than was unconjugated CPS. The CPS-specific immune responses in various organs were dependent on the route of immunization. Generally, the highest levels of IgA and IgG were generated in the regions or sites of the conjugate exposure. Thus, intranasal vaccination elicited the highest anti-CPS IgA and IgG antibody levels in the lungs, whereas peroral administration in the intestinal site and vaginal vaccination elicited the highest antibody levels in the vagina. Rectal vaccination was superior to the other routes in inducing high antibody levels in the rectum. The four routes of mucosal vaccination also induced distant antibody responses to CPS. Rectal vaccination induced high specific IgA levels in the vagina and intestine, and oral administration induced high specific IgA levels in the lungs and rectum. All four routes of vaccination with the conjugate elicited similarly high levels of anti-CPS IgG in serum. Intranasal vaccination with different doses of the conjugate (10, 30, and 80 μg of CPS) did not have a significant influence on the anti-CPS specific antibody responses. Intranasal immunization induced better antibody responses when one dose of the conjugate was divided and given on three consecutive days compared to administration of the full dose on one occasion. In conclusion, rectal and vaginal vaccination may be the best way of stimulating anti-CPS immune responses in the rectal and vaginal tracts, while high levels of anti-CPS antibodies in the lungs can be achieved after intranasal administration. The vaccination regimen thus might influence the mucosal immune response to CPS. This conjugate may serve as an effective mucosal vaccine for preventing mucosal colonization and invasive infection caused by GBS.

Mucosal and systemic antibody responses after peroral or intranasal immunization: Effects of conjugation to enterotoxin B subunits and/or of co-administration with free toxin as adjuvant

The mucosa‐binding molecules cholera toxin (CT) from Vibrio cholerae and heat‐labile enterotoxin (LT) from Escherichia coli have previously been used as mucosal adjuvants and carriers for many types of antigen. However, since these molecules are toxic and cannot be used in human vaccines, it is important to study whether their non‐toxic mucosa‐binding B subunits, CTB and LTB, can be used as alternative safe mucosal adjuvants and/or carrier molecules. We have as a model protein antigen used human gammaglobulin (HGG) for admixture with or chemical conjugation to recombinantly produced CTB and LTB, respectively, and measured antigen‐specific local secretory IgA antibodies in saponin extracts from intestine and lung tissue by ELISA following intra‐nasal (i.n.) or per‐oral (p.o.) immunization. The results show that local antibody formation against HGG was increased after immunization with conjugated as compared to free HGG. However, while the conjugates alone gave rise to significant immune responses in the lung and also, to a lesser degree, in the intestine after i.n. immunization, co‐administration of a small amount of free CT/LT as adjuvant was needed to induce a significant immune response in the intestine after p.o. immunization. We also found that following i.n. immunization, the addition of CTB to HGG, without coupling, increased the mucosal immune response to some extent, indicating that CTB by itself can work as an adjuvant by the i.n. route of immunization. A striking finding was that, as a carrier, CTB was superior to LTB when the conjugates were used by the oral but not by the i.n. route of immunization. In conclusion, conjugation of an antigen to mucosa‐binding molecules such as CTB and/or LTB can dramatically increase their mucosal immunogenicity. This approach may thus be useful in the preparation of mucosal vaccines.

Preparation and preclinical evaluation of experimental group B streptococcus type III polysaccharide-cholera toxin B subunit conjugate vaccine for intranasal immunization.

Streptococcus group B (GBS) is usually carried asymptomatically in the vaginal tract of women and can be transferred to the newborn during parturition. Serum antibodies to the capsular polysaccharide (CPS) can prevent invasive diseases, whereas immunity acting at the mucosal surface may be more important to inhibit the mucosal colonization of GBS and thus the risk of infection for the newborn. We prepared different GBS type III CPS-protein conjugate vaccines and evaluated their systemic and mucosal immunogenicity in mice. GBS type III CPS was conjugated to tetanus toxoid (TT) or recombinant cholera toxin B subunit (rCTB) either directly or to rCTB indirectly via TT. The conjugation was performed by different methods: (1) CPS was coupled to TT with 1-ethyl-3 (3-dimethylaminopropyl)-carbodiimide (EDAC), using adipic acid dihydrazide (ADH) as a spacer; (2) CPS was conjugated with rCTB using reductive amination; or, (3) N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) was used to bind rCTB to the TT of the CPS-TT conjugate. Mice were immunized with these conjugates or purified CPS by subcutaneous (s.c.) and intranasal (i. n.) routes. Antibodies to GBS III in serum, lungs and vagina were measured with ELISA. All of the CPS-protein conjugates were superior to unconjugated CPS in eliciting CPS-specific immune responses in serum and mucosal tissue extracts. The conjugates, when administrated s.c., induced only IgG responses in serum, lung and vagina, while i.n. vaccination also elicited IgA responses in the lungs and vagina. The CPS-TT conjugate administrated i.n. induced a strong serum IgG, but only a weak mucosal IgA response, while the CPS-rCTB conjugate elicited high IgG as well as IgA antibodies in the lungs after i.n. immunization. GBS III CPS-TT conjugated with rCTB produced a strong systemic and local anti-CPSIII response after i.n. administration. Co-administration of CT as adjuvant enhanced the anti-CPS systemic and mucosal immune responses further after i.n. administration with the CPS conjugates. These findings indicate that: (i) i.n. immunization with GBS CPS-protein conjugates was more effective than s.c immunization for stimulating serum as well as mucosal immune responses; (ii) rCTB as a carrier protein for GBS III CPS could markedly improve the mucosal immune response; and (iii) the experimental GBS type III CPS conjugates containing rCTB should be investigated as mucosal vaccine to prevent GBS infection in humans.

Group B Streptococcus capsular polysaccharide-cholera toxin B subunit conjugate vaccines prepared by different methods for intranasal immunization

ABSTRACT Group B Streptococcus (GBS) type III capsular polysaccharide (CPS III) was conjugated to recombinant cholera toxin B subunit (rCTB) using three different methods which employed (i) cystamine and N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), (ii) carbodiimide with adipic acid dihydrazide (ADH) as a spacer, or (iii) reductive amination (RA). The CPS III-rCTB conjugates were divided into large- and small-molecular-weight (Mr) fractions, and the immunogenicities of the different preparations after intranasal (i.n.) immunization were studied in mice. Both large- and small-Mrconjugates of CPS III-rCTBRA or CPS III-rCTBADHinduced high, almost comparable levels of CPS-specific immunoglobulin G (IgG) in serum, lungs, and vagina that were generally superior to those obtained with CPS III-rCTBSPDP conjugates or a CPS III and rCTB mixture. However, the smaller-Mrconjugates of CPS III-rCTBRA or CPS III-rCTBADHin most cases elicited a lower anti-CPS IgA immune response than the large-Mr conjugates, and the highest anti-CPS IgA titers in both tissues and serum were obtained with the large-Mr CPS III-rCTBRA conjugate. Serum IgG anti-CPS titers induced by the CPS III-rCTBRAconjugate had high levels of specific IgG1, IgG2a, IgG2b, and IgG3 antibodies. Based on the effectiveness of RA for coupling CPS III to rCTB, RA was also tested for conjugating GBS CPS Ia with rCTB. As for the CPS III-rCTB conjugates, the immunogenicity of CPS Ia was greatly increased by conjugation to rCTB. Intranasal immunization with a combination of CPS Ia-rCTB and CPS III-rCTB conjugates was shown to induce anti-CPS Ia and III immune responses in serum and lungs that were fully comparable with the responses to immunization with the monovalent CPS Ia-rCTB or CPS III-rCTB conjugates. These results suggest that the GBS CPS III-rCTB and CPS Ia-rCTB conjugates prepared by the RA method may be used in bivalent and possibly also in multivalent mucosal GBS conjugate vaccines.

Coupling of antigen to cholera toxin for dendritic cell vaccination promotes the induction of MHC class I-restricted cytotoxic T cells and the rejection of a cognate antigen-expressing model tumor

We previously demonstrated that cholera toxin (CT) is highly efficient as a combined carrier and adjuvant for dendritic cell (DC) vaccination, inducing strong Th1‐dominated B cell and CD4+ T cell responses. In this study we show that vaccination with DC pre‐pulsed ex vivo with CT‐conjugated OVA (OVA‐CT) gives rise to OVA‐specific CD8+ T cells that produce IFN‐γ and are cytotoxic for OVA‐expressing E.G7 tumor cells both in vitro and in vivo. The induction of specific CD8+ CTL by OVA‐CT‐treated DC was associated with enhanced presentation of OVA peptide (SIINFEKL) on MHC class I in combination with an overall activation of the pulsed DC. Vaccination of mice with OVA‐CT‐pulsed DC resulted in rejection of already established MHC class I‐positive, MHC class II‐negative, OVA‐expressing E.G7 tumors in an antigen‐specific, CD8+ T cell‐dependent fashion and was associated with high numbers of tumor‐infiltrating CD8+ T cells. Conjugation of antigen to CT facilitated DC uptake of the linked antigen through the GM1 receptor‐binding B subunit and induced strong activation‐maturation signals through the biologically active A subunit. These results have interesting implications for DC vaccination aimed at inducing CTL immune responses.

Cholera toxin B subunit (CTB) entrapped in microparticles shows comparable immunogenicity to CTB mixed with whole cholera toxin following oral immunization

Abstract Cholera toxin B subunit (CTB) was entrapped in poly(lactide-co-glycolide) microparticles and the responses induced by microencapsulated CTB were comparable to those induced by oral immunization with CTB mixed with whole cholera toxin. In addition, the CTB was released from microparticles intact following in vitro incubation, as determined by the ability of the CTB to bind to its cellular receptor, GM1. Moreover, microencapsulated CTB induced serum antibodies against intact CTB and not against monomeric fragments.

Purification of El tor cholera enterotoxins and comparisons with classical toxin

In 55 clinical isolates of Vibrio cholerae biotype El Tor, cholera toxin (CT) production was higher after growth in liquid medium first under relatively anaerobic conditions followed by excessive aeration (AKI conditions) as compared with growth under the optimal conditions for CT production from V. cholerae of classical biotype (median toxin level being 400 ng ml-1 and 1 ng ml-1 respectively, for the two different growth conditions). Large growth volumes further enhanced El Tor toxin production to levels at or above 3-5 micrograms ml-1 from several strains, which allowed for easy purification of toxin by salt precipitation, aluminium hydroxide adsorption and/or GM1 ganglioside affinity chromatography. However, such purified El Tor CT completely lacked the A subunit when examined by SDS-PAGE or by monoclonal anti-A subunit antibody GM1-ELISA. In contrast, when El Tor CT was prepared from bacteria grown in the presence of specific antiserum against soluble haemagglutinin/protease it contained the A subunit (unnicked) in the same proportion to the B subunit (1A:5B) as classical CT. Immunodiffusion-in-gel tests revealed that the B subunits of El Tor and classical CTs share major epitopes but also have one or more weaker biotype-specific epitopes. The two types of toxin were practically indistinguishable in various GM1-ELISA tests, and antisera raised against El Tor and classical CT, respectively, could also completely neutralize the heterologous as well as the homologous toxin activity in vivo. The results indicate that CTs from El Tor and classical V. cholerae, despite demonstrable epitope differences, are predominantly cross-reactive and give rise to antisera with strong cross-neutralizing activity.

Novel Immunostimulatory Agent Based on CpG Oligodeoxynucleotide Linked to the Nontoxic B Subunit of Cholera Toxin

In this study, we report the development of a novel, rationally designed immunostimulatory adjuvant based on chemical conjugation of CpG oligodeoxynucleotide (ODN) to the nontoxic B subunit of cholera toxin (CTB). We demonstrate that the immunostimulatory effects of CpG can be dramatically enhanced by conjugation to CTB. Thus, CpG ODN linked to CTB (CTB-CpG) was shown to be a more potent stimulator of proinflammatory cytokine and chemokine responses in murine splenocytes and human PBMCs than those of CpG ODN alone in vitro. The presence of CpG motif, but not modified phosphorothioate ODN backbone, was found to be critical for the enhanced immunostimulatory effects of CTB-CpG. Our mode-of-action studies, including studies on cells from specifically gene knockout mice suggest that similar to CpG, CTB-CpG exerts its immunostimulatory effects through a TLR9/MyD88- and NF-κB-dependent pathway. Surprisingly, and as opposed to CpG ODN, CTB-CpG-induced immunity was shown to be independent of endosomal acidification and resistant to inhibitory ODN. Furthermore, preincubation of CTB-CpG with GM1 ganglioside reduced the immunostimulatory effects of CTB-CpG to those of CpG ODN alone. Interestingly, conjugation of CpG ODN to CTB confers an enhanced cross-species activity to CpG ODN. Furthermore, using tetanus toxoid as a vaccine Ag for s.c. immunization, CTB-CpG markedly enhanced the Ag-specific IgG Ab response and altered the specific pattern of Ab isotypes toward a Th1 type response. To our knowledge, CTB is the first nontoxic derivative of microbial toxins discovered that when chemically linked to CpG remarkably augments the CpG-mediated immune responses.

Effect of pre-existing immunity for systemic and mucosal immune responses to intranasal immunization with group B Streptococcus type III capsular polysaccharide-cholera toxin B subunit conjugate

The effects of priming with a group B Streptococcus type III capsular polysaccharide (GBS CPS III)-recombinant cholera toxin B subunit (rCTB) conjugate, purified GBS CPS III or rCTB alone on the systemic and mucosal immune responses to CPS III after intranasal (i.n.) immunization were investigated in mice. Priming with purified GBS CPS III followed by boosting with GBS CPS III-rCTB conjugate or priming with the conjugate followed by boosting with free CPS induced comparable levels of specific IgG and IgA in both serum and in lungs and vagina. However, i.n. immunization comprising both priming and boosting with conjugate was superior to priming with CPS and boosting with conjugate or the reverse, especially with regard to inducing mucosal IgA anti-CPS responses. All the immunization schemes, except priming and boosting with free CPS, induced high and similar levels of IgG1 in serum. In contrast, mice primed with free CPS III and then boosted with CPS III-rCTB conjugate by the i.n. route failed to produce significant levels of IgG2a, IgG2b and IgG3 in serum, at difference from mice primed with the conjugate and boosted with either conjugate or free CPS. Pre-immunization with rCTB either i.n. or i.p. did not suppress specific serum IgG responses induced by GBS CPS III-rCTB conjugate intranasally, but did inhibit serum and especially mucosal IgA responses. Our findings suggest that priming with CPS affects the distribution of IgG subclasses to GBS CPS and that pre-existing anti-carrier rCTB immunity can have an inhibitory effect on mucosal immune responses elicited by the conjugate vaccine given by the i.n. route.