Carbohydrate Moieties as Vaccine Candidates: meeting summary.

In September 2007, a meeting entitled 'Carbohydrate Moieties as Vaccine Candidates' was held at the National Institutes of Health (Bethesda, MD). This meeting brought together scientists from a number of disciplines to address issues concerning carbohydrate moieties as targets for vaccines for a variety of pathogens and tumors. In addition, the meeting participants addressed fundamental topics of glycoimmunology including the recognition of glycotopes by B and T lymphocytes, the ontogeny of anti-carbohydrate immune responses, peptide mimicry, carbohydrate antigen processing pathways and adjuvants. One session reported progress in the development of new tools such as computational algorithms, glycan arrays and oligosaccharide synthesis and their application to carbohydrate vaccine research. The session titles were: (1) immune response to bacterial carbohydrate antigens; (2) immune response to glycolipids; (3) immune response to carbohydrate antigens on other microbes and on tumors; (4) novel vaccine approaches; (5) novel tools in carbohydrate vaccine research; (6) bench to bedside: carbohydrate moieties as vaccine immunopotentiators.

Genetic variation influences the B-cell response to immunization with a pneumococcal polysaccharide conjugate vaccine.

CBA/J mice immunized with pneumococcal 23F-CRM(197) vaccine produce significantly lower titers of 23F-specific antibodies and fewer 23F-specific antibody-secreting cells (ASC) than did BALB/c or (CBA/J x BALB/c)F(1) (CCBAF(1)) mice. The reduced 23F-specific titers of CBA/J versus BALB/c or CCBAF(1) mice are presumably related to lower frequencies of 23F-specific ASC influenced by genetic variation.

HLA-G genotypes and pregnancy outcome in couples with unexplained recurrent miscarriage.

HLA-G is a non-classical human leukocyte antigen expressed primarily in fetal tissues at the maternal-fetal interface. This expression pattern is unique among HLA genes and suggests that HLA-G may be involved in interactions that are critical in establishing and/or maintaining pregnancy. To evaluate the role of polymorphisms at this locus in maternal-fetal interactions, 113 couples with unexplained recurrent miscarriage were genotyped for seven polymorphisms that define 12 HLA-G alleles. Logistic regression analysis was used to assess whether HLA-G genotypes were associated with an increased risk for a subsequent miscarriage. The presence of an HLA-G*0104 or HLA-G*0105N allele in either partner was significantly associated with an increased risk for miscarriage, after adjustment for maternal age, number of previous miscarriages, history of a previous liveborn, and treatment with paternal mononuclear cells. The *0104 and *0105N alleles are defined by polymorphisms in the alpha-2 domain and encode protein variants that are present only in the full-length HLA-G1 protein. The significant genotype-specific risk in this population suggests that allelic variation in the alpha-2 domain of the HLA-G1 isoforms contributes to recurrent miscarriage.

Immunization with Haemophilus influenzae type b-CRM(197) conjugate vaccine elicits a mixed Th1 and Th2 CD(4+) T cell cytokine response that correlates with the isotype of antipolysaccharide antibody.

Haemophilus influenzae type b (Hib) capsular polysaccharide (PS) induces protective antibodies but is T independent and poorly immunogenic in infants. Conjugate vaccines of Hib PS linked to proteins, such as CRM(197), increase the PS antibody titer and elicit immunologic memory. To define the conjugate-induced memory T cell response, 19 adults were immunized with Hib-CRM(197), and antibody titers, carrier protein-specific CD4(+) T cell proliferation, and cytokine production were measured. Hib-CRM(197) induced PS and CRM(197) antibodies, vigorous T cell recall responses, and production of cytokines, including interleukin (IL)-2, IL-5, IL-10, and interferon-gamma. There was marked variability in PS antibody titer, despite consistent CRM(197)-specific recall responsiveness, which correlated with peak IgM and IgA PS antibody titers. Correlations were also found between IL-2 and IL-5 and IgA PS antibody levels. Hib-CRM(197) induced a rapid increase in CRM(197)-specific memory T cells and mixed Th1/Th2 cytokines, which may regulate the isotype and quantity of PS antibody.

Human monoclonal antibodies against Pseudomonas aeruginosa lipopolysaccharide derived from transgenic mice containing megabase human immunoglobulin loci are opsonic and protective against fatal pseudomonas sepsis.

Pseudomonas aeruginosa is a significant human pathogen, and no vaccine is commercially available. Passive antibody prophylaxis using monoclonal antibodies (MAb) against protective P. aeruginosa epitopes is an alternative strategy for preventing P. aeruginosa infection, but mouse MAb are not suitable for use in humans. Polyclonal human antibodies from multiple donors have variable antibody titers, and human MAb are difficult to make. We used immunoglobulin-inactivated transgenic mice reconstituted with megabase-size human immunoglobulin loci to generate a human MAb against the polysaccharide (PS) portion of the lipopolysaccharide O side chain of a common pathogenic serogroup of P. aeruginosa, 06ad. The anti-PS human immunoglobulin G2 MAb made from mice immunized with heat-killed P. aeruginosa was specific for serogroup 06ad pseudomonas. The MAb was highly opsonic for the uptake and killing of P. aeruginosa by human polymorphonuclear leukocytes in the presence of human complement. In addition, 25 microg of the MAb protected 100% of neutropenic mice from fatal P. aeruginosa sepsis. DNA sequence analysis of the genes encoding the MAb revealed V(H)3 and Vkappa2/A2 variable-region genes, similar to variable-region genes in humans immunized with bacterial PS and associated with high-avidity anti-PS antibodies. We conclude that human MAb to P. aeruginosa made in these transgenic mice are highly protective and that these mice mimic the antibody response seen in humans immunized with T-cell-independent antigens such as bacterial PS.

CpG oligodeoxynucleotides act as adjuvants for pneumococcal polysaccharide-protein conjugate vaccines and enhance antipolysaccharide immunoglobulin G2a (IgG2a) and IgG3 antibodies.

Pneumococcal polysaccharide-protein conjugate vaccines elicit antipolysaccharide antibodies, but multiple doses are required to achieve protective antibody levels in children. In addition, the immunogenicity of experimental multivalent pneumococcal conjugate vaccines varies with different polysaccharide serotypes. One strategy to improve these vaccines is to incorporate an adjuvant to enhance their immunogenicity. Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) are adjuvants that promote T-cell and T-dependent antibody responses to protein antigens, but it has been unclear whether CpG ODN can enhance polysaccharide-specific antibody responses. The present studies demonstrate significant adjuvant activity of CpG ODN for antibody responses against Streptococcus pneumoniae polysaccharide types 19F and 6B induced by conjugates of 19F and 6B with the protein carrier CRM(197). BALB/c ByJ mice were injected with 19F-CRM(197) or 6B-CRM(197) with or without CpG ODN, and sera were tested for anti-19F or anti-6B antibodies by enzyme-linked immunosorbent assay. The polysaccharide-specific antibody response to 19F-CRM(197) alone was predominantly of the immunoglobulin G1 (IgG1) and IgM isotypes, but addition of CpG ODN markedly increased geometric mean titers of total anti-19F antibody (23-fold), anti-19F IgG2a (26-fold), and anti-19F IgG3 (>246-fold). The polysaccharide-specific antibody response to 6B-CRM(197) alone consisted only of IgM, but addition of CpG ODN induced high titers of anti-6B IgG1 (>78-fold increase), anti-6B IgG2a (>54-fold increase), and anti-6B IgG3 (>3,162-fold increase). CpG ODN also increased anti-CRM(197) IgG2a and IgG3. Adjuvant effects were not observed with control non-CpG ODN. Thus, CpG ODN significantly enhance antipolysaccharide IgG responses (especially IgG2a and IgG3) induced by these glycoconjugate vaccines.

Severe La Crosse encephalitis with significant neurologic sequelae.

La Crosse encephalitis, a member of the California arbovirus group, is the most common cause of reported mosquito-borne illness in the United States. Approximately 70 cases of La Crosse encephalitis are reported each year. The principal vector is the mosquito Aedes triseriatus. During the summer the virus is amplified horizontally in a cycle among small mammals such as chipmunks and squirrels. Infected female A. triseriatus deposit eggs in the basal holes of hardwood trees, although man-made containers and old tires containing water also supply a suitable breeding site. Some of these eggs infected with La Crosse virus hatch the next spring and give rise to infected adult A. triseriatus, and the host-vector cycle is renewed. Only a minority of children infected with the virus become ill. Clinical disease caused by La Crosse is usually mild, and neurologic sequelae are relatively uncommon. In this report we describe six patients with severe La Crosse meningoencephalitis diagnosed within a 4-week period. All patients required intensive care management, and there was a high rate of neurologic sequelae, suggesting that La Crosse is not necessarily a benign meningoencephalitis.

A polyclonal outbreak of predominantly VanB vancomycin-resistant enterococci in northeast Ohio. Northeast Ohio Vancomycin-Resistant Enterococcus Surveillance Program.

We studied the molecular epidemiology of vancomycin-resistant enterococci (VRE) isolated in northeast Ohio during 1996 and examined the association between isolation of VRE from samples other than stool and antimicrobial purchases for five Cleveland hospitals. Susceptibility testing and pulsed-field gel electrophoresis were used to analyze 363 isolates from individual patients from 13 hospitals. Susceptibility testing indicated that 287 strains (79%) expressed the VanB phenotype and 76 (21%) expressed the VanA phenotype. The outbreak was polyclonal, with 30 total genotypes. Both VanA and VanB VRE demonstrated multiple genotypes. One genotype was present in all hospitals, suggesting spread between hospitals. For five teaching hospitals, rates of isolation from non-stool sources and from blood correlated positively with purchases of ticarcillin/clavulanic acid (P = .005). In summary, this outbreak demonstrates transmission of VRE between several hospitals in a geographic region and suggests that use of certain beta-lactam antibiotics may be associated with an increased prevalence of VRE.

B- and T-cell immune responses to pneumococcal conjugate vaccines: divergence between carrier- and polysaccharide-specific immunogenicity.

Conjugation of various serotypes of pneumococcal polysaccharide (PnPS) to carrier protein enhances the magnitude of the polysaccharide-specific antibody response, presumably by eliciting T-cell help. However, variability in PnPS serotype-specific immunogenicity has been observed. CBA/J mice immunized with either 6B or 19F PnPS conjugated to the protein carrier Cross Reactive Material(197) (CRM(197)) produce a strong anti-PnPS antibody response; however, when mice are immunized with 23F PnPS conjugated to CRM(197), they fail to produce a significant anti-PnPS response. In order to determine whether this difference was related to alterations in antigen processing of the carrier protein and the subsequent T-cell responses, we studied proliferation of lymphocytes from CBA/J mice immunized with CRM(197) alone or conjugated to 6B, 19F, or 23F PnPS. T-cell proliferative responses to synthetic peptides demonstrated that lymph node cells elicited by the poorly immunogenic conjugate 23F-CRM(197) recognized many, but not all, of the epitopes recognized by lymph node cells elicited by 6B- and 19F-CRM(197) as well as additional epitopes. Despite marked differences in PnPS-specific immunogenicity, all mice made high titers of CRM(197) antibodies of the immunoglobulin G(1) isotype. Cells from mice immunized with any of the conjugates yielded vigorous T-cell responses to whole antigen. We conclude that the serotype of PnPS can alter the peptide specificities of T-cell responses, but even a poorly immunogenic PnPS conjugate can elicit a significant T-cell response. Thus, conjugation of PnPS to a carrier protein that elicits carrier-specific T- and B-cell responses does not necessarily enhance PnPS immunogenicity.

Mononuclear-cell immunisation in prevention of recurrent miscarriages: a randomised trial.

BACKGROUND: Couples with unexplained recurrent miscarriage may have an alloimmune abnormality that prevents the mother from developing immune responses essential for the survival of the genetically foreign conceptus. Immunisation with paternal mononuclear cells is used as a treatment for such alloimmune-mediated pregnancy losses. However, the published results on this treatment are conflicting. In this study (the Recurrent Miscarriage [REMIS] Study), we investigated whether paternal mononuclear cell immunisation improves the rate of successful pregnancies. METHODS: Women who had had three or more spontaneous abortions of unknown cause were enrolled in a double-blind, multicentre, randomised clinical trial. 91 were assigned immunisation with paternal mononuclear cells (treatment) and 92 immunisation with sterile saline (control). The primary outcomes were the inability to achieve pregnancy within 12 months of randomisation, or a pregnancy which terminated before 28 weeks of gestation (failure); and pregnancy of 28 or more weeks of gestation (success). Two analyses were done: one included all women (intention to treat), and the other included only those who became pregnant. FINDINGS: Two women in each group received no treatment, and eight (three treatment, five control) were censored after an interim analysis. In the analysis of all randomised women who completed the trial, the success rate was 31/86 (36%) in the treatment group and 41/85 (48%) in the control group (odds ratio 0.60 [95% CI 0.33-1.12], p=0.108). In the analysis of pregnant women only, the corresponding success rates were 31/68 (46%) and 41/63 (65%; odds ratio 0.45 [0.22-0.91], p=0.026). The results were unchanged after adjustment for maternal age, number of previous miscarriages, and whether or not the couple had had a previous viable pregnancy. Similar results were obtained in a subgroup analysis of 133 couples with no previous livebirth. INTERPRETATION: Immunisation with paternal mononuclear cells does not improve pregnancy outcome in women with unexplained recurrent miscarriage. This therapy should not be offered as a treatment for pregnancy loss.

In vitro transport of active alpha(1)-antitrypsin to the apical surface of epithelia by targeting the polymeric immunoglobulin receptor.

In cystic fibrosis (CF), the intense host inflammatory response to chronic infection largely accounts for the progressive pulmonary disease, and ultimately death. Neutrophils are the prominent inflammatory cells in the lungs of patients with CF, and large amounts of neutrophil elastase (NE) are released during phagocytosis. Besides having direct effects on structural elastin, NE stimulates the release of proinflammatory mediators from the respiratory epithelium and is a potent secretogogue. Therapeutic use of elastase inhibitors in CF has been complicated by difficulties in delivery to the critical site in the airway-the surface of the epithelium. We describe a unique strategy to protect the respiratory epithelial cell surface directly by capitalizing on the nondegradative transcytotic pathway of the polymeric immunoglobulin receptor (pIgR). A recombinant fusion protein was constructed consisting of an antihuman pIgR single-chain Fv (scFv) antibody linked to human alpha(1)-antitrypsin (A1AT), an inhibitor of NE. The recombinant scFv-A1AT fusion protein bound specifically to the pIgR on the basolateral surface of an epithelial cell monolayer, and was transported and released into the apical medium where the A1AT domain was capable of forming an inactivation complex with NE. Thus, A1AT linked to an antihuman pIgR scFv was delivered in receptor-specific fashion from the basolateral to apical surface and was released as an active antiprotease, indicating that it is feasible to deliver therapeutic proteins to the apical surface of epithelia by targeting the pIgR.

Low yield of bacterial stool culture in children with nosocomial diarrhea.

OBJECTIVE: To determine whether bacterial stool cultures (BSC) are useful in initial evaluation of children with symptoms of nosocomial diarrhea. To answer this question we performed a retrospective record review to determine the yield of BSC in children who developed diarrhea after the third hospital day (HD-3). METHODS: The hospital computer record keeping system was utilized to compile the result of BSC collected from children and adolescents ages 0 to 20 years between January 1, 1988, and October 31, 1996. All specimens were analyzed for Salmonella, Shigella, Yersinia and Campylobacter. We reviewed hospital charts of all children who developed a positive BSC beyond HD-3 to determine the time of onset of diarrhea and clinical circumstances. RESULTS: A total of 11 516 BSCs were submitted from 9262 children during the 8 1/2-year period. Five hundred sixty-eight (6.6%) of 9262 children had at least 1 positive BSC. Two thousand five hundred seventy-two children had the first BSC submitted after HD-3 and 13 (0.5%) of these children had a positive result. Chart review of these 13 children demonstrated that 6 had onset of diarrhea during the first 3 hospital days. Therefore only 7 children met our criteria for having nosocomially acquired diarrhea caused by a bacterial pathogen. Children whose first BSC was submitted after HD-3 accounted for 3767 (46%) of the total 8126 inpatient BSCs and in excess of $21000 annually in patient billing charges. CONCLUSION: In the absence of a known exposure the isolation of a bacterial pathogen from the stool of children with onset of diarrhea beyond HD-3 is a rare event. Under most circumstances BSC should not be part of the initial evaluation of children with symptoms of nosocomial diarrhea.

Gamma 3 gene-disrupted mice selectively deficient in the dominant IgG subclass made to bacterial polysaccharides undergo normal isotype switching after immunization with polysaccharide-protein conjugate vaccines.

Bacterial polysaccharides (PS) are T-independent type 2 Ags that elicit restricted Ab responses of IgM and IgG3 in mice and IgM and predominantly IgG2 in humans. Immunodeficiency in the dominant IgG subclass made to PS is associated with chronic sinus and pulmonary infections with PS-encapsulated bacteria. To elucidate the biologic role of the dominant IgG subclass in the immune response to PS and to make an animal model of human IgG subclass deficiency, we generated mice with a targeted disruption of the exon encoding the CH1 domain of the gamma 3 heavy-chain constant region gene. Homozygotes had no detectable serum IgG3, and their splenocytes did not produce IgG3 after LPS stimulation. IgG3(-/-) mice immunized with PS from Pseudomonas aeruginosa LPS O-side chain or Streptococcus pneumoniae type 19F capsule did not produce any IgG3 anti-PS Abs, in contrast to wild-type mice in which IgG3 was the major IgG subclass. Immunizing both wild-type and IgG3(-/-) mice with 19F PS-protein conjugate elicited IgG1 Abs. We conclude that IgG3(-/-) mice have a selective deficiency in the dominant murine IgG subclass made to T-independent type 2 Ags and may be a useful animal model of IgG subclass deficiency. In addition, we show that the anti-PS Ab class switching to IgG1 that occurs when mice are immunized with a PS-protein conjugate vaccine does not require sequential Ig expression or an intact, upstream gamma 3 heavy-chain gene.

Mitogenic synthetic polynucleotides suppress the antibody response to a bacterial polysaccharide.

Unmethylated bacterial DNA containing a high frequency of the CpG motif, is mitogenic and induces T-cell independent, murine B-cell proliferation. These stimulatory effects are also induced by synthetic oligonucleotides that contain one or more unmethylated CpG dinucleotides (CpG oligo). Such mitogenicity is not seen with highly methylated vertebrate DNA, which has a lower prevalence of the CpG motif than bacterial DNA. Due to their stimulatory effects, CpG oligo have been proposed for use as vaccine adjuvants. In order to determine if a synthetic CpG oligo that was stimulatory for B-cell proliferation could augment the murine antibody response to protective bacterial polysaccharide epitopes (Pseudomonas aeruginosa LPS-O polysaccharide side chain; high-molecular-weight polysaccharide or high-MW PS), BALB/c mice were injected with mitogenic doses of CpG oligo simultaneously with high-MW PS, and antibody titers were measured by ELISA weekly for 4 weeks. Controls received PBS, a nonstimulatory control oligo plus PS, CpG alone, or PS alone. Despite evidence of B-cell mitogenicity and an increase in total IgM in CpG oligo-treated mice, CpG oligo treatment plus PS significantly decreased the high-MW PS antibody response compared to PS alone. The blunting of the anti-PS antibody response could be eliminated by vaccinating the animals with PS prior to CpG oligo. We conclude that despite in vitro and in vivo evidence of B-cell proliferation, this CpG oligo reduces PS-specific antibody responses in an animal model when given simultaneously with a bacterial polysaccharide. Based on results in this model, oligonucleotides containing stimulatory unmethylated CpG dinucleotides may not be useful adjuvants when given simultaneously with bacterial PS vaccines.

Transmission disequilibrium of maternally-inherited CTLA-4 microsatellite alleles in idiopathic recurrent miscarriage.

To elucidate the mechanisms that facilitate tolerance at the maternal-fetal interface, we are investigating the role of genes that are involved in peripheral self-tolerance in couples with idiopathic recurrent miscarriage. CTLA-4 is a negative regulator of T-cell proliferation and has been associated with human autoimmune disease. An AT(n) polymorphism in the 3'-untranslated region (UTR) of the human gene results in AT stretches that vary in length from 16 to 46 bp. We hypothesized that long stretches of AT repeats would result in mRNA instability, and reduced fetal survival in humans. We examined the transmission of AT(n) alleles in 60 couples with a history of > or = 3 unexplained spontaneous abortions to their 51liveborn children and 10 abortuses. The shorter allele was transmitted from heterozygous mothers to 26 of 35 liveborn children (chi2 = 8.3, P = 0.0040) and to three of nine aborted fetuses (chi2 = 1.0, P = 0.317). The shorter allele was transmitted from heterozygous fathers to 15 of 32 liveborn children (chi2 =0.12, P=0.726) and to five of eight aborted fetuses (chi2 = 0.5, P = 0.480). Furthermore, liveborn fetuses who inherited smaller alleles were more likely to represent the first successful pregnancy than liveborn fetuses who inherited larger maternal alleles (Pexact = 0.044) and fetuses of first pregnancies that inherited the smaller allele were significantly more likely to survive to term (Pexact = 0.0086). The preferential transmission of maternally-inherited shorter alleles to liveborn children, but random transmission of paternally-inherited alleles, suggests that CTLA-4 may be imprinted in humans and that this gene may play a role in inducing or maintaining tolerance at the maternal-fetal interface.

Bispecific antibodies overcome the opsonin-receptor mismatch of cystic fibrosis in vitro: restoration of neutrophil-mediated phagocytosis and killing of Pseudomonas aeruginosa.

Inflammation and infection associated with bacterial pathogens, primarily Pseudomonas aeruginosa (Pa), are the primary causes of morbidity and mortality for cystic fibrosis (CF) patients. CF patients may be predisposed to these bacterial infections by a defect in phagocytosis due to "opsonin-receptor mismatch," in which a complement receptor (CR1) and an important opsonin (iC3b) are destroyed by proteolytic enzymes. We show that opsonin-receptor mismatch can be mitigated in vitro using a bispecific Ab (bsAb) to cross-link neutrophils via the beta-chain of leukocyte integrins (CD18) to bacterial epitopes or C3d on opsonized Pa. Two chemically cross-linked bsAb were constructed with mAb specific for C3d (or the O-specific side chain of Fisher Devlin Immunotype 1 Pa) and CD18. Using an in vitro model of elastase-mediated opsonin-receptor mismatch, these bsAb specifically enhanced Pa phagocytosis and killing, with the anti-C3d-containing bsAb restoring the levels of phagocytosis to approximately those for the non-elastase-treated opsonic control. These results encourage the further investigation of bsAb as therapeutic agents for bacterial infection in the lungs of CF patients.

Cross-sectional and longitudinal studies of naturally occurring antibodies to Pseudomonas aeruginosa in cystic fibrosis indicate absence of antibody-mediated protection and decline in opsonic quality after infection.

Most patients with cystic fibrosis (CF) develop chronic endobronchial infection with mucoid Pseudomonas aeruginosa. It has been suggested that opsonic antibodies to the mucoid exopolysaccharide of P. aeruginosa protect older CF patients (> 12 years of age) who have remained free of colonization by this organism. Serum antibodies from chronically infected CF patients had greater total complement-dependent opsonic activity than did those of older noncolonized patients (P < .02), but when bound antibody was equalized, opsonic quality was greater for the latter group (P < .03). In longitudinal studies, antibody titers to mucoid P. aeruginosa rose greatly after initial infection, but opsonic quality declined (P = .002). Twenty CF patients who passed age 12 free of P. aeruginosa colonization developed chronic P. aeruginosa lung infection at ages 14-35 years. Thus, naturally occurring antibodies do not protect CF patients from P. aeruginosa infection, and opsonic quality of serum antibodies deteriorates as infection becomes established.