Receptor-Dependent Immune Responses in Pigs after Oral Immunization with F4 Fimbriae

F4 receptor-positive (F4R⁺) and F4 receptor-negative (F4R⁻) pigs were orally vaccinated with purified F4 fimbriae of enterotoxigenic Escherichia coli (ETEC). Serum immunoglobulin G (IgG) and IgA responses were readily detected in F4R⁺ animals, whereas immune responses were not detected in F4R⁻ animals. Even after a subsequent oral infection with virulent F4⁺ ETEC and a booster immunization with F4, the F4R⁻ animals remained F4 seronegative whereas the unvaccinated F4R⁺ pigs exhibited clear IgA and IgG responses. These results clearly demonstrate that F4Rs are a prerequisite for an immune response following oral immunization. Furthermore, indications that oral F4 vaccination can induce mucosal protection were obtained, since the experimental ETEC infection did not induce a systemic booster response or fecal ETEC excretion in orally vaccinated F4R⁺ pigs, in contrast to the clear immune response and ETEC excretion of unvaccinated F4R⁺ animals. F4-specific IgA antibodies could be found in the feces of the vaccinated F4R⁺ pigs. They are secreted at the intestinal mucosal surface and appear to prevent ETEC infection. The F4R-dependent induction of a mucosal immune response can be used as a model to better understand mucosal immunization and mucosal immune responses and can contribute to the development of oral vaccines in veterinary as well as in human medicine.     

Expression of Mucin-Type Glycoprotein K88 Receptors Strongly Correlates with Piglet Susceptibility to K88+ Enterotoxigenic Escherichia coli, but Adhesion of This Bacterium to Brush Borders Does Not

Three antigenic variants of the K88 fimbrial adhesin exist in nature, K88ab, K88ac, and K88ad. Enterotoxigenic Escherichia coli (ETEC) strains that produce these fimbriae cause life-threatening diarrhea in some but not all young pigs. The susceptibility of pigs to these organisms has been correlated with the adherence of bacteria to isolated enterocyte brush borders. Whether that correlation holds for multiple K88 variants and over a broad genetic base of pigs is unknown and was the impetus for this study. We also desired to examine the correlation of the expression of a porcine intestinal brush border mucin-type glycoprotein (IMTGP) which binds K88ab and K88ac with the susceptibility of piglets to K88⁺ ETEC. Of 31 neonatal gnotobiotic pigs inoculated with K88ab⁺ or K88ac⁺ ETEC, 13 developed severe diarrhea, became dehydrated, and died or became moribund. Another pig became severely lethargic but not dehydrated. In vitro brush border adherence analysis was not possible for 10 of the severely ill pigs due to colonization by challenge strains. However, of the 17 pigs that did not become severely ill, 8 (47%) had brush borders that supported the adherence of K88ab⁺ and K88ac⁺ bacteria in vitro, suggesting a poor correlation between in vitro brush border adherence and piglet susceptibility to K88⁺ ETEC. By contrast, the expression of IMTGP was highly correlated with susceptibility to K88⁺ ETEC. Of the 12 pigs that produced IMTGP, 11 developed severe diarrhea. The other pig that produced IMTGP became lethargic but not severely diarrheic. Only 2 of 18 pigs that did not produce IMTGP became severely diarrheic. Colonizing bacteria were observed in histologic sections of intestines from all pigs that expressed IMTGP except for the one that did not develop severe diarrhea. However, colonizing bacteria were observed in histologic sections from only one pig that did not produce IMTGP. The bacterial concentration in the jejuna and ilea of pigs expressing IMTGP was significantly greater (P < 0.005) than that in pigs not expressing IMTGP. These observations suggest the IMTGP is a biologically relevant receptor for K88ab⁺ and K88ac⁺ E. coli or a correlate for expression for such a receptor.     

Maternal Vaccination with a Fimbrial Tip Adhesin and Passive Protection of Neonatal Mice against Lethal Human Enterotoxigenic Escherichia coli Challenge

Globally, enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood and travelers'' diarrhea, for which an effective vaccine is needed. Prevalent intestinal colonization factors (CFs) such as CFA/I fimbriae and heat-labile enterotoxin (LT) are important virulence factors and protective antigens. We tested the hypothesis that donor strand-complemented CfaE (dscCfaE), a stabilized form of the CFA/I fimbrial tip adhesin, is a protective antigen, using a lethal neonatal mouse ETEC challenge model and passive dam vaccination. For CFA/I-ETEC strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407, which has been extensively studied in volunteers, an inoculum of 2 × 10⁷ bacteria resulted in 50% lethal doses (LD₅₀) in neonatal DBA/2 mice. Vaccination of female DBA/2 mice with CFA/I fimbriae or dscCfaE, each given with a genetically attenuated LT adjuvant (LTK63) by intranasal or orogastric delivery, induced high antigen-specific serum IgG and fecal IgA titers and detectable milk IgA responses. Neonates born to and suckled by dams antenatally vaccinated with each of these four regimens showed 78 to 93% survival after a 20× LD₅₀ challenge with {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407, compared to 100% mortality in pups from dams vaccinated with sham vaccine or LTK63 only. Crossover experiments showed that high pup survival rates after ETEC challenge were associated with suckling but not birthing from vaccinated dams, suggesting that vaccine-specific milk antibodies are protective. In corroboration, preincubation of the ETEC inoculum with antiadhesin and antifimbrial bovine colostral antibodies conferred a dose-dependent increase in pup survival after challenge. These findings indicate that the dscCfaE fimbrial tip adhesin serves as a protective passive vaccine antigen in this small animal model and merits further evaluation.     

Detection of Human Toxoplasma-Specific Immunoglobulins A, M, and G with a Recombinant Toxoplasma gondii Rop2 Protein

The Toxoplasma gondii rhoptry protein Rop2 was expressed in Escherichia coli as a fusion protein containing 44 kDa of the 55-kDa mature Rop2, supplied with six histidyl residues at the N-terminal end (Rop2_196–561). Humoral response during Toxoplasma infection of humans was analyzed by immunoglobulin G (IgG), IgA, and IgM enzyme-linked immunosorbent assay with Rop2_196–561 as the antigen substrate. The analyzed sera were divided according to T. gondii-specific serological tests (IgG, IgA, or IgM indirect immunofluorescence and IgA or IgM immunosorbent agglutination assay) as group A (IgG⁺ IgA⁻ IgM⁻; n = 35), group B (IgG⁺ IgA⁺ IgM⁺; n = 21), group C (IgG⁺ IgA⁺ IgM⁻; n = 5), and group D (IgG⁺ IgA⁻ IgM⁺; n = 16). Twenty-six T. gondii-seronegative sera from individuals with other infections were also included (group E). Anti-Rop2 IgG antibodies were detected in 82.8% of group A sera and in 97.6% of the sera with acute-phase marker immunoglobulins (groups B, C, and D). The percentage of IgA antibody reactivity against Rop2_196–561 was 17.1% in group A, 50% in group D, and 80.8% in groups B and C. The percentage of IgM antibody reactivity was 0% in groups A and C and 62% in groups B and D. Sera from group E failed to show IgA, IgM, or IgG antibody reactivity. Since T. gondii Rop2 elicits a strong humoral response from an early stage of infection, it is suggested that recombinant Rop2_196–561 would be suitable for use in diagnostic systems, in combination with other T. gondii antigens, to detect specific IgG, IgA, and IgM antibodies.     

Peyer's patches are required for intestinal immunoglobulin A responses to Salmonella spp

Previous studies have shown that Peyer's patches (PP) are not required for intestinal immunoglobulin A (IgA) responses to orally administered soluble protein. However, the roles of PP in regulation of mucosal immune responses against bacterial antigen remain to be clarified. In the present study, we generated several gut-associated lymphoreticular tissue-null mice by treatment with anti-interleukin-7 receptor antibody, the fusion protein of lymphotoxin β receptor and IgG Fc, and/or tumor necrosis factor receptor p55 and IgG Fc. These mice were then immunized with recombinant Salmonella expressing the C fragment of the tetanus toxin (rSalmonella-Tox C). Orally immunized PP-null mice as well as isolated lymphoid follicle (ILF)-null, PP/ILF-null, and PP/ILF/mesenteric lymph node-null mice induced identical levels of tetanus toxoid (TT)-specific systemic IgG responses to those of control mice. However, PP-null mice, but not ILF-null mice, failed to induce TT-specific intestinal IgA antibodies. Analysis of TT-specific CD4⁺ T-cell responses showed a reduction of gamma interferon (IFN-γ) synthesis in the intestinal lamina propriae of PP-null mice given oral rSalmonella-Tox C. In contrast, TT-specific IFN-γ responses in the spleen and delayed-type hypersensitivity responses were intact in those immunized mice. Interestingly, Salmonella lipopolysaccharide (LPS)-specific fecal IgA responses were not elicited in PP-null mice, while serum IgG anti-LPS antibodies were identical to those of control mice. These results suggest that while none of the gut-associated lymphoreticular tissues are required for the induction of systemic immune responses, PP are an essential lymphoid tissue for induction and regulation of intestinal IgA immunity against orally administered rSalmonella.     

T- and B-Cell Immune Responses of Patients Who Had Undergone Colectomies to Oral Administration of Salmonella enterica Serovar Typhi Ty21a Vaccine

The capacity of an oral live attenuated Salmonella enterica serovar Typhi Ty21a vaccine to induce immune responses in patients who had undergone colectomies because of ulcerative colitis was evaluated, and these responses were compared with those of healthy volunteers. Purified CD4⁺ and CD8⁺ T cells from peripheral blood were stimulated in vitro by using the heat-killed Ty21a vaccine strain, and the proliferation and gamma interferon (IFN-γ) production were measured before and 7 or 8 days after vaccination. Salmonella-specific immunoglobulin A (IgA) and IgG antibody responses in serum along with IgA antibody responses in ileostomy fluids from the patients who had undergone colectomies were also evaluated. Three doses of vaccine given 2 days apart failed to induce proliferative T-cell responses in all the six patients who had undergone colectomies, and increases in IFN-γ production were found only among the CD8⁺ cells from three of the patients. In contrast, both proliferative responses and increased IFN-γ production were observed among CD4⁺ and CD8⁺ T cells from 3 and 6 of 10 healthy volunteers, respectively. Salmonella-specific IgA and/or IgG antibody responses in serum were observed for five (56%) of nine patients who had undergone colectomies and in 15 (88%) of 17 healthy volunteers. In ileostomy fluids, significant anti-Salmonella IgA antibody titer increases were detected in six (67%) of nine patients who had undergone colectomies. The impaired T- and B-cell immune responses found after vaccination in the circulation of patients who have undergone colectomies may be explained by a diminished colonization of the Ty21a vaccine strain due to the lack of a terminal ileum and colon.     

Humoral and Cellular Immunity in Children with Mycoplasma pneumoniae Infection: a 1-Year Prospective Study

To determine whether children have persistent abnormalities in cellular and humoral immunity development after acute Mycoplasma pneumoniae infection, serum immunoglobulin G (IgG), IgA, IgM, and IgE levels and lymphocyte phenotypes were determined. There were no changes in the levels of IgG, IgM, IgA, or CD4⁺ or CD19⁺ lymphocytes that were measured in M. pneumoniae-positive patients after 3 months or after 12 months, but there were increases in these in M. pneumoniae-negative patients. Serum IgE increased in M. pneumoniae-positive patients. We have shown alterations in immunity development after M. pneumoniae infection.     

Differentiation of F18ab+ from F18ac+ Escherichia coli by Single-Strand Conformational Polymorphism Analysis of the Major Fimbrial Subunit Gene (fedA)

Toxin-producing Escherichia coli expressing F18 fimbriae colonizes the small intestines of weaned pigs and causes diarrhea, edema disease, or both. The F18 family is composed of two antigenic variants, F18ab and F18ac. Because many strains do not express F18 fimbriae in vitro, identification and differentiation of these two variants are difficult. Single-strand conformational polymorphism (SSCP) analysis is a rapid method for identifying genetic mutations and polymorphisms. The F18 major fimbrial subunit genes (fedA) of 138 strains were amplified by PCR, and genetic differences were detected by SSCP analysis. The SSCP analysis of the fedA gene differentiated F18ab⁺ strains from F18ac⁺ strains. Most strains classified as F18ab⁺ by SSCP analysis contained Shiga toxin 2e and enterotoxin genes. Most strains classified as F18ac⁺ by SSCP analysis contained only enterotoxin genes. The SSCP analysis was a useful method for predicting the antigenicity of F18⁺ E. coli and could also be used for analysis of other virulence genes in E. coli and other pathogenic bacteria.Enterotoxigenic Escherichia coli (ETEC) and E. coli organisms that produce Shiga toxin 2e (STEC) colonize the porcine small intestine and cause diarrhea and edema disease, respectively. The fimbrial adhesins of K99, F41, K88, and 987P fimbriae mediate adherence and promote ETEC colonization of the neonatal pig’s small intestine. Of these four fimbriae, only K88 is frequently detected in ETEC isolated from both weaned and neonatal pigs (24). The F18 fimbria mediates colonization of both ETEC and STEC in weaned, but not neonatal, pigs. The F18 fimbrial family is composed of two antigenic variants, F18ab and F18ac, and has been previously referred to as F107, 2134P, Av24, and 8813 (1–4, 11, 14, 15, 19, 20, 25).Differentiation of strains expressing F18ab from those expressing F18ac may be important in development and selection of effective vaccines for ETEC and STEC infections in weaned pigs. Differentiation of strains producing F18ab and F18ac is also important because of a correlation between the type of toxin produced and clinical sequelae in infected swine. F18ab⁺ strains are generally STEC and are associated with edema disease, while F18ac⁺ strains are generally ETEC and are associated with diarrhea (4, 15, 26). Monospecific polyclonal antisera and the monoclonal antibody 6C7/C1, which is specific for F18ac⁺ strains, can differentiate between these two antigenic variants (3, 4, 15, 19). However, serologic differentiation is not always possible because many strains do not express F18 when cultured in vitro under standard culture conditions (1, 8, 25, 26). The gene encoding the major fimbrial subunit of F18 (fedA) in both F18ab⁺ and F18ac⁺ strains has been sequenced, and differences have been found (9). A PCR-restriction fragment length polymorphism (RFLP) test consisting of amplification of the fedA gene followed by digestion with the restriction enzyme NgoMI has been used to differentiate F18ab⁺ from F18ac⁺ strains (9, 15, 19). This PCR-RFLP test avoided the problems of serologic differentiation, which requires in vitro pilus expression, and was based upon the DNA sequences of seven different fedA genes, fedA and fedA.1 to fedA.6 (9). These seven different sequences were determined by sequencing of the fedA genes of only 10 unique strains (9), demonstrating that the fedA gene is highly polymorphic.Single-strand conformational polymorphism (SSCP) analysis can rapidly identify polymorphisms in a gene and is useful when a large number of samples are being analyzed. Single-strand DNA migrates according to size and shape in a nondenaturing gel. The shape is dependent upon folding due to intermolecular interactions which are DNA sequence dependent (5). The major objective of this study was to determine if SSCP analysis of the fedA gene could differentiate F18ab⁺ from F18ac⁺ strains.     

Cryptococcus neoformans-Reactive and Total Immunoglobulin Profiles of Human Immunodeficiency Virus-Infected and Uninfected Ugandans

We determined total and Cryptococcus neoformans glucuronoxylomannan (GXM)-reactive antibody repertoires of human immunodeficiency virus (HIV)-infected and HIV-uninfected Ugandans in a retrospective, case-control study of participants in a randomized controlled trial of pneumococcal vaccination. The study included 192 adults: 48 who subsequently developed cryptococcal meningitis (CM); (HIV⁺ CM⁺); 2 individuals who matched them in CD4⁺ T-cell level, stage of HIV disease, and age but did not develop CM (HIV⁺ CM⁻); and 48 HIV-uninfected individuals. Total serum immunoglobulin concentrations and titers of immunoglobulin M (IgM), IgG, and IgA to GXM, pneumococcal polysaccharides, and antibodies expressing certain V_H3 idiotypes were determined with banked sera obtained before the development of cryptococcosis for HIV⁺ CM⁺ subjects. The results showed that HIV-infected subjects had significantly lower levels of IgM to GXM but higher levels of total immunoglobulin and IgG and IgA to GXM than those of HIV-uninfected subjects. HIV-infected subjects with a history of pneumonia had higher levels, and those with a history of herpes zoster had lower levels of GXM-binding antibodies than subjects with no history of either disease. Minimal to no cross-reactivity was demonstrated between antibodies to GXM and polysaccharides in a pneumococcal vaccine. No significant differences between the antibody repertoires of HIV⁺ CM⁺ and HIV⁺ CM⁻ subjects were identified, but among subjects without a history of pneumonia, there was a trend towards lower V_H3-positive antibody levels among HIV⁺ CM⁺ than among HIV⁺ CM⁻ subjects. Our findings demonstrate an association between previous infectious diseases and differences in the total and GXM-reactive antibody repertoires of HIV-infected subjects and suggest the question of whether certain microbes modulate subsequent antibody responses to GXM deserves further study.     

Immunoglobulin G, A, and M Responses in Serum and Circulating Immune Complexes Elicited by the 16-Kilodalton Antigen of Mycobacterium tuberculosis

The 16-kDa cytosolic antigen of M. tuberculosis was purified to homogeneity by molecular sieving chromatography, and the diagnostic potential of the antigen was evaluated in various categories of patients by enzyme-linked immunosorbent assay (ELISA). The immunoglobulin G (IgG), IgA, and IgM antibody levels to 16-kDa antigen were estimated in the two polar groups, namely, smear- and culture-positive pulmonary tuberculosis (S⁺C⁺) patients and healthy subjects (HS). Sensitivities of 62, 52 and 11% with specificities of 100, 97, and 95% were obtained for the three isotypes, respectively. The total number of positives by a combination of the three isotypes was analyzed in the polar groups, and the sensitivity improved to 83% with a specificity of 93%. Even when a combination of IgG and IgA alone was considered, the sensitivity was 82% with a specificity of 97%. Polyethylene glycol precipitation of the circulating immune complex (CIC) in sera was carried out. The CIC-bound antibodies to 16-kDa antigen were assessed by ELISA in the S⁺C⁺, S⁻C⁺, and S⁻C⁻ categories of patients. Measuring the IgG-IgA-IgM combination positivities of the CIC-bound antibodies gave sensitivities of 97.5, 100, and 45.3%, respectively. The specificity of the assay with these combinations was maintained at 95.4%.     

A Live Recombinant Avirulent Oral Salmonella Vaccine Expressing Pneumococcal Surface Protein A Induces Protective Responses against Streptococcus pneumoniae

A live oral recombinant Salmonella vaccine strain expressing pneumococcal surface protein A (PspA) was developed. The strain was attenuated with Δcya Δcrp mutations. Stable expression of PspA was achieved by the use of the balanced-lethal vector-host system, which employs an asd deletion in the host chromosome to impose an obligate requirement for diaminopimelic acid. The chromosomal Δasd mutation was complemented by a plasmid vector possessing the asd⁺ gene. A portion of the pspA gene from Streptococcus pneumoniae Rx1 was cloned onto a multicopy Asd⁺ vector. After oral immunization, the recombinant Salmonella-PspA vaccine strain colonized the Peyer’s patches, spleens, and livers of BALB/cByJ and CBA/N mice and stimulated humoral and mucosal antibody responses. Oral immunization of outbred New Zealand White rabbits with the recombinant Salmonella strain induced significant anti-PspA immunoglobulin G titers in serum and vaginal secretions. Polyclonal sera from orally immunized mice detected PspA on the S. pneumoniae cell surface as revealed by immunofluorescence. Oral immunization of BALB/cJ mice with the PspA-producing Salmonella strain elicited antibody to PspA and resistance to challenge by the mouse-virulent human clinical isolate S. pneumoniae WU2. Immune sera from orally immunized mice conferred passive protection against otherwise lethal intraperitoneal or intravascular challenge with strain WU2.     

Partially assembled K99 fimbriae are required for protection

Antibodies to K99 fimbriae afford protection to F5+ bovine enterotoxigenic Escherichia coli (ETEC). Previous studies show that murine dams immunized with Salmonella vaccine vectors stably expressing K99 fimbriae confer protection to ETEC-challenged neonatal pups. To begin to address adaptation of the K99 scaffold to display heterologous B- and T-cell epitopes, studies were conducted to determine how much of the assembled K99 fimbria is required to maintain protective immunity. Sequential deletions in the K99 gene clusters were made, resulting in diminished localization of the K99 fimbrial subunit in the outer membrane. As placement of the K99 fimbrial subunit became progressively contained within the vaccine vector, diminished immunoglobulin A (IgA) and IgG1 antibody titers, as well as diminished Th2-type cytokine responses, were observed in orally immunized mice. Deletion of fanGH, which greatly reduced the export of the fimbrial subunit to the outer membrane, showed only partial reduction in protective immunity. By contrast, deletion of fanDEFGH, which also reduced the export of the fimbrial subunit to the outer membrane but retained more subunit in the cytoplasm, resulted in protective immunity being dramatically reduced. Thus, these studies showed that retention of K99 fimbrial subunit as native fimbriae or with the deletion of fanGH is sufficient to confer protection.     

Prevalence of Pilus Antigens, Enterotoxin Types, and Enteropathogenicity Among K88-Negative Enterotoxigenic Escherichia coli from Neonatal Pigs

Enterotoxigenic Escherichia coli (ETEC) that were isolated from neonatal pigs and that did not react in preliminary tests for pilus antigen K88 were subjected to additional tests for K88 and for pilus antigens K99 and 987P. Four such isolates produced K88, 9 isolates produced K99, 55 isolates produced 987P, and the remaining 43 isolates produced none of the three pilus antigens (3P⁻). Immunofluorescence tests of ileal sections from pigs were more sensitive for 987P detection than was serum agglutination of bacteria grown from the ileum. Most ETEC that produced K88, K99, or 987P were enteropathogenic (adhered to ileal villi, colonized intensively, and caused profuse diarrhea) when given to neonatal pigs. In contrast, only 3 of the 43 ETEC that produced none of the pilus antigens were enteropathogenic. The isolates were also tested for the type of enterotoxin produced. The K88⁺ isolates all produced heat-labile enterotoxin (LT) detectable in cultured adrenal cells (i.e., were LT⁺). None of the 987P⁺, K99⁺, or enterpathogenic 3P⁻ isolates produced LT. However (except for a single K99⁺ isolate), they all produced heat-stable enterotoxin detectable in infant mice (STa⁺). Sixteen isolates produced neither LT nor STa but did produce enterotoxin detectable in ligated intestinal loops of pigs (STb). Most of these LT⁻ STa⁻ STb⁺ isolates were also K88⁻, K99⁻, and 987P⁻ and non-enteropathogenic. One of them was K99⁺ and enteropathogenic. Our conclusions are as follows. (i) Most enteropathogenic ETEC from neonatal pigs produce either K88, 987P, or K99; however, there are some that produce none of the three antigens. (ii) Immunofluorescence tests for pilus antigens produced in vivo are recommended for the diagnosis of ETEC infections. (iii) Reports of LT⁻ STa⁻ STb⁺ swine ETEC are confirmed; furthermore, such isolates can be enteropathogenic.     

Magnitude of Serum and Intestinal Antibody Responses Induced by Sequential Replicating and Nonreplicating Rotavirus Vaccines in Gnotobiotic Pigs and Correlation with Protection

A sequential mucosal prime-boost vaccine regimen of oral attenuated (Att) human rotavirus (HRV) priming followed by intranasal (i.n.) boosting with rotavirus protein VP2 and VP6 rotavirus-like particles (2/6-VLPs) has previously been shown to be effective for induction of intestinal antibody-secreting cell (ASC) responses and protection in gnotobiotic pigs. Because serum or fecal antibody titers, but not intestinal ASC responses, can be used as potential markers of protective immunity in clinical vaccine trials, we determined the serum and intestinal antibody responses to this prime-boost rotavirus vaccine regimen and the correlations with protection. Gnotobiotic pigs were vaccinated with one of the two sequential vaccines: AttHRV orally preceding 2/6-VLP (VLP2x) vaccination (AttHRV/VLP2x) or following VLP2x vaccination (VLP2x/AttHRV) given i.n. with a mutant Escherichia coli heat-labile toxin (mLT) as adjuvant. These vaccines were also compared with three i.n. doses of VLP+mLT (VLP3x) and one and three oral doses of AttHRV (AttHRV1x and AttHRV3x, respectively). Before challenge all pigs in the AttHRV/VLP2x group seroconverted to positivity for serum immunoglobulin A (IgA) antibodies. The pigs in this group also had significantly higher (P < 0.05) intestinal IgA antibody titers pre- and postchallenge and IgG antibody titers postchallenge compared to those in the other groups. Statistical analyses of the correlations between serum IgM, IgA, IgG, and virus-neutralizing antibody titers and protection demonstrated that each of these was an indicator of protective immunity induced by the AttHRV3x and the AttHRV/VLP2x regimens. However, only IgA and not IgM or IgG antibody titers in serum were highly correlated (R² = 0.89; P < 0.001) with the corresponding isotype antibody (IgA) titers in the intestines among all the vaccinated groups, indicating that the IgA antibody titer is probably the most reliable indicator of protection.     

Children with the Le(a+b?) Blood Group Have Increased Susceptibility to Diarrhea Caused by Enterotoxigenic Escherichia coli Expressing Colonization Factor I Group Fimbriae

Recent studies have shown that children with blood group A have increased susceptibility to enterotoxigenic Escherichia coli (ETEC) diarrhea and that Lewis blood group “a” antigen (Le^a) may be a candidate receptor for ETEC colonization factor (CF) antigen I (CFA/I) fimbriae. Based on these findings, we have attempted to determine if children with the Le(a+b−) phenotype may be more susceptible to diarrhea caused by ETEC, in particular ETEC expressing CFA/I and related fimbriae of the CFA/I group, than Le(a−b+) children. To test this hypothesis, we have determined the Lewis antigen expression in 179 Bangladeshi children from a prospective birth cohort study in urban Dhaka in which ETEC expressing major CFs such as CFA/I, CS3, CS5, and CS6 was the most commonly isolated diarrhea pathogen during the first 2 years of life. The Lewis blood group phenotypes were determined by a dot blot immunoassay using saliva samples and by a tube agglutination test using fresh red blood cells. The results indicate that Le(a+b−) children more often had symptomatic than asymptomatic ETEC infections (P < 0.001), whereas symptomatic and asymptomatic ETEC infections were equally frequent in Le(a−b+) children. We also show that children with the Le(a+b−) blood type had significantly higher incidences of diarrhea caused by ETEC expressing fimbriae of the CFA/I group than Le(a−b+) children (P < 0.001). In contrast, we did not find any association between the Lewis blood group phenotype and diarrhea caused by ETEC expressing CS6 or rotavirus.Expression of Lewis or ABO histo-blood group types has been shown to be associated with different risks of enteric infections (4, 5, 12, 15, 24, 27), presumably through differential expression of cell surface glycoconjugates that are used as receptors for pathogens of the intestinal mucosa. The Lewis blood group antigens on the intestinal mucosa are synthesized through a group of glycosyltransferases, which insert fucose residues in type 1 and type 2 oligosaccharide precursors (21, 29, 30). The synthesis of Lewis antigens is dependent on the FUT2 and FUT3 genes. If both genes are functional, the phenotype will be Le(a−b+), i.e., the secretor type, whereas individuals in whom the FUT2 gene is not expressed will have the Le(a+b−) phenotype, i.e., the nonsecretor type. Failure to express both FUT2 and FUT3 will result in Le(a−b−) (9).A predisposition for obtaining dehydrating cholera has been seen in blood group O individuals (8, 12, 14, 19, 28). In contrast, our recent study showed that enterotoxigenic Escherichia coli (ETEC) diarrheal episodes were more common in children with blood group AB or A than in individuals with blood group O (24). We have also shown that colonization factor (CF) antigen I (CFA/I) expressed by ETEC binds to glycosphingolipids that are associated with blood group antigens, e.g., Le^a, that may be expressed on epithelial cells in the small intestine in humans (16). The glycosphingolipid binding capacity of CFA/I fimbriae resides in the major CfaB subunit protein (3, 8, 16). CFA/I was the first identified human-specific CF of ETEC bacteria (11). Subsequently, seven other genetically related fimbriae, CS1, CS2, CS4, CS14, CS17, CS19, and putative CF O71, denoted as the CFA/I group (1), have been shown to be related to CFA/I both in the structural subunits (26) and tip-localized minor adhesive subunits (1). A glycosphingolipid binding pattern similar to that of CFA/I has been demonstrated for CS1 and CS4 that might be due to related N-terminal sequences (3, 8, 16). In addition, in another study (25) we have also shown that the conserved regions of the CF subunit proteins (shared by the CFA/I group fimbriae) are likely to be responsible for the receptor binding, since monoclonal antibodies against this region prevented enterocyte binding and protected against challenge with ETEC expressing CFA/I and CS4.In a recent longitudinal birth cohort (BC) study in Dhaka, we showed that ETEC was a major pathogen in children up to 2 years old and that a high proportion of symptomatic infections were caused by ETEC expressing the CFA/I group fimbriae (24). In this study, we present additional data to determine whether children with specific Lewis blood group antigen phenotypes, e.g., Le(a+b−) or Le(a−b+), have different susceptibilities to diarrhea caused by ETEC, in particular ETEC expressing the CFA/I group fimbriae, as well as susceptibilities to diarrhea caused by rotavirus.     

Analysis of Human Serum Immunoglobulin G against O-Acetyl-Positive and O-Acetyl-Negative Serogroup W135 Meningococcal Capsular Polysaccharide

The capsular polysaccharide of Neisseria meningitidis serogroup W135 is expressed in both O-acetyl-positive (OA⁺) and O-acetyl-negative (OA⁻) forms. This study investigates the impact of OA status (OA⁺ versus OA⁻) on serological measurements of anti-W135 immunoglobulin G (IgG) antibodies in immunized adults. W135-specific serum antibody assignments were made for 28 postimmunization sera from adults by enzyme-linked immunosorbent assay using the meningococcal standard reference serum CDC1992. The established IgG concentration in micrograms per milliliter ([IgG]μg/ml) for CDC1992 against OA⁺ antigen (16.2 μg/ml) was used as a reference to assign a concentration of 10.13 μg/ml IgG against OA⁻ antigen by cross-standardization. Overall, the IgG assignments for these sera were higher against OA⁺ antigen (geometric mean concentration [GMC] = 7.16 μg/ml) than against OA⁻ antigen (GMC = 2.84 μg/ml). However, seven sera showed higher specific [IgG]μg/ml values against the OA⁺ antigen than against the OA⁻ antigen. These sera were also distinguished by the inability of fluid-phase OA⁻ antigen to compete for antibody binding to OA⁺ solid-phase antigen. Although there was no overall difference in functional activity measured by complement-mediated serum bactericidal assay (SBA) against OA⁺ and OA⁻ target bacteria (geometric mean titers of 9,642 and 9,045, respectively), three serum specimens showed a large difference in SBA antibody titers against OA⁺ versus OA⁻ W135 target bacteria, which may reflect different epitope specificities for these sera. Our data indicate that, for some sera, the agreement in anti-OA⁺ versus anti-OA⁻ W135 IgG assignments is serum specific and does not reflect the functional (killing) activity in vitro.     

Induction of cross-serovar protection against genital chlamydial infection by a targeted multisubunit vaccination approach

An important consideration for antichlamydial vaccine development is the induction of cross-serovar protection, since multiple serovars (D to L) of Chlamydia trachomatis cause genital infections. We have shown previously that vaccination with C. trachomatis-derived recombinant chlamydial protease-like activity factor (rCPAF) induced significant earlier resolution of Chlamydia muridarum infection and reduced oviduct pathology. However, the vaccinated mice continued to shed chlamydiae for up to 2 weeks after challenge. In this study, C. trachomatis serovar D recombinant proteins, such as recombinant major outer membrane protein (rMOMP), recombinant inclusion membrane protein A (rIncA), and rCPAF were administered intranasally, individually or in combinations, with murine interleukin-12 (IL-12) as an adjuvant, and cross-species immunity against intravaginal C. muridarum infection was examined. Immunization with rCPAF plus IL-12 (rCPAF+IL-12), compared to immunization with rIncA+IL-12 or rMOMP+IL-12, induced the greatest antigen-specific gamma interferon production from purified CD4⁺ T cells and concurrently enhanced serum antibody production. All (100%) the animals vaccinated with rCPAF+IL-12 alone or in any combination completely resolved the infection by day 18 after challenge compared to animals vaccinated with rIncA+IL-12 (50%), rMOMP+IL-12 (33%), or phosphate-buffered saline (mock vaccinated; 0%). Moreover, oviduct pathology in mice vaccinated by any regimen that included rCPAF, but not rMOMP+IL-12 or rIncA+IL-12 alone, was markedly reduced compared to mock-immunized animals. The addition of rMOMP and/or rIncA did not significantly enhance the rCPAF+IL-12-induced effect on bacterial clearance or oviduct pathology. These results suggest a greater conservation of protective linear antigenic epitopes within CPAF than MOMP or IncA across the examined serovars and the need to identify other highly conserved antigens for use with rCPAF in a multisubunit recombinant vaccine.     

Humoral Immunity against Capsule Polysaccharide Protects the Host from magA+ Klebsiella pneumoniae-Induced Lethal Disease by Evading Toll-Like Receptor 4 Signaling

Klebsiella pneumoniae magA (for mucoviscosity-associated gene A) is linked to the pathogenesis of primary pyogenic liver abscess, but the underlying mechanism by which magA increases pathogenicity is not well elucidated. In this study, we investigated the role of the capsular polysaccharides (CPS) in the pathogenesis of magA⁺ K. pneumoniae by comparing host immunity to magA⁺ K. pneumoniae and a ΔmagA mutant. We found that Toll-like receptor 4 recognition by magA⁺ K. pneumoniae was hampered by the mucoviscosity of the magA⁺ K. pneumoniae CPS. Interestingly, monoclonal antibodies (MAbs) against magA⁺ K. pneumoniae CPS recognized all of the K1 strains tested but not the ΔmagA and non-K1 strains. Moreover, the anti-CPS MAbs protected mice from magA⁺ K. pneumoniae-induced liver abscess formation and lethality. This indicates that the K1 epitope is a promising target for vaccine development, and anti-CPS MAbs has great potential to protect host from K1 strain-induced mortality and morbidity in diabetic and other immunocompromised patients in the future.     

Fimbria-Mediated Enhanced Attachment of Nontypeable Haemophilus influenzae to Respiratory Syncytial Virus-Infected Respiratory Epithelial Cells

Respiratory syncytial virus (RSV) infection is known to predispose children to otitis media and sinusitis due to bacteria such as nontypeable Haemophilus influenzae (NTHI). In this study, we investigated the role of NTHI surface outer membrane protein P5-homologous fimbriae (P5-fimbriae) in attachment to RSV-exposed A549 epithelial cells. Analysis by fluorescence flow cytometry showed that a live P5-fimbriated NTHI strain (NTHI^F+) attached to a higher proportion of RSV-exposed A549 cells than to control cells (mean, 68% for RSV versus 29% for control; P = 0.008), while attachment of the P5-fimbriae-deficient isogenic mutant strain (NTHI^F−) was significantly lower than in control cells and rose only slightly following RSV exposure (mean, 17% for RSV versus 10% for control, P = 0.229). Attachment of NTHI^F+ did not correlate with the amount of RSV antigen expressed by A549 cells. Furthermore, paraformaldehyde-fixed NTHI^F+ also demonstrated an enhanced binding to RSV-exposed cells. Observations by transmission electronic microscopy showed that the mean number of bacteria attached per 100 RSV-exposed A549 cells was higher for NTHI^F+ than NTHI^F− (99 versus 18; P < 0.001). No intracellular bacteria were identified. UV-irradiated conditioned supernatants collected from RSV-infected A549 cultures (UV-cRSV) also enhanced the attachment of NTHI^F+ to A549, suggesting the presence of a preformed soluble mediator(s) in UV-cRSV that enhances the expression of receptors for P5-fimbriae on A549 cells. In summary, RSV infection significantly enhances NTHI attachment to respiratory epithelial cells. P5-fimbria is the critical appendage of NTHI that participates in this attachment. In clinical settings, blocking of the P5-fimbria-mediated attachment of NTHI^F+ by passive or active immunity may reduce the morbidity due to NTHI during RSV infection.     

A sopB deletion mutation enhances the immunogenicity and protective efficacy of a heterologous antigen delivered by live attenuated Salmonella enterica vaccines

SopB is a virulence factor of Salmonella encoded by SPI-5. Salmonella sopB deletion mutants are impaired in their ability to cause local inflammatory responses and fluid secretion into the intestinal lumen and also can enhance the immunogenicity of a vectored antigen. In this study, we evaluated the effects on immunogenicity and the efficacy of a sopB deletion mutation on two Salmonella enterica serovar Typhimurium vaccine strains with different attenuating mutations expressing a highly antigenic α-helical region of the Streptococcus pneumoniae surface protein PspA from an Asd⁺-balanced lethal plasmid. After oral administration to mice, the two pairs of strains induced high levels of serum antibodies specific for PspA as well as to Salmonella antigens. The levels of antigen-specific serum immunoglobulin G (IgG) and mucosal IgA were higher in mice immunized with sopB mutants. Enzyme-linked immunospot assay results indicated that the spleen cells from mice immunized with a sopB mutant showed higher interleukin-4 and gamma interferon secretion levels than did the mice immunized with the isogenic sopB⁺ strain. The sopB mutants also induced higher numbers of CD4⁺ CD44^hi CD62L^hi and CD8⁺ CD44^hi CD62L^hi central memory T cells. Eight weeks after primary oral immunization, mice were challenged with 100 50% lethal doses of virulent S. pneumoniae WU2. Immunization with either of the sopB mutant strains led to increased levels of protection compared to that with the isogenic sopB⁺ parent. Together, these results demonstrate that the deletion of sopB leads to an overall enhancement of the immunogenicity and efficacy of recombinant attenuated Salmonella vaccine strains.