Methicillin-resistant Staphylococcus aureus alters cell wall glycosylation to evade immunity.

Methicillin-resistant Staphylococcus aureus (MRSA) is a frequent cause of difficult-to-treat, often fatal infections in humans1,2. Most humans have antibodies against S. aureus, but these are highly variable and often not protective in immunocompromised patients3. Previous vaccine development programs have not been successful4. A large percentage of human antibodies against S. aureus target wall teichoic acid (WTA), a ribitol-phosphate (RboP) surface polymer modified with N-acetylglucosamine (GlcNAc)5,6. It is currently unknown whether the immune evasion capacities of MRSA are due to variation of dominant surface epitopes such as those associated with WTA. Here we show that a considerable proportion of the prominent healthcare-associated and livestock-associated MRSA clones CC5 and CC398, respectively, contain prophages that encode an alternative WTA glycosyltransferase. This enzyme, TarP, transfers GlcNAc to a different hydroxyl group of the WTA RboP than the standard enzyme TarS7, with important consequences for immune recognition. TarP-glycosylated WTA elicits 7.5-40-fold lower levels of immunoglobulin G in mice than TarS-modified WTA. Consistent with this, human sera contained only low levels of antibodies against TarP-modified WTA. Notably, mice immunized with TarS-modified WTA were not protected against infection with tarP-expressing MRSA, indicating that TarP is crucial for the capacity of S. aureus to evade host defences. High-resolution structural analyses of TarP bound to WTA components and uridine diphosphate GlcNAc (UDP-GlcNAc) explain the mechanism of altered RboP glycosylation and form a template for targeted inhibition of TarP. Our study reveals an immune evasion strategy of S. aureus based on averting the immunogenicity of its dominant glycoantigen WTA. These results will help with the identification of invariant S. aureus vaccine antigens and may enable the development of TarP inhibitors as a new strategy for rendering MRSA susceptible to human host defences.

Immunoassays for riboflavin and flavin mononucleotide using antibodies specific to d-ribitol and d-ribitol-5-phosphate.

Riboflavin (vitamin B2), a water-soluble vitamin, plays a key role in maintaining human health. Though, numerous methods have been reported for the determination of total riboflavin (TRF) content in foods and biological samples, very few methods are reported for quantifying riboflavin and its coenzymes [flavin mononucleotide (FMN); flavin adenine dinucleotide (FAD)] individually. Recently, we have demonstrated that antibodies specific to d-ribitol and d-ribitol-5-phosphate also recognize riboflavin and FMN, respectively, and not vice-versa. In this study, we have evaluated these two antibodies for the analysis of riboflavin and FMN by indirect competitive ELISA (icELISA) in selected foods and pharmaceuticals. Under the optimal assay conditions, 50% inhibition concentration (IC50) and limit of detection (LOD, IC10) were 3.41ng/mL and 0.02ng/mL for riboflavin, and 7.84ng/mL and 0.24ng/mL for FMN, respectively, with detectable concentration range between 0.1 and 100ng of analytes and <0.1% cross-reactivity with other water-soluble vitamins. The amounts of TRF in food samples, as analyzed by icELISA using ribitol antibody, were 90-95% of the reported values in the literature or label values. Quantification of individual flavins (riboflavin and FMN) from the same food samples showed variation in their values compared to TRF, and were in good agreement with values obtained from HPLC and AOAC methods. Further, spiking and recovery analysis of food samples and pharmaceuticals showed no significant matrix effects. The immunoassays were validated in terms of accuracy and precision using inter- and intra-assays. The immunoassays developed in this study are sensitive and appears feasible for screening a large number of samples in the quantification of riboflavin and FMN in various biological samples, pharmaceuticals and natural/processed foods.

Recognition of flavin mononucleotide, Haemophilus influenzae type b and its capsular polysaccharide vaccines by antibodies specific to D-ribitol-5-phosphate.

D-Ribitol-5-phosphate (Rbt-5-P) is an important metabolite in the pentose phosphate pathway and an integral part of bacterial cell wall polysaccharides, specifically as polyribosyl ribitol phosphate (PRP) in Haemophilus influenzae type b (Hib). The major objective of this study was to investigate whether an antibody specific to Rbt-5-P can recognize the PRP of Hib. D-Ribose-5-phosphate was reacted with proteins in the presence of sodium cyanoborohydride to obtain Rbt-5-P epitopes; 120 h reaction resulted in conjugation of ~30 and ~17 moles of Rbt-5-P/mole of BSA and OVA, respectively, based on decrease in amino groups, MALDI-TOF analyses, an increase in apparent molecular weight (SDS-PAGE) and glycoprotein staining. Immunization of rabbits with Rbt-5-P-BSA conjugate generated antibodies to Rbt-5-P as demonstrated by dot immunoblot and non-competitive ELISA. Homogeneous Rbt-5-P-specific antibody was purified from Rbt-5-P-BSA antiserum subjected to caprylic acid precipitation followed by hapten-affinity chromatography; its affinity constant is 7.1 × 10(8) M(-1). Rbt-5-P antibody showed 100 % specificity to Rbt-5-P, ~230 %, 10 % and 3.4 % cross-reactivity to FMN, riboflavin and FAD, respectively; the antibody showed ~4 % cross-reactivity to D-ribitol and <3 % to other sugars/sugar alcohols. Rbt-5-P-specific antibody recognized Hib conjugate vaccines containing PRP which was inhibited specifically by Rbt-5-P, and also detected Hib cell-surface capsular polysaccharides by immunofluorescence. In conclusion, Rbt-5-P-protein conjugate used as an immunogen elicited antibodies binding to an epitope also present in PRP and Hib bacteria. Rbt-5-P-specific antibody has potential applications in the detection and quantification of free/bound Rbt-5-P and FMN as well as immunological recognition of Hib bacteria and its capsular polysaccharide.

Immunogenicity and safety of four different doses of Haemophilus influenzae type b-tetanus toxoid conjugated vaccine, combined with diphtheria-tetanus-pertussis vaccine (DTP-Hib), in Indonesian infants.

Widespread use of Haemophilus influenzae type b (Hib) conjugated vaccine in industrialized countries has resulted in a dramatic decline in the incidence of invasive Hib diseases, but the vaccine's cost has prevented its inclusion in basic immunization programs in developing countries. To overcome this problem, combination with diphtheria-tetanus-pertussis (DTP) vaccine or reduction in the dose of Hib vaccine has been proposed. To evaluate the immunogenicity and adverse reactions from lower doses of Hib-polyribosylphosphate (PRP) conjugated with tetanus toxoid (PRP-T), a double-blind study was conducted in Jakarta, Indonesia, and its suburbs. A total of 1048 infants 6 weeks to 6 months of age received three doses of DTP vaccine combined with the usual 10 microg dose or with a reduced dose of 5, 2.5 or 1.25 microg of PRP-T at two-monthly intervals. Antibodies were measured prior to the first dose and 4-6 weeks following the third dose. Adverse reactions were similar among all four groups. The only significant difference was a higher rate of irritability (p<0.02) and of temperature elevation >38 degrees C (p<0.009) after doses 1 and 2 in the lowest dose group (1.25 microg PRP-T) compared to the other groups. All participants tested had a 4-fold increase in antibodies against all DTP antigens. In addition, after a fourth booster dose of Hib, 99.6% of infants produced >or=0.15 microg/ml of antibody to Hib-PRP, and 96.4% showed levels >or=1.0 microg/ml after primary immunization, level that correlate with short- and long-term immunity, respectively. Antibody titers to the PRP antigen showed no significant differences among dosage groups with the exception of the 5.0 microg group, which had a significantly higher GMC than the 1.25 microg group (p<0.012). This study demonstrates that primary vaccination with half, one-fourth, or one-eighth of the usual dose of PRP-T, combined with DTP vaccine, produces protective immune responses, and has side effects that are comparable to DTP vaccination alone. In these lower dosages, PRP-T conjugate vaccine can lower vaccine costs to a level that is affordable for infant immunization programs in developing countries.

Antigenicity and Immunogenicity of a Synthetic Oligosaccharide-Protein Conjugate Vaccine against Haemophilus influenzae Type b

Polysaccharide-protein conjugates as vaccines have proven to be very effective in preventing Haemophilus influenzae type b infections in industrialized countries. However, cost-effective technologies need to be developed for increasing the availability of anti-H. influenzae type b vaccines in countries from the developing world. Consequently, vaccine production with partially synthetic antigens is a desirable goal for many reasons. They may be rigidly controlled for purity and effectiveness while at the same time being cheap enough that they may be made universally available. We describe here the antigenicity and immunogenicity of several H. influenzae type b synthetic oligosaccharide-protein conjugates in laboratory animals. The serum of H. influenzae type b-immunized animals recognized our synthetic H. influenzae type b antigens to the same extent as the native bacterial capsular polysaccharide. Compared to the anti-H. influenzae type b vaccine employed, these synthetic versions induced similar antibody response patterns in terms of titer, specificity, and functional capacity. The further development of synthetic vaccines will meet urgent needs in the less prosperous parts of the world and remains our major goal(AU)

Conjugados proteína-polisacárido como vacunas han demostrado ser muy eficaz en la prevención de Haemophilus influenzae tipo b infecciones en los países industrializados. Sin embargo, tecnologías rentables necesitan ser desarrolladas para incrementar la disponibilidad de anti-H. influenzae tipo b, las vacunas en los países del mundo en desarrollo. En consecuencia, la producción de vacunas con antígenos sintéticos en parte es un objetivo deseable por muchas razones. Pueden ser rígidamente controlada por la pureza y la eficacia al mismo tiempo ser lo suficientemente baratas para que sean universalmente disponibles. Se describe aquí la antigenicidad e inmunogenicidad de varias H. influenzae tipo b-oligosacáridos sintéticos conjugados proteína en animales de laboratorio. El suero de H. influenzae tipo b-reconocido nuestros animales inmunizados sintéticas H. influenzae tipo b antígenos en la misma medida que los nativos bacteriana polisacárido capsular. En comparación con el anti-H. influenzae tipo b vacuna empleada, estas versiones sintéticas similares respuesta de anticuerpos inducida por los patrones en términos de títulos, la especificidad y la capacidad funcional. El ulterior desarrollo de vacunas sintéticas satisfacer las necesidades urgentes en las regiones menos prósperas del mundo y sigue siendo nuestro principal objetivo

Antigenicity and immunogenicity of a synthetic oligosaccharide-protein conjugate vaccine against Haemophilus influenzae type b.

Polysaccharide-protein conjugates as vaccines have proven to be very effective in preventing Haemophilus influenzae type b infections in industrialized countries. However, cost-effective technologies need to be developed for increasing the availability of anti-H. influenzae type b vaccines in countries from the developing world. Consequently, vaccine production with partially synthetic antigens is a desirable goal for many reasons. They may be rigidly controlled for purity and effectiveness while at the same time being cheap enough that they may be made universally available. We describe here the antigenicity and immunogenicity of several H. influenzae type b synthetic oligosaccharide-protein conjugates in laboratory animals. The serum of H. influenzae type b-immunized animals recognized our synthetic H. influenzae type b antigens to the same extent as the native bacterial capsular polysaccharide. Compared to the anti-H. influenzae type b vaccine employed, these synthetic versions induced similar antibody response patterns in terms of titer, specificity, and functional capacity. The further development of synthetic vaccines will meet urgent needs in the less prosperous parts of the world and remains our major goal.

Defective anticarbohydrate antibody responses to naturally occurring bacteria following bone marrow transplantation.

The long-term recipients of allogeneic bone marrow transplantation (BMT) are at an increased risk of death due to bacterial infections. We evaluated the anticarbohydrate antibody responses of BMT recipients to a naturally occurring bacterial carbohydrate, polyribose phosphate (PRP). The recipients of autologous BMT achieved protective anti-PRP levels (>100 ng/mL) by 3 years after transplantation, with a pattern consistent with a recapitulation of the ontogeny of anticarbohydrate antibody responses. None of the six recipients of unrelated BMT who were off immunosuppressive therapy had protective anti-PRP levels, though their response to a protein antigen (tetanus toxoid) was normal. Of 48 recipients of histocompatible BMT, 22 (46%) had protective anti-PRP antibody levels, whereas 13 (27%) recipients who were >3 years post-BMT did not have protective levels. Therefore, all unrelated recipients and a significant proportion of histocompatible recipients without clinical graft-vs.-host disease had persistent and prolonged defects in their capacity to produce antibodies to naturally occurring bacterial carbohydrate antigens. These results suggest that allogeneic BMT recipients should be longitudinally evaluated for their anticarbohydrate antibody responses and that patients with defective antibody responses should receive prophylactic antibiotics or replacement immunoglobulin therapy or both to reduce their risk of late bacterial infections.

The immunogenicity of Haemophilus influenzae type b conjugate vaccines in children born to human immunodeficiency virus-infected women. Women and Infants Transmission Study Group.

BACKGROUND: Immunocompromise caused by HIV-1 infection increases the importance of receipt of routine childhood vaccines to prevent infections such as invasive Haemophilus influenzae type B (Hib) disease. The objectives of the study were to evaluate the immunogenicity of Hib conjugate vaccines among HIV-infected children according to clinical and immunologic disease progression as well as viral load. METHODS: The concentration of antibody to polyribosylribitol phosphate (PRP) was measured at approximately 9 and 24 months of age in plasma specimens from children of HIV-infected women enrolled in the Women and Infants Transmission Study. RESULTS: Among 227 children (35 HIV-infected, 192 uninfected) at the 9-month study visit who were known to have received age-appropriate immunization with CRM197 mutant Corynebacterium diphtheriae protein-conjugated Hib vaccine, geometric mean antibody concentrations were lower among HIV-infected children (1.64 microg/ml) than among uninfected children (2.70 microg/ml), although the difference was not statistically significant. Anti-PRP antibody concentrations did not vary significantly among these HIV-infected children with predominantly mild-moderate disease progression according to clinical category, immunologic stage or viral load (P > or = 0.48). The proportion of children with antibody concentrations > or = 1.0 microg/ml did not vary significantly according to HIV infection status (73% uninfected, 74% infected) or, if infected, clinical or immunologic disease progression or viral load. Similar results were obtained among 127 children (17 HIV-infected, 110 uninfected) eligible for analysis at the 24-month study visit. Changes in antibody concentrations over time (between 9 and 24 months of age) did not differ significantly among 10 HIV-infected as compared with 72 uninfected children (P=0.81). CONCLUSIONS: These results suggest that HIV-infected children with predominantly mild-moderate disease progression respond reasonably well in terms of a quantitative antibody response to Hib conjugate vaccines during the first 2 years of life. Research to further characterize the immune response to Hib conjugate vaccines and to further delineate the "durability" of anti-PRP antibody concentrations beyond 2 years of life should be pursued.

Safety and immunogenicity of a bivalent Haemophilus influenzae type b/hepatitis B vaccine in healthy infants. Hib-HB Vaccine Study Group.

OBJECTIVE: To assess the safety, tolerability and immunogenicity of COMVAX, a liquid, bivalent Haemophilus influenzae type b-hepatitis B vaccine, containing the polyribosylribitol phosphate (PRP)-Neisseria meningitidis outer membrane protein complex conjugate used in the Hib vaccine, PedvaxHIB, and the yeast-derived hepatitis B surface antigen (HBsAg) used in the HB vaccine, RECOMBIVAX HB. DESIGN: Eight hundred eighty-two healthy infants, approximately 2 months of age, were enrolled in an open, multicenter (n = 11) clinical trial and randomized to receive either COMVAX (7.5 micrograms of PRP/5 micrograms of HBsAg in 0.5 ml) or concurrent injections of the liquid formulation of PedvaxHIB (P) (7.5 micrograms of PRP in 0.5 ml) and RECOMBIVAX HB (R) (5 micrograms of HBsAg in 0.5 ml) at 2, 4 and 12 or 15 months of age. Safety and tolerability were monitored after each injection. The serum concentrations of anti-PRP and anti-HBs were determined at the time of each vaccination, 2 months after the second vaccination and 1 month after the third vaccination. RESULTS: COMVAX was well-tolerated and proved to be immunologically comparable with a series of concomitant P+R injections. There were no serious adverse experiences attributable to the study vaccines. The most commonly reported nonserious adverse experiences were all events prelisted on diary cards given to parents. These included generally mild and transient signs of inflammation at the injection site (pain/ soreness, erythema, swelling/induration), somnolence and irritability. Because children are at peak risk of invasive Hib disease during the first year of life, 6 months of age (2 months after the second dose of vaccine) was designated the time of primary interest with regard to the development of anti-PRP. At that time 94.8% of the infants given COMVAX had > 0.15 microgram/ml of anti-PRP and 72.4% had > 1.0 microgram/ ml, with a geometric mean concentration (GMC) of 2.5 micrograms/ml, compared with 95.2%, 76.3% and 2.8 micrograms/ml, respectively, in recipients of P+R. The third injection given at 12 or 15 months of age induced a secondary rise in antibody. The proportions with > 0.15 microgram/ml and > 1.0 microgram/ml of anti-PRP increased to 99.3 and 92.6%, respectively, and the GMC rose to 9.5 micrograms/ml among COMVAX recipients, compared with 98.9%, 92.3% and 10.2 micrograms/ml in children given concurrent injections of P+R. In contrast to Hib few infants in countries with low endemicity of HBV infection are at near term risk of exposure to virus. Consequently the anti-HBs response after the last dose of vaccine was designated the outcome of primary interest. At 13 to 16 months of age (1 month after the third dose of vaccine) 98.4% of children given COMVAX had a protective anti-HBs concentration of > or = 10 mIU/ml with a GMC of 4468 mIU/ml, compared with 100% and a GMC of 6944 mIU/ml among children given P+R. CONCLUSIONS: COMVAX is well-tolerated by healthy infants and can induce immunity against invasive Hib disease and HBV infection using only three injections compared with six injections if separate courses of monovalent PedvaxHIB and RECOMBIVAX HB are given.

Haemophilus influenzae type b-specific antibody in infants after maternal immunization.

OBJECTIVE: To study the kinetics of Haemophilus influenzae type b (Hib)-specific antibody in infants born to mothers immunized with an Hib polysaccharide or one of two Hib conjugate vaccines. STUDY DESIGN: Serum antibody to the polyribosylribitol (PRP) moiety of Hib was measured by radioimmunoassay and enzyme-linked immunosorbent assay at birth and at 2 and 6 months of age in infants born to women immunized with Hib polysaccharide or conjugate vaccine (PRP-D and HbOC). A subset of infants > or = 6 months of age was immunized with Hib conjugate vaccine after licensure of this vaccine for infants. A comparison group of 18 infants born to unimmunized women received the same Hib conjugate vaccine on a similar schedule. RESULTS: Total PRP antibody concentrations were 1.50, 14.4 and 20.4 microg/ml in 2-month-old infants born to mothers immunized with polysaccharide, PRP-D and HbOC vaccines, respectively, and 2.54, 1.35 and 2.46 microg/ml in 6-month-old infants. Infants born to mothers immunized with polysaccharide vaccine had significantly less PRP antibody at 2 months of age but similar antibody concentrations at 6 months of age. Persistence or increases in total PRP antibody during 6 months were noted in 21 of 47 (44.6%) study infants. A subset of study and comparison infants was immunized with a mean of 2.6 doses of Hib vaccines between 6 months and 2 years of age, and all infants had total PRP antibody concentrations > or = 0.15 microg/ml. CONCLUSION: Conjugate Hib vaccines administered during the last trimester of pregnancy resulted in significantly higher PRP antibody titers in infants at birth and 2 months of age than did polysaccharide vaccine. A subset of infants born to immunized mothers was subsequently immunized with Hib conjugate vaccine and had antibody concentrations similar to those in infants born to nonimmunized women.

Immunogenicity of Haemophilus influenzae type b polysaccharide-tetanus protein conjugate vaccine in children with sickle hemoglobinopathy or malignancies, and after systemic Haemophilus influenzae type b infection.

To determine the immunogenicity of Haemophilus influenzae type b polysaccharide-tetanus protein conjugate vaccine in specific populations at risk, we administered vaccine to children with sickle cell anemia (n = 19; mean age, 18.3 months, malignancies (n = 18; mean age, 43.1 months), or a recent history of systemic H. influenzae type b infection (n = 17; mean age, 11.9 months). After one dose of polyribosylribitol phosphate-tetanus toxoid conjugate vaccine the geometric mean titers for polyribosylribitol phosphate antibody were 4.8 micrograms/ml (14/19 greater than 1 microgram/ml), 1.4 micrograms/ml (9/18 greater than 1 microgram/ml), and 5.6 micrograms/ml (15/17 greater than 1 microgram/ml) in these three groups, respectively. Children with sickle cell anemia or a recent history of systemic H. influenzae type b infection had polyribosylribitol phosphate antibody levels comparable to those of normal children of similar age after one or two doses of polyribosylribitol phosphate-tetanus toxoid conjugate vaccine. We conclude that this vaccine is immunogenic in children with underlying conditions associated with an increased risk of H. influenzae type b infection.

Safety and immunogenicity of subcutaneous H. influenzae vaccines in 15-17 month-old children.

To evaluate the safety and immunogenicity of subcutaneously administered PRP and PRP-D, one hundred and one healthy 15 to 17 month-old children were randomly assigned to receive two subcutaneous doses, 2 months apart, of either vaccine. The incidence of local and systemic reactions did not differ significantly between the two groups. PRP antibody levels in the pre-immunization sera of 95% of subjects were below the level associated with immediate protection from Hib disease (less than 0.15 microgram/ml). After the first dose, 32% of PRP and 70% of PRP-D (p less than 0.001) recipients achieved antibody levels of greater than or equal to 0.15 microgram/ml, and 8% and 26% (p = 0.003) respectively achieved levels greater than or equal to 1 microgram/ml, a level believed to predict long-term protection. After the second dose, antibody levels greater than or equal to 0.15 microgram/ml were reached in 38% of PRP and 88% of PRP-D (p less than 0.001) recipients and levels of greater than or equal to 1 microgram/ml were reached in 22% and 78% respectively (p less than 0.001). The geometric mean levels of PRP antibody after immunization were significantly higher in the PRP-D group after both the first and second doses (p less than 0.001). These results suggest that subcutaneous administration of both vaccines is safe, but PRP-D results in significantly higher antibody levels compared to PRP. The levels were lower than what has been reported following intramuscular administration.

Safety and immunogenicity of oligosaccharide conjugate Haemophilus influenzae type b (HbOC) vaccine in infancy. The Northern California Kaiser Permanente Vaccine Study Center Pediatrics Group.

The safety and immunogenicity of the HbOC conjugate vaccine have been evaluated in a study population of 61,080 children in the Kaiser Permanente Medical Care Program of Northern California. Half of the population served as controls. The vaccine was given as part of a three-dose regimen in the first year of life with the first dose given between 6 weeks and 6 months of age, a minimum interval of 1 month between doses and with the third dose given by 1 year of age. The vaccine was highly immunogenic with 97% of infants developing 1 microgram/ml or more of anti-polyribosyl phosphate antibody as measured by Farr assay 1 month after completion of the three-dose series and with 71% maintaining this concentration until a booster dose was given at approximately 18 months of age. There were three cases of Haemophilus influenzae type b disease after the first dose of vaccine and two of these children died. However, there was no statistically significant difference between either the incidence of H. influenzae type b disease or the mortality rate in infants after one dose of HbOC vaccine when compared with H. influenzae type b disease incidence and mortality in unvaccinated children of the same age. The rate of sudden infant death syndrome following HbOC vaccine was lower than that observed within the Kaiser Permanente Medical Care Program as a whole or in the three counties in which sudden infant death syndrome surveillance was tabulated. Immediate local and systemic reactions were evaluated by telephone interviews of 6887 infants within 72 hours of vaccine.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of crossreactive idiotypes by human antibodies specific for the capsular polysaccharide of Hemophilus influenzae B.

Human antibodies specific, for polyribosyl-ribitol-phosphate (PRP), the capsular polysaccharide of Hemophilus influenzae b, were studied using idiotypic analysis. Antisera were prepared against purified F(ab')2 anti-PRP from two unrelated adults, H.H. and P.T. After repeated absorption with IgG myeloma proteins and with PRP-absorbed normal human Ig and donor Ig, anti-idiotypic (anti-Id) sera were obtained that specifically reacted with anti-PRP antibodies. Anti-IdHH and anti-IdPT reciprocally crossreacted with H.H. and P.T. anti-PRP antibodies and F(ab')2 fragments, and also reacted with the serum anti-PRP antibodies from three additional adults unrelated to P.T. and H.H. Both anti-Id sera partially inhibited anti-PRP paratopes but not anti-tetanus toxoid paratopes. PRP did not inhibit anti-Id recognition of shared or crossreactive idiotypic (CRI) determinants. Naturally occurring and PRP immunization-induced anti-PRP antibodies expressed CRI. While CRI titer increased after immunization, the increase was usually less than the rise in total anti-PRP antibody. Quantitative differences in CRI expression were also apparent between natural and immunization-induced H.H. and P.T. anti-PRP antibodies as shown by their differential inhibitability by anti-Id. Our data demonstrate that anti-PRP antibodies from five unrelated adults express CRI determinants that are probably distant from the PRP combining site. Naturally occurring and immunization-induced anti-PRP antibodies share CRI and therefore appear to be clonally related, although immunization apparently induces the expression CRI-negative antibodies as well. These results, taken with previous studies showing restricted and identical anti-PRP isoelectric focusing spectrotypes in unrelated adults, suggest that some PRP-specific V domains are structurally conserved and probably germ-line encoded.

Altered antibody isotype in cystic fibrosis: impaired natural antibody response to polysaccharide antigens.

Patients with cystic fibrosis (CF) have impaired natural (preinfection) IgG2 antibody responses to Pseudomonas aeruginosa lipopolysaccharide. To investigate the basis for this defect, we measured natural IgG and IgG1-4 antibody levels to Haemophilus influenzae type b polyribophosphate (PRP) and tetanus toxoid by enzyme-linked immunosorbent assay in 24 adult CF patients and 20 normal controls. Immunoglobulin heavy- and light-chain allotypes were determined on 146 Caucasian CF patients and 96 controls. The tetanus toxoid-specific IgG response was predominantly IgG1. CF and control subjects had similar IgG and IgG1 antibody levels. The PRP-specific IgG response was predominantly IgG2. In contrast to tetanus toxoid results, CF patients had lower geometric mean level of PRP-specific IgG compared to normal controls (p = 0.0036). ELISA results were confirmed by liquid-phase 3H-PRP-binding assay: CF patients had a geometric mean serum antibody level of 395 versus 922 ng/ml in controls (p = 0.0044). PRP-specific IgG2 levels were also depressed in CF patients (p = 0.03). CF patients had a lower prevalence of the A2m(2) allotype than the local racially matched control sample (p less than 0.025). Other allotype prevalences including G2m(n) and Km(1) were similar. Impaired IgG2 antibody responses to microbial polysaccharide surface antigens in CF patients might predispose them to persistent endobronchial infection and lead to production of nonopsonizing isotype responses. The potential role of A2m(2), coded for in the H chain locus on chromosome 14, is unknown, but could be related to mucosal IgA2 antibody responses.

Fetal-neonatal passive immunization against Hemophilus influenzae, type b.

Twenty-one pregnant women were vaccinated with the polyribophosphate capsular antigen of Hemophilus influenzae, type b, at 34 to 36 weeks of gestation. All women experienced a large boost in their own antibody levels of anti-polyribophosphate capsular antigen, and 30% was transferred to their newborn infants. The newborn serum anti-polyribophosphate capsular antigen level at birth was 100-fold greater than that of control newborn infants, and the antibody persisted at a protective level for 12 months. Since newborn infants lose significant antibody by 3 months of age, they are susceptible to infection by Hemophilus influenzae, type b, such that it is the leading cause of meningitis in infants. The passive levels of anti-polyribophosphate capsular antigen achieved in these fetuses-neonates by active immunization of their mothers should theoretically lead to less disease caused by Hemophilus influenzae, type b, during infancy.

Isoelectric focusing of human antibody to the Haemophilus influenzae b capsular polysaccharide: restricted and identical spectrotypes in adults.

Serum antibody to the capsular polysaccharide of Haemophilus influenzae b of human adults was analyzed by isoelectric focusing. Restricted antibody spectrotype patterns were commonly observed with as few as one spectrotype in some subjects after immunization with the isolated capsular polysaccharide. Some patterns were as restricted as human hybridoma antibody. There was no correlation of antibody titer and heterogeneity of patterns. The dominant spectrotype persisted unchanged for over 2 yr after immunization, and the pattern detected in preimmunization serum samples persisted unchanged after immunization. Indistinguishable patterns were commonly observed in genetically unrelated adults. Adults immunized with conjugate vaccines, which were composed of oligosaccharides prepared from the capsular polysaccharide that were covalently linked to protein carriers, also produced restricted serum antibody spectrotype patterns. Immunization with the cross-reactive polysaccharide of E. coli K100 induced a spectrotype pattern that was restricted but different from that induced by the H. influenzae b capsular polysaccharide.

Immunization of 2-month-old infants with protein-coupled oligosaccharides derived from the capsule of Haemophilus influenzae type b.

We studied an immunogen consisting of oligosaccharides derived from Haemophilus influenzae type b capsular polysaccharide (PRP) coupled to CRM197, a nontoxic relative of diphtheria toxin. Subcutaneous injections were given to eight subjects at ages 2, 4, and 6 months, simultaneously with conventional diphtheria-tetanus-pertussis (DTP) vaccine. After the first immunization, total serum anti-PRP antibodies declined in all subjects, but increased in most after the second immunization and after the third in seven of seven subjects analyzed. In these seven infants, the geometric mean level at age 9 months (0.73 micrograms/ml) exceeded by at least 40 times the means of historical control groups given DTP only or DTP plus (uncoupled) PRP vaccine. An isotype-specific assay showed that IgM antibodies increased after the first immunization with the coupled vaccine in all eight infants. Against the background of declining maternal IgG antibody, elevations in IgG antibody were detected after the second or third immunization in six of the eight. These six at age 9 to 11 months were immunized with (uncoupled) PRP vaccine, and a "boost" in anti-PRP antibody, including an IgG component, was found.