NCT00231907.

Combinatorial cloning and expression library analysis were used to isolate human antibody Fab fragments specific for the capsular polysaccharide of Streptococcus pneumoniae serotype 23F. Thirty 23F-specific Fabs were isolated from seven vaccinated donors, and the sequences of the heavy (H)-and light (L)-chain variable regions were determined. All individuals utilized either the V A23 L chain, the V L6 L chain, or both chains in forming the 23F-specific combining site. V A23 L chains paired primarily with VH3-23 H chains. V L6 L chains were more promiscuous in heavy-chain usage between individuals. Both H and L chains were mutated, primarily in the complementarity-determining regions, compared to their closest germ line counterpart, suggesting a recall response that has undergone affinity maturation. H-chain isotypes were reflective of those found in the serum. Shared somatic modifications demonstrated that immunoglobulin G2 (IgG2) and IgA antibodies arose from the same somatically matured B cell. Our results indicate that the response to the serotype 23F pneumococcal capsular polysaccharide is oligoclonal within the individual, with one or two paratope families accounting for the majority of expressed antibody. We also determined that, in spite of the combinatorial diversity available to the immune system, the 23F-specific response is highly restricted at the population level, with the same two L-chain-determined paratope families recurring in all individuals. Lastly, analysis of the isolated Fabs indicate all have undergone extensive somatic mutation, as well as class switch, maturational events that presumably require the participation of T cells.Streptococcus pneumoniae is a significant human pathogen causing pneumonia, bacteremia, meningitis, and otitis media. The pathogenic pneumococci are surrounded by a complex capsule composed of polymeric sugars, C polysaccharide, peptidoglycan, and surface proteins. The pneumococcal capsular polysaccharides (PPS), are heterogeneous in structure, with at least 90 different serotypes occurring within the species S. pneumoniae. PPS epitopes are immunogenic in adults and elicit antibodies that protect against infection. A vaccine containing capsular polysaccharides from 23 pneumococcal serotypes (23-valent) is available and is currently recommended for persons over 65 years of age and for other adults considered to be at increased risk of developing pneumococcal disease. Purified capsular polysaccharides do not, however, induce a protective antibody response in infants, who comprise one of the primary populations at risk. Consequently, a 7-valent polysaccharide-protein conjugate vaccine has been developed and shown to be efficacious in infants and has recently been licensed for use in this age group. Capsular polysaccharides are defined as TI-2 (T-cell-independent) antigens based on their repetitive structure and lack of immunogenicity in xid mice and human infants. The serum response to these polysaccharides in adults is oligoclonal and restricted in isotype, with immunog...

Both live attenuated influenza vaccines (LAIV) and inactivated influenza vaccines (IIV) induce protective immunity against influenza. There is evidence that LAIV induces superior protection in children, whereas IIV may induce superior protection in adults. The immune mechanisms responsible for these differences have not been identified. We previously compared LAIV and IIV in young children of 6 to 36 months of age, and we demonstrated that while both induced similar hemagglutination inhibition (HAI) antibody responses, only LAIV induced significant increases in T cell responses. In the present study, 37 healthy adult subjects of 18 to 49 years of age were randomized to receive seasonal influenza vaccination with LAIV or IIV. Influenza virus-specific HAI, T cell, and secretory IgA (sIgA) responses were studied pre- and postvaccination. In contrast to the responses seen in young children, LAIV induced only minimal increases in serum HAI responses in adults, which were significantly lower than the responses induced by IIV. Both LAIV and IIV similarly induced only transient T cell responses to replication-competent whole virus in adults. In contrast, influenza virus-specific sIgA responses were induced more strongly by LAIV than by IIV. Our previous studies suggest that LAIV may be more protective than IIV in young children not previously exposed to influenza virus or influenza vaccines due to increased vaccine-induced T cell and/or sIgA responses. Our current work suggests that in adults with extensive and partially cross-reactive preexisting influenza immunity, LAIV boosting of sIgA responses to hemagglutinin (HA) and non-HA antigenic targets expressed by circulating influenza virus strains may be an important additional mechanism of vaccine-induced immunity.

Combinatorial cloning and expression library analysis were used to determine the expressed human antibody repertoire specific for the capsular polysaccharide (PS) of Streptococcus pneumoniae serotype 6B. Sequence analysis of 55 6B-specific antibody Fab fragments isolated from six vaccinated donors reveal that different individuals used a variety of heavy and light chain germ line variable (V) region genes to form pneumococcal capsular PS (PPS) 6B-specific paratopes. Within each donor, however, the response was more restricted, with five of the six donors using at most one or two gene pairs to form combining sites. Analysis also indicated that although the response in each donor was oligoclonal in terms of variable gene usage, the combination of extensive somatic hypermutation, deletion of germ line-encoded residues, insertion of non-germ line-encoded residues, and intraclonal isotype switching generated a surprising degree of paratope diversity within the individuals analyzed. In contrast to previously studied PS-specific responses, we find that the PPS 6B repertoire makes use of a diverse collection of heavy-chain and light-chain V region gene products to form specific paratopes, with no apparent tendency for conservation of immunoglobulin gene usage between individuals.Streptococcus pneumoniae is a significant human pathogen causing pneumonia, bacteremia, meningitis, and otitis media. The primary determinants of virulence for the many strains of S. pneumoniae are the pneumococcal capsular polysaccharides (PPS). The PPS are heterogeneous in structure, and at least 90 different serotypes occur within the species (10). PPS epitopes are immunogenic in adults, and immunization with the polysaccharides (PS) provides serotype-specific protection against infection (26). PPS-protein conjugates are immunogenic in infants and provide protection for this age group as well (31). Both plain PS and PS-conjugate vaccines are available and are currently recommended for the appropriate age groups.In addition to providing protection against disease, these vaccines offer an opportunity to explore several aspects of basic immunobiology in humans. The carbohydrate epitopes are structurally defined, the vaccines are routinely and safely administered to adults and children, and specific B cells circulate in the periphery following vaccination, thereby facilitating minimally invasive access to the cellular components of interest. Although the serology of the response to various PPS antigens has been studied in detail (13,19,20,24,27,30), the difficulty in constructing stable human heterohybridomas has limited the degree to which the PPS-specific antibody response could be studied at the level of immunoglobulin (Ig) gene usage.In this report we use repertoire cloning to examine the paratopic repertoire of human antibodies specific for the capsular PS of S. pneumoniae serotype 6B. Heavy (H)-and light (L)-chain variable (V) (V H and V L , respectively) region sequences are reported for 55 PPS 6B-specific Fab fragments isolated from six i...

Inactivated poliovirus vaccine (IPV) is believed to induce significantly lower mucosal immunity than oral poliovirus vaccine (OPV). Most of the data supporting this were generated before enhanced IPV (eIPV) was introduced. Excretion of poliovirus by OPV recipients can be used to assess intestinal immunity. We studied polymerase chain reaction amplification of viral complementary DNA from the stool of children vaccinated with either OPV alone or eIPV. Of first-time OPV recipients, 92% excreted virus after 1 week, and 81% excreted virus after 3 weeks. Prior vaccination with OPV reduced the number to 22% and shortened the duration of virus excretion (to 5% after 3 weeks). Two doses of IPV reduced the number of poliovirus-positive 1-week samples (to 76%), the duration of shedding (to 37% at 3 weeks), and the quantity of excreted virus. This suggests that IPV-vaccinated communities are partially protected from the spread of poliovirus. Further enhancement of IPV potency may lead to even higher levels of mucosal immunity.