Immunogenicity in a mouse model of a conjugate vaccine made with a synthetic single repeating unit of type 14 pneumococcal polysaccharide coupled to CRM197

Oligosaccharides (OSs) related to the pneumococcal type 14 capsular polysaccharide (Pn14PS) were studied for their ability to inhibit the binding between anti-PS14 antisera and native PS14. A synthetic tetrasaccharide corresponding to the repeating unit of the Pn14PS, a hexasaccharide mimic, and an octasaccharide fragment obtained by Pn14PS depolymerization were good inhibitors. CRM197 conjugates of the tetrasaccharide and an octasaccharide mimic were prepared by using either adipic acid diester or diethyl squarate linkers. The conjugate with the tetrasaccharide chains induced anti-Pn14PS antibodies when injected subcutaneously into mice, as determined by an enzyme-linked immunosorbent assay, and antibody titers increased with oligosaccharide loading. The adipic acid-linked tetrasaccharide conjugates elicited higher antibody titers than those prepared with a squarate spacer. The lower anti-Pn14PS antibody response of the octasaccharide mimic conjugate indicates the importance of the backbone galactose residue for an appropriate antibody response. The OS-CRM197 conjugate prepared from a single repeat unit of the Pn14PS is a potential vaccine candidate.     

Avidity, potency, and cross-reactivity of monoclonal antibodies to pneumococcal capsular polysaccharide serotype 6B

Many pneumococcal capsular polysaccharides (PSs) are similar in structure, and a pneumococcal antibody often binds to all of the PSs with a similar structure. Yet, these cross-reactive antibodies may bind to the structurally related pneumococcal capsular PSs with an avidity too low to be effective. If memory B cells producing such weakly cross-reactive antibodies are elicited with pneumococcal conjugate vaccines, the memory cells for low-avidity antibodies could compromise the subsequent immune responses to the cross-reactive PS (original antigenic sin). To investigate these issues, we produced 14 hybridomas secreting monoclonal antibodies (MAbs) to the capsular PS of Streptococcus pneumoniae serotype 6B by immunizing BALB/c mice with antigens containing 6B PS and studied their epitope, avidity, in vitro opsonizing capacity, in vivo protective capacity, and "antigen binding titer" by enzyme-linked immunosorbent assay (ELISA) of 6A and 6B capsular PSs. Six MAbs bound to the non-cross-reactive 6B-specific epitope, and seven MAbs bound to the cross-reactive epitope present in both 6A and 6B PSs One MAb (Hyp6BM6) revealed a novel epitope. This epitope was found on 6A PS in solution, but not on 6A PS adsorbed onto the plastic surface of the ELISA plates. The avidity of the MAb for 6A or 6B PS ranged from 7.8 x 10(6) M(-1) to 4.1 x 10(11) M(-1). No MAbs were weakly cross-reactive, since none of the cross-reactive MAbs showed any tendency toward having less avidity to 6A PS (the cross-reactive PS) than to 6B PS. Avidity influenced the results of several antibody assays. When all of the hybridomas were examined, avidity strongly correlated with the titer of a unit amount of MAb to bind antigen-coated ELISA plates (r = 0.91) or to opsonize pneumococci in vitro (r = -0.85). Because both assay results are avidity dependent, the ELISA and the opsonization assay results were strongly correlated (r = 0.91), regardless of avidity. Avidity also correlated with the potency of a MAb to passively protect mice against pneumococcal infections. When only the immunoglobulin G hybridomas were examined, little increase in opsonizing capacity and in vivo protective potency was observed above 10(9) M(-1). Taken together, an ELISA measuring antigen binding titer may be an adequate measure of the protective immunity induced with pneumococcal vaccines, and the absence of a partially cross-reactive MAb suggests that antigenic sin may not be significant in responses to vaccines against the S. pneumoniae 6B serotype.     

Identification of the smallest structure capable of evoking opsonophagocytic antibodies against Streptococcus pneumoniae type 14

Synthetic overlapping oligosaccharide fragments of Streptococcus pneumoniae serotype 14 capsular polysaccharide (Pn14PS), [6)-[beta-D-Galp-(1-->4)-]beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->](n), were conjugated to CRM(197) protein and injected into mice to determine the smallest immunogenic structure. The resulting antibodies were then tested for Pn14PS specificity and for their capacity to promote the phagocytosis of S. pneumoniae type 14 bacteria. Earlier studies have reported that the oligosaccharide corresponding to one structural repeating unit of Pn14PS, i.e., Gal-Glc-(Gal-)GlcNAc, induces a specific antibody response to Pn14PS. The broader study described here, which evaluated 16 oligosaccharides, showed that the branched trisaccharide element Glc-(Gal-)GlcNAc is essential in inducing Pn14PS-specific antibodies and that the neighboring galactose unit at the nonreducing end contributes clearly to the immunogenicity of the epitope. Only the oligosaccharide conjugates that produce antibodies recognizing Pn14PS were capable of promoting the phagocytosis of S. pneumoniae type 14. In conclusion, the branched tetrasaccharide Gal-Glc-(Gal-)GlcNAc may be a serious candidate for a synthetic oligosaccharide conjugate vaccine against infections caused by S. pneumoniae type 14.     

Preparation, characterization, and immunogenicity of meningococcal lipooligosaccharide-derived oligosaccharide-protein conjugates.

A method was developed for coupling carboxylic acid-containing oligosaccharides (OS) to proteins. An OS was isolated from Neisseria meningitidis group A strain A1 lipooligosaccharide (LOS). This LOS has no human glycolipid-like lacto-N-neotetraose structure and contains multiple immunotypes, including L8, found in group B and C strains. The carboxylic acid at 2-keto-3-deoxyoctulosonic acid of the OS was linked through adipic acid dihydrazide to tetanus toxoid. The molar ratio of the OS to tetanus toxoid in three conjugates ranged from 11:1 to 19:1. The antigenicity of the OS was conserved in these conjugates, as measured by an enzyme-linked immunosorbent assay (ELISA) and an inhibition ELISA with polyclonal and monoclonal antibodies to A1 LOS. These conjugates induced immunoglobulin G antibodies to A1 LOS in mice and rabbits. The immunogenicity of the conjugates in rabbits was enhanced by use of monophosphoryl lipid A plus trehalose dimycolate as an adjuvant. The resulting rabbit antisera cross-reacted with most of 12 prototype LOSs and with LOSs from two group B disease strains, 44/76 and BB431, in an ELISA and in Western blotting (immunoblotting), which revealed a 3.6-kDa reactive band in these LOSs. The rabbit antisera showed bactericidal activity against homologous strain A1 and heterologous strains 44/76 and BB431. These results indicate that conjugates derived from A1 LOS can induce antibodies against many LOS immunotypes from different organism serogroups, including group B. OS-protein conjugates derived from meningococcal LOSs may therefore be candidate vaccines to prevent meningitis caused by meningococci.     

Characterization of a recombinant pneumolysin and its use as a protein carrier for pneumococcal type 18C conjugate vaccines.

Pneumolysin from Streptococcus pneumoniae was expressed in Escherichia coli as a glutathione S-transferase fusion protein and purified by affinity and hydroxylapatite chromatography. The purified recombinant pneumolysin (rPL), with a molecular mass of 53 kDa, had a specific activity of 3 x 10(5) hemolytic units per mg of protein on rabbit erythrocytes and reacted identically in immunodiffusion with the antisera against native pneumolysin. The rPL was used as a protein carrier to prepare conjugate vaccine with pneumococcal type 18C polysaccharide (PS18C). The PS18C was directly coupled to rPL by reductive animation or was indirectly coupled to rPL via a spacer molecule, adipic acid dihydrazide. The conjugates were nontoxic for mice and guinea pigs at 100 micrograms per dose. The immunogenicity and protective efficacy of both conjugates were tested in mice. A single dose of either of the vaccines elicited a rise in immunoglobulin G antibody production; after two booster injections of the vaccines, statistically significant booster responses (P < 0.001) to both rPL and PS18C were produced. The sera containing the antibodies to rPL were capable of neutralizing the hemolytic activity of rPL to rabbit erythrocytes and the cytotoxicity of rPL to bovine pulmonary endothelial cells. Immunization with the conjugate vaccines conferred statistically significant protection in mice against lethal challenge with type 18C pneumococci.     

Streptococcus pneumoniae Type 14 Polysaccharide-Conjugate Vaccines: Length Stabilization of Opsonophagocytic Conformational Polysaccharide Epitopes

A simple and convenient method was developed for the preparation of Streptococcus pneumoniae type 14 polysaccharide (Pn14PS)-tetanus toxoid (TT) conjugate vaccines, using terminally linked Pn14PS fragments of different lengths. Native Pn14PS was simultaneously depolymerized and activated for conjugation by partial N-deacetylation followed by nitrous acid deamination which yielded fragments (1.4 to 150.0 kDa) having a free aldehyde at the reducing end. These were then conjugated to TT through their terminal aldehydic groups, using the reductive amination procedure. All of the above conjugates, when injected in rabbits, induced anti-Pn14PS antibodies, whereas the native Pn14PS did not. The amounts of anti-Pn14PS antibodies elicited by these conjugates, as determined by enzyme-linked immunosorbent assay, followed a trend with conjugates containing the highest-molecular-weight Pn14PS eliciting the highest titers. The same trend was also observed in the ability of the antibodies to opsonize and kill live type 14 pneumococci, although the increase in opsonophagocytic activity was more pronounced and did not correlate linearly with increases in antibody titer. Competitive inhibition of the binding of different conjugate antisera to the native Pn14PS, using Pn14PS fragments as inhibitors, established that the conjugates induced antibodies with specificities for different lengths of Pn14PS beginning at 2 repeating units (RU). It was also established, both immunologically and antigenically, that at least 4 RU of Pn14PS were required to form an extended conformational epitope and that approximately 22 RU of Pn14PS were required to duplicate the same epitope on the same saccharide chain. The conformational epitope was found to be essential for the induction of antibodies with high opsonophagocytic activity and that augmentation of opsonophagocytic activity was also dependent on further chain extension.     

Protective Immunity of Pneumococcal Glycoconjugates

Pneumococcal polysaccharides (PSs), designated as T-cell independent type 2 (TI-2) antigens, induce poor immune responses in young children. Splenic marginal zone B cells, associated with CD21, CD19 and C3d, play an important role in TI-2 antibody responses, and provide host defense against bacterial pathogens. Antibody response, avidity, and opsonophagocytic activity of antisera were examined in mice immunized with type 9V PS conjugated to inactivated pneulmolysin (Ply) or to autolysin (Aly). Compared to mice given 9V PS alone, serum IgG and IgM concentrations against the 9V PS were higher in mice immunized with conjugates. High concentrations of serum antibodies were maintained for over 12 weeks. The relative avidities of IgG and IgM antibodies and opsonophagocytic activity against 9V pneumococci were high in mice immunized with conjugates. Thus, conjugate vaccines can induce high as well as long duration of antibody response and effective functional activity. In another study, mice received intranasal immunization with type 9V conjugate or 9V PS. These animals produced 9V PS IgG and IgA antibodies in their serum, spleen, intestine, lung, Peyer's patch and fecal extract samples. Mice immunized with these glycoconjugates exhibited opsonophagocytic activity and rapid bacterial clearance from blood and provided homologous and cross-protection against challenge with virulent pneumococci. These results indicate that intranasal immunization with glycoconjugate vaccines may serve as an alternative and convenient approach for prevention of pneumococcal infection.     

Protective immunity of pneumococcal glycoconjugates

Pneumococcal polysaccharides (PSs), designated as T-cell independent type 2 (TI-2) antigens, induce poor immune responses in young children. Splenic marginal zone B cells, associated with CD21, CD19 and C3d, play an important role in TI-2 antibody responses, and provide host defense against bacterial pathogens. Antibody response, avidity, and opsonophagocytic activity of antisera were examined in mice immunized with type 9V PS conjugated to inactivated pneulmolysin (Ply) or to autolysin (Aly). Compared to mice given 9V PS alone, serum IgG and IgM concentrations against the 9V PS were higher in mice immunized with conjugates. High concentrations of serum antibodies were maintained for over 12 weeks. The relative avidities of IgG and IgM antibodies and opsonophagocytic activity against 9V pneumococci were high in mice immunized with conjugates. Thus, conjugate vaccines can induce high as well as long duration of antibody response and effective functional activity. In another study, mice received intranasal immunization with type 9V conjugate or 9V PS. These animals produced 9V PS IgG and IgA antibodies in their serum, spleen, intestine, lung, Peyer's patch and fecal extract samples. Mice immunized with these glycoconjugates exhibited opsonophagocytic activity and rapid bacterial clearance from blood and provided homologous and cross-protection against challenge with virulent pneumococci. These results indicate that intranasal immunization with glycoconjugate vaccines may serve as an alternative and convenient approach for prevention of pneumococcal infection.     

Comparative immunogenicity of group 6 pneumococcal type 6A(6) and type 6B(26) capsular polysaccharides.

The comparative immunogenicity of the two cross-reacting group 6 pneumococcal capsular polysaccharides, type 6A(6) and type 6B(26), was studied with hyperimmune rabbit typing antisera and with sera from adult volunteers injected with polyvalent pneumococcal vaccines containing either 50 mug of type 6A (U.S. designation, type 6) or 50 mug each of type 6A and type 6B (U.S. designation, type 26) polysaccharides. Both group 6 polysaccharides were linear copolymers composed of 1 mol each of d-galactose, d-glucose, l-rhamnose, and d-ribitol phosphate. They differed only in that type 6A had a rhammopyranosyl-(1 --> 3)-d-ribitol bond and the type 6B had a rhamnopyranosyl-(1 --> 4)-d-ribitol bond. Quantitative precipitation and absorption analyses with rabbit hyperimmune antisera induced by simultaneous injection with type 6A and type 6B organisms revealed extensive cross-reactions between the two group 6 polysaccharides. There was less, although still quite extensive, cross-reactivity between the two group 6 polysaccharides examined with antisera from rabbits injected with only one of the group 6 pneumococci. In a radioimmunoassay, using (14)C internally labeled type 6A or type 6B polysaccharide antigens, there was no difference in the serum antibody level to either type of volunteer injected with polyvalent pneumococcal vaccines containing type 6A or both type 6A and type 6B polysaccharides. These studies indicate that the structural similarity of the pneumococcal group 6 polysaccharides confers extensive cross-reactivity with hyperimmune typing antisera prepared with whole organisms or after injection of purified polysaccharides in adult volunteers. With our current polysaccharides, it appears that a polyvalent pneumococcal vaccine formulation that contains only type 6A will serve to induce the maximum amount of serum antibodies to both group 6 organisms.     

Epitope specificity of rabbit immunoglobulin G (IgG) elicited by pneumococcal type 23F synthetic oligosaccharide- and native polysaccharide-protein conjugate vaccines: comparison with human anti-polysaccharide 23F IgG.

Streptococcus pneumoniae type 23F capsular polysaccharide (PS23F) consitss of a repeating glycerol-phosphorylated branched tetrasaccharide. The immunogenicities of the following related antigens were investigated: (i) a synthetic trisaccharide comprising the backbone of one repeating unit, (ii) a synthetic tetrasaccharide comprising the complete repeating unit, and (iii) native PS23F (all three conjugated to keyhole limpet hemocyanin [KLH]) and (iv) formalin-killed S. pneumoniae 23F. All antigens except the trisaccharide-KLH conjugate induced relatively high anti-PS23F antibody levels in rabbits. The epitope specificity of such antibodies was then studied by means of an inhibition immunoassay. The alpha(1-->2)-linked L-rhamnose branch was shown to be immunodominant for immunoglobulin G (IgG) induced by tetrasaccharide-KLH, PS23F-KLH, and killed S. pneumoniae 23F: in most sera L-rhamnose totally inhibited the binding of IgG to PS23F. Thus, there appears to be no major difference in epitope specificity between IgG induced by tetrasaccharide-KLH and that induced by antigens containing the polymeric form of PS23F. Human anti-PS23F IgG (either vaccine induced or naturally acquired) had a different epitope specificity: none of the inhibitors used, including L-rhamnose and tetrasaccharide-KLH, exhibited substantial inhibition. These observations suggest that the epitope recognized by human IgG on PS23F is larger than the epitope recognized by rabbit IgG. Both human and rabbit antisera efficiently opsonized type 23F pneumococci, as measured in a phagocytosis assay using human polymorphonuclear leukocytes.     

Serum antibody responses of juvenile and infant rhesus monkeys injected with Haemophilus influenzae type b and pneumococcus type 6A capsular polysaccharide-protein conjugates

Juvenile and infant rhesus monkeys were injected subcutaneously with saline solutions of Haemophilus influenzae type b (Hib) and pneumococcus type 6A (Pn6A) capsular polysaccharides conjugated to either tetanus toxoid (TT), horseshoe crab hemocyanin, or cholera toxin (CT), and the antibody responses of the monkeys to both bacterial components were measured. All three Hib conjugates were immunogenic and elicited booster responses; their comparative immunogenicity was Hib-CT greater than Hib-TT greater than Hib-horseshoe crab hemocyanin. Hib alone did not elicit antibodies in the juveniles. Juveniles responded earlier and with higher levels of antibodies than did infants. TT, as well as diphtheria-tetanus toxoids-pertussis vaccine adsorbed injected concurrently at a separate site, increased both Hib and TT antibody responses in juveniles (P less than 0.05). Concurrent injection of 5 Lf of fluid TT with a nonimmunogenic 5-micrograms dose in infants elicited levels of Hib antibodies comparable to those elicited by 50 micrograms of Hib-TT. Hib antibodies elicited by the conjugates remained at protective levels in both juveniles and infants 2 months after the last injection, were bactericidal, and conferred passive immunity against bacteremia in infant rats. Passive immunization of juveniles with tetanus immune globulin before each injection of Hib-TT did not suppress Hib antibodies. Hib-TT and Hib-CT elicited increases of Hib antibodies of the immunoglobulin M and G isotypes in the infants. The Pn6A-TT conjugate was considerably less immunogenic than the Hib-TT conjugate; only a few of the juveniles or infants responded with protective levels of Pn6A antibodies. Pn6A antibodies from responders conferred protection in mice against intraperitoneal challenge with Pn6A organisms. TT antibodies were elicited in both juvenile and infant animals after one injection of 50 micrograms of Hib-TT and in the infants injected with 5 micrograms of Hib-TT plus 5 Lf of TT; 5 micrograms of Hib-TT and Pn6A-TT in combination alone did not elicit TT antibodies. Hib-CT elicited CT antibodies in both juveniles and infants.     

Synthesis and characterization of lipooligosaccharide-based conjugate vaccines for serotype B Moraxella catarrhalis

Moraxella catarrhalis is an important cause of otitis media in children and respiratory tract infections in the elderly. Lipooligosaccharide (LOS) is a major surface antigen of the bacterium that elicits bactericidal antibodies. Serological studies show that three major LOS types (A, B, and C) have been identified among clinical isolates. Our previous studies demonstrated that the type A LOS-based conjugates were immunogenic in animals. In this study, LOS from type B strain 26397 was detoxified and conjugated to tetanus toxoid (TT) or a cross-reactive mutant (CRM) of diphtheria toxin to form detoxified LOS (dLOS)-TT and dLOS-CRM, respectively, as vaccine candidates. The molar ratios of dLOS to TT and CRM in the conjugates were 43:1 and 19:1, respectively, while both weight ratios were around 0.9. The antigenicity of the conjugates was similar to that of the LOS, as determined by enzyme-linked immunosorbent assay using a rabbit antiserum to strain 26397. Subcutaneous immunization with each conjugate elicited a 180- to 230-fold rise of serum anti-LOS immunoglobulin G in mice and >2,000-fold rise in rabbits. In addition, both mouse and rabbit antisera showed elevated complement-mediated bactericidal activity against the homologous strain, and a representative rabbit antiserum showed bactericidal activity against nine of twelve clinical isolates studied. The bactericidal activity of the rabbit antiserum can be fully inhibited by the type B LOS but not the A or C LOS. These results indicate that the type B LOS-based conjugates can be used as vaccine components for further investigation.     

Rabbit antibodies to the cell wall polysaccharide of Streptococcus pneumoniae fail to protect mice from lethal challenge with encapsulated pneumococci.

A conjugate, composed of the cell wall polysaccharide (C polysaccharide) of Streptococcus pneumoniae and bovine serum albumin (BSA), was prepared with the bifunctional agent N-succinimidyl-3-(2-pyridyldithio)-propionate. Analysis with monoclonal antibodies provided evidence that the phosphocholine (PC) moiety of the C polysaccharide was retained during the conjugation procedure. The C polysaccharide-BSA conjugate elicited antibodies to C polysaccharide in rabbits; no PC-specific antibodies were detected in globulins prepared from these hyperimmune sera obtained early and late after a second immunization. Rabbit hyperimmune sera were taken after multiple intravenous injections of the pneumococcus strain SRC-2, which has a capsulelike structure composed of the C polysaccharide. Globulin prepared from these antisera had both C polysaccharide- and PC-specific antibodies. Antibodies to C polysaccharide elicited by the C polysaccharide-BSA conjugate failed to protect mice against intraperitoneal challenge with a strain of type 3 or type 6A pneumococci. The anti-SRC-2 globulin conferred protection against both of these pneumococcal strains. Absorption of the SRC-2 globulin with C polysaccharide, however, failed to change its protective activity. These data provide evidence that antibodies to the C polysaccharide do not confer immunity against infection of mice with encapsulated pneumococci inoculated by the intraperitoneal route.     

Relationship between surface accessibility for PpmA, PsaA, and PspA and antibody-mediated immunity to systemic infection by Streptococcus pneumoniae

Antibodies to capsular polysaccharide (PS) are protective against systemic infection by Streptococcus pneumoniae, but the large number of pneumococcal serogroups and the age-related immunogenicity of pure PS limit the utility of PS-based vaccines. In contrast, cell wall-associated proteins from different capsular serotypes can be cross-reactive and immunogenic in all age groups. Therefore, we evaluated three pneumococcal proteins with respect to relative accessibility to antibody, in the context of intact pneumococci, and their ability to elicit protection against systemic infection by encapsulated S. pneumoniae. Sequences encoding pneumococcal surface adhesin A (PsaA), putative protease maturation protein A (PpmA), and the N-terminal region of pneumococcal surface protein A (PspA) from S. pneumoniae strain A66.1 were cloned and expressed in Escherichia coli. The presence of genes encoding PsaA, PpmA, and PspA in 11 clinical isolates was examined by PCR, and the expression of these proteins by each strain was examined by Western blotting with antisera raised to the respective recombinant proteins. We used flow cytometry to demonstrate that PspA was readily detectable on the surface of the pneumococcal strains analyzed, whereas PsaA and PpmA were not. Consistent with these observations, mice with passively or actively acquired antibodies to PspA or type 3 PS were equivalently protected from homologous systemic challenge with type 3 pneumococci, whereas mice with passively or actively acquired antibodies to PsaA or PpmA were not effectively protected. These experiments support the hypothesis that the extent of protection against systemic pneumococcal infection is influenced by target antigen accessibility to circulating host antibodies.     

Isotypes and Opsonophagocytosis of Pneumococcus Type 6B Antibodies Elicited in Infants and Adults by an Experimental Pneumococcus Type 6B-Tetanus Toxoid Vaccine

Streptococcus pneumoniae is a major respiratory pathogen of infants, children, and the elderly. Polysaccharide vaccines have been useful in adult populations but do not elicit protective immunity in infants and young children. To enhance their immunogenicity, vaccines of pneumococcal polysaccharides conjugated to proteins are being developed. In this study antibody levels and opsonic activities were compared in sera of infants and adults injected with pneumococcal polysaccharide type 6B (Pn6B) conjugated to tetanus toxoid (TT) (Pn6B-TT). Healthy infants were injected with Pn6B-TT; group A was injected at 3, 4, and 6 months of age, and group B was injected at 7 and 9 months of age. A booster injection was given at 18 months. Adults were injected once. Antibodies were measured by enzyme-linked immunosorbent assay and radioimmunoassay, and their functional activities were measured by opsonophagocytosis of radiolabelled pneumococci. In adults, increases in immunoglobulin M (IgM), IgG, IgA, IgG1, and IgG2 to Pn6B were observed. Infants reached adult levels of IgG1 anti-Pn6B after the primary injections. After the booster injection the infant groups had total IgG- and IgM-Pn6B antibody levels similar to those of adults. After the booster injection, IgG1 was the dominant infant anti-Pn6B isotype and at a level higher than in vaccinated adults, but IgA and IgG2 antibodies remained at very low levels. Opsonic activity increased significantly after Pn6B-TT injections; the highest infant sera showed opsonic activity comparable to that of vaccinated adults. Overall, opsonic activity correlated best with total and IgG anti-Pn6B antibodies (r = 0.741, r = 0.653, respectively; n = 35) and was highest in sera with high levels of all Pn6B antibody isotypes. The results indicate the protective potential of a pneumococcal 6B polysaccharide protein conjugate vaccine for young infants.     

Modified Opsonization,Phagocytosis, and Killing Assays To Measure Potentially Protective Antibodies against Pneumococcal Surface Protein A

The standard opsonophagocytosis killing assay (OPKA) for antibodies to pneumococcal capsular polysaccharide was modified to permit an evaluation of the protection-mediating antibodies to pneumococcal surface protein A (PspA). We found that by increasing the incubation time with the complement and phagocytes from 45 min to 75 min, the protective activity was readily detected. In another modification, we used a capsule type 2 target strain that expressed PspA but not pneumococcal surface protein C (PspC). With these modifications separately or in combination, rabbit antisera to the recombinant α-helical or proline-rich domains of PspA mediated >50% killing of the target strain. The ability of normal human sera to mediate the killing of pneumococci in this modified OPKA correlated with their levels of antibodies to PspA and their ability to protect mice against fatal infection with a type 3 strain. Passive protection of mice against pneumococci and killing in the modified OPKA were lost when normal human sera were adsorbed with recombinant PspA (rPspA) on Sepharose, thus supporting the potential utility of the modified OPKA to detect protective antibodies to PspA. In the standard OPKA, monoclonal antibodies to PspA were strongly protective in the presence of subprotective amounts of anti-capsule. Thus, the currently established high-throughput OPKA for antibodies to capsule could be modified in one of two ways to permit an evaluation of the opsonic efficacy of antibodies to PspA.     

Further studies on the immunogenicity of Haemophilus influenzae type b and pneumococcal type 6A polysaccharide-protein conjugates

Conjugates were prepared by carbodiimide-mediated coupling of adipic acid hydrazide derivatives of Haemophilus influenzae type b (Hib), Escherichia coli K100, and pneumococcal 6A (Pn6A) polysaccharides with tetanus toxoid (TT), as an example of a “useful” carrier, and horseshoe crab hemocyanin (HCH), as an example of a “nonsense” carrier. These conjugates were injected into NIH mice, and their serum antibody responses to the polysaccharides and proteins were characterized. As originally reported, Hib conjugates increased the immunogenicity of the capsular polysaccharide and elicited greater than the estimated protective levels of anti-Hib antibodies in most recipients after one injection and in all after the third injection (Schneerson et al., J. Exp. Med. 152:361-376, 1980). Both Hib conjugates induced similar anti-Hib responses. The K100-HCH conjugate was more immunogenic than the K100-TT conjugate and elicited anti-Hib responses similar to the Hib conjugates after the third injection. Simultaneous injection of the K100 and the Hib conjugates did not enhance the anti-Hib response. The Pn6A-TT conjugate induced low levels of anti-Hib antibodies; when injected simultaneously with the Hib conjugates, the anti-Hib response was enhanced, as all mice responded after the first injection and with higher levels of anti-Hib than observed with the Hib conjugates alone (P < 0.05). The Pn6A conjugates were not as immunogenic as the Hib conjugates. Pn6A-TT was more effective than was Pn6A-HCH; it elicited anti-Pn6A (>100 ng of antibody nitrogen per ml) in 6 of 10 mice after the third injection. The addition of the Hib-HCH conjugate to the Pn6A-TT conjugate increased the anti-Pn6A response with a higher geometric mean antibody titer, and 9 of 10 mice responded after the third injection. A preparation of diphtheria toxoid, TT, and pertussis vaccine increased the anti-Hib antibody levels after the first injection only in mice receiving Hib-TT, but not in mice receiving Hib-HCH, suggesting that additional carrier protein (TT) enhanced the anti-polysaccharide response. Simultaneous injection of Hib and Pn6A conjugates with the same or different carriers resulted in an enhanced serum antibody response to each polysaccharide. The anti-tetanus toxin response reached protective levels (>0.01 U/ml) in most mice after the first injection and in all mice after the second and third injections of TT conjugates. A progressive increase in the anti-HCH response with each additional injection was noted in animals receiving HCH conjugates. Animals receiving the diphtheria toxoid-TT-pertussis vaccine preparation responded with a greater increase in anti-carrier antibody than those receiving the conjugates alone. This method of synthesis provided conjugates capable of inducing protective levels of antibodies to both the polysaccharides and carrier proteins.     

Artificial Salmonella vaccines: Salmonella typhimurium O-antigen-specific oligosaccharide-protein conjugates elicit opsonizing antibodies that enhance phagocytosis.

Outbred NMRI mice and rabbits were vaccinated with different artificial Salmonella typhimurium immunogens and the specificity and activity of elicited antibodies were studied in in vivo and in vitro phagocytosis assays. The Salmonella immunogens used were: (i) the synthetic disaccharide, abequose (formula see text) D-mannose, representative of Salmonella O antigen 4, covalently linked to bovine serum albumin (BSA); (ii) the octa- and dodecasaccharides, (formula see text) covalently linked to BSA; and (iii) whole heat-killed Salmonella. Rabbit antibodies passively administered to mice significantly enhanced the clearance of intravenously injected S. typhimurium challenge bacteria from the bloodstream. The clearance rate and the titer of anti-O-antigen-specific antibodies correlated. The clearance rate of an S. thompson (O6,7) strain, which has a different O antigen, was the same irrespective of the rabbit serum given. NMRI mice actively immunized with the various oligosaccharide-BSA conjugates had a significantly increased clearance rate of S. typhimurium only. In the in vitro assay, mouse antioligosaccharide-BSA sera promoted phagocytosis of S. typhimurium, but not S. thompson, when incubated with complement and mouse peritoneal exudate cells activated with Freund complete adjuvant.     

Peptide mimics of two pneumococcal capsular polysaccharide serotypes (6B and 9V) protect mice from a lethal challenge with Streptococcus pneumoniae

Anti‐polysaccharide immunity is a key facet of protection against several bacterial pathogens. Problems exist with current polysaccharide vaccines and alternative strategies that deliver a protective response are needed. We have identified immunological peptide mimics of type 6B and 9V pneumococcal capsular polysaccharides that could be used as vaccine antigens. Peptides mimicking antigenic properties of serotype 6B capsular polysaccharide were obtained from a phage‐displayed peptide library expressing dodecameric peptides, using a human monoclonal antibody (Db3G9). A murine monoclonal antibody (206, F‐5) against the serotype 9V capsular polysaccharide identified three peptide mimotopes from the dodecameric peptide library and one from a random pentadecameric peptide library. In ELISA, binding of 206, F‐5 and Db3G9 to phage displaying the selected mimotopes was significantly inhibited by type‐specific pneumococcal polysaccharide. Peptides were conjugated to keyhole limpet haemocyanin and were used to immunise mice. Two peptides, MP13 and MP7, induced specific anti‐6B and 9V polysaccharide antibodies, respectively. Mice immunised with MP7‐keyhole limpet hemocyanin or MP13‐keyhole limpet hemocyanin conjugate were significantly and specifically protected against a lethal challenge with pneumococci of the appropriate serotype. This study provides strong in vivo evidence that peptide mimics are alternatives to polysaccharide vaccines.     

Immunogenicity of conjugate vaccines consisting of pneumococcal capsular polysaccharide types 6B, 14, 19F, and 23F and a meningococcal outer membrane protein complex.

In an effort to prepare pneumococcal (Pn) capsular polysaccharide (Ps) vaccines that would be immunogenic in infants, covalent conjugates were prepared for Pn types 6B, 14, 19F, and 23F. Each Ps type was covalently bound to an outer membrane protein complex from Neisseria meningitidis serogroup B and evaluated for immunogenicity in mice and infant monkeys. The conjugates induced specific anti-Ps antibody responses in mice and in infant rhesus and African green monkeys; a conjugate of 6B and outer membrane protein complex was immunogenic at Ps doses as low as 20 ng. Although low levels of the Pn group-common cell wall polysaccharide were present in all type-specific Ps preparations, anti-cell wall polysaccharide responses induced by covalent conjugates were < 1% of the total anti-Ps response after two doses of vaccine. In contrast, the anti-cell wall polysaccharide response of a noncovalent conjugate represented 41% of the anti-Ps response after two doses. Relative T-cell dependence, a requirement for the human target population of infants less than 18 months old, was demonstrated for all four Pn Ps conjugates in an athymic mouse model. Therefore, these Pn Ps-outer membrane protein complex conjugate vaccines are excellent candidates for evaluation in human infants.