Arthur Karpas

Phase 1 and phase 2 studies of Salmonella enterica serovar Paratyphi A O-specific polysaccharide-tetanus toxoid conjugates in adults, teenagers, and 2- to 4-year-old children in Vietnam.

ABSTRACT Salmonella enterica serovar Paratyphi A O-specific polysaccharide (O-SP) was activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) and bound to tetanus toxoid (TT) with adipic acid dihydrazide as a linker (SPA-TT1) or directly (SPA-TT2). In mice, these two conjugates elicited high levels of immunoglobulin G (IgG) anti-lipopolysaccharide (LPS) in serum with bactericidal activity (E. Konadu, J. Shiloach, D. A. Bryla, J. B. Robbins, and S. C. Szu, Infect. Immun. 64:2709–2715, 1996). The safety and immunogenicity of the two conjugates were then evaluated sequentially in Vietnamese adults, teenagers, and 2- to 4-year-old children. None of the vaccinees experienced significant side effects, and all had preexisting LPS antibodies. At 4 weeks after injection, there were significant increases of the geometric mean IgG and IgM anti-LPS levels in the adults and teenagers: both conjugates elicited a greater than fourfold rise in the IgG anti-LPS level in serum in ≥80% of the volunteers. SPA-TT2 elicited slightly higher, though not statistically significantly, levels of IgG anti-LPS than did SPA-TT1 in these age groups. Accordingly, only SPA-TT2 was evaluated in the 2- to 4-year-old children. On a random basis, one or two injections were administered 6 weeks apart to the children. No significant side effects were observed, and the levels of preexisting anti-LPS in serum were similar in children of all ages. A significant rise in the IgG anti-LPS titer was elicited by the first injection (P = 0.0001); a second injection did not elicit a booster response. Representative sera from all groups had bactericidal activity that could be adsorbed by S. enterica serovar Paratyphi A LPS.

An avidin-biotin based ELISA for quantitation of antibody to bacterial polysaccharides

A solid phase immunoassay utilizing avidin-biotin binding has been developed for measuring anticapsular polysaccharide antibodies. Capsular polysaccharides of Escherichia coli K1, Haemophilus influenzae type b, Staphylococcus aureus types 5 and 8, and levan from Aerobacter levanicum have been biotinylated through -OH or COOH groups with retention of antigenicity. Polysaccharides were immobilized on avidin-coated microtiter wells for use in an enzyme-linked immunosorbent assay (ELISA) to detect antibody. Two preparations of biotinylated S. aureus type 8 polysaccharide were equivalent as antigens in ELISA. Specificity was demonstrated by absorption of antisera, by competitive inhibition with purified antigens, and by reaction with specific monoclonal or myeloma antibodies. Reproducibility of the assay for H. influenzae type b and S. aureus type 8 antibody was demonstrated by replicate titrations of high and low level antisera.

Safety and Immunogenicity of Shigella sonnei and Shigella flexneri 2a O-Specific Polysaccharide Conjugates in Children

O-specific polysaccharide conjugates of shigellae were safe and immunogenic in young adults, and a Shigella sonnei conjugate conferred protection [1-3]. Shigellosis is primarily a disease of children; therefore, the safety and immunogenicity of S. sonnei and Shigella flexneri 2a conjugates were studied in 4- to 7-year-old children. Local and systemic reactions were minimal. The first injection of both conjugates elicited significant rises in geometric mean levels of serum IgG only to the homologous lipopolysaccharide (LPS) (S. sonnei, 0.32-8.25 ELISA units [EU]; S. flexneri 2a, 1.15-20.5 EU; P<.0001). Revaccination at 6 weeks induced a booster response to S. flexneri 2a LPS (20.5-30.5 EU, P=.003). Six months later, the geometric mean levels of IgG anti-LPS for both groups were higher than the prevaccination levels (P<.0001). Similar, but lesser, rises were observed for IgM and IgA anti-LPS. The investigational Shigella conjugates were safe and immunogenic in children and merit evaluation of their efficacy.

Phase 1 Study of a Recombinant Mutant Protective Antigen of Bacillus anthracis

ABSTRACT A phase 1 study of a recombinant mutant protective antigen (rPA) vaccine was conducted in 186 healthy adults aged 18 to 45 years. Volunteers were randomized to receive one of three formulations of rPA (formalin treated, alum adsorbed, or both), in 10- or 20-μg dosages each, or the licensed vaccine, AVA. Three injections were given at 2-month intervals and a 4th 1 year after the 3rd. Vaccinees were examined at the clinic once following each injection, at 48 to 72 h postinjection. Adverse reactions were recorded in diaries for 7 days. Sera were collected before each injection and 1 week after the 1st, 2 weeks after the 3rd and 4th, and 1 year after the 4th. Serum anti-PA IgG was assayed by enzyme-linked immunosorbent assay (ELISA) and toxin neutralization assay (TNA). All formulations at both dosages were safe and immunogenic, inducing booster responses, with the highest antibody levels following the 4th injection (354 to 732 μg/ml). The lowest levels were induced by the formalin-only-treated rPA; there was no statistical difference between levels induced by alum-adsorbed and formalin-treated/alum-adsorbed rPA or by the two dosages. The antibody levels declined in all groups during the 1-year intervals after the 3rd and 4th injections but less so during the 2nd year, after the 4th injection (fold decreases were 10 to 25 versus 3.4 to 7.0, P < 0.001). There were too few AVA recipients for statistical comparisons, but their antibody levels followed those of rPA. Anti-rPA measured by ELISA correlated with TNA titers (r = 0.97). These data support studying alum-adsorbed rPA in children.

Of four murine, anti-Shigella itdysenteriae type 1 O-polysaccharide antibodies, three employ V-genes that differ extensively from those of the fourth

Three murine, monoclonal antibodies, IgM 5286 F2, IgM 5297 C1, and IgG 5338 H4 were generated against Shigella dysenteriae type 1 O-specific polysaccharide (O-SP)-conjugate. They are specific for the O-SP, which is a poly-[alpha-L-rhamnopyranosyl-(1-->3)-alpha-L-rhamnopyranosyl-(1-->2)-al pha-D-galactopyranosyl-(1-->3)-2-deoxy-2-amino-N-acetyl-alpha-D-glucopyr anosyl]. The VH and VL genes of these antibodies were cloned and their sequences determined. They showed 93% homology, but were quite different to the primary sequence of IgM 3707 E9, of the same O-SP-specificity, previously reported. The fine-specificities of both IgG 5338 H4 and IgM 3707 E9 were for the same disaccharide moiety in the O-SP, while IgMs 5286 F2 and 5297 C1 showed fine-specificity for the entire repeating unit of the O-SP. Therefore, divergent sequences can confer upon antibodies similar-, or even identical-carbohydrate-epitope fine-specificity. In addition, close primary sequence-homology does not preclude differences in antibody fine-specificity.

A murine antibody to Shigella dysenteriae type 1 employs V-genes that contain a rearranged codon for the λ light chain

The cDNA coding for a hybridoma anti Shigella dysenteriae type 1 antibody (3707 E9) has been cloned, and sequenced. Binding patterns with fragments of the bacterial polysaccharide antigen had already been studied in detail. The VH sequence utilizes the VH441 gene, first identified amongst beta-(1,6)galactan-binding antibodies, while the VL is closely related to the V lambda 1 gene. We found that the VL3707 E9 gene employed a VL-J combinatorial joining leading to a rare Trp-->Leu substitution at position L96.