Identification of a human monoclonal antibody to replace equine diphtheria antitoxin for treatment of diphtheria intoxication.

Diphtheria antitoxin (DAT) has been the cornerstone of the treatment of Corynebacterium diphtheriae infection for more than 100 years. Although the global incidence of diphtheria has declined steadily over the last quarter of the 20th century, the disease remains endemic in many parts of the world, and significant outbreaks still occur. DAT is an equine polyclonal antibody that is not commercially available in the United States and is in short supply globally. A safer, more readily available alternative to DAT would be desirable. In the current study, we obtained human monoclonal antibodies (hMAbs) directly from antibody-secreting cells in the circulation of immunized human volunteers. We isolated a panel of diverse hMAbs that recognized diphtheria toxoid, as well as a variety of recombinant protein fragments of diphtheria toxin. Forty-five unique hMAbs were tested for neutralization of diphtheria toxin in in vitro cytotoxicity assays with a 50% effective concentration of 0.65 ng/ml for the lead candidate hMAb, 315C4. In addition, 25 µg of 315C4 completely protected guinea pigs from intoxication in an in vivo lethality model, yielding an estimated relative potency of 64 IU/mg. In comparison, 1.6 IU of DAT was necessary for full protection from morbidity and mortality in this model. We further established that our lead candidate hMAb binds to the receptor-binding domain of diphtheria toxin and physically blocks the toxin from binding to the putative receptor, heparin-binding epidermal growth factor-like growth factor. The discovery of a specific and potent human neutralizing antibody against diphtheria toxin holds promise as a potential therapeutic.

[Development of monoclonal latex diagnostic kit for diphtheria toxin and toxoid detection].

Latex diagnostic kit with high specificity and sensitivity of diphtheria toxin and toxoid detection has been developed on the basis of protective monoclonal antibodies to diphtheria toxin and polyacrolein microspheres. Diagnostic kit was stable during 1 year of shelf-life (time of the study).

Collaborative study for establishment of the European Pharmacopoeia BRP batch 1 for diphtheria toxin.

A stable liquid candidate Biological Reference Preparation (BRP) for diphtheria toxin was prepared in peptone buffer (nominal content of diphtheria toxin: 1 Lf/ml, 0.4 micro g/ml), filled in ampoules (filling volume: 1 ml) and characterised in a collaborative study. The toxin is to be used in the test "Absence of toxin and irreversibility of toxoid" as described in the current European Pharmacopoeia (Ph. Eur.) monograph Diphtheria Vaccine (Adsorbed) (2002:0443). Eleven laboratories assessed the specific activity of the preparation by in vivo and in vitro assays. The material is assumed to have satisfactory stability with a calculated predicted loss of activity of <1% per year at 4-8 degrees C. From the collaborative study, the specific activity was calculated as 77.6 (45-113) LD( 50)/ml (lethal challenge) and >75 000 Lr/Lf (intradermal challenge). The candidate BRP was successfully used in nine laboratories and confirmed suitable for use in the Vero cell test for "Absence of toxin and irreversibility of toxoid" as described in the Ph. Eur. monograph 2002:0443; i.e., concentrations of 5 x 10( -5) Lf/ml and below caused cytotoxic effects in the Vero cell test. Due to its liquid nature, the stability of the material will be monitored at regular intervals and preparation of a stable freeze-dried formulation will be considered for long-term use. Additional studies will be performed to confirm suitability of this BRP for other applications. The candidate BRP was adopted as the Ph. Eur. reference material for Diphtheria Toxin Batch 1 by the Ph. Eur. Commission at its session in March 2003.

Employment of toxins bound to porous silica beads for isolation of pure antibodies by immunoadsorption.

A simple technique for the isolation of pure antibodies by use of bacterial toxins or toxoids bound to porous silica beads "Spherosil" is described. Employment of toxins instead of toxoids has the advantage of resulting higher yields of purified antibodies.

Immunity to tetanus and diphtheria in various age groups of the Polish population.

The concentration of tetanus and diphtheria antitoxins was determined in 279 sera of 18 to 81-year-old women and 509 sera of 22 to 46-year-old men. Tetanus antitoxin content was determined also in 246 samples of fluid from human placentas and 428 lots of commercial human normal immunoglobulins. Immunity to tetanus was clearly age-dependent: in younger age groups the percentage of immunized persons amounted to 90%-100%, in middle-age groups to about 80%, and at age above 60 to about 25%. The findings are in agreement with age-dependent incidence of tetanus, which has become now in Poland a disease primarily of older people. Two groups of the population are now protected against diphtheria: a younger group under 20, and an older one above 40. Immunity in younger age groups was induced by artificial immunization, and among older persons by natural immunization through contact with diphtheria bacilli during severe diphtheria epidemics in the past. Between these groups, gaps exist including 20-30 years of age, who are sensitive to diphtheria.

Susceptibility to diphtheria.

Schick tests and antitoxin titrations have been carried out to investigate what percentage of individuals in each of 9 small groups were susceptible to diphtheria. Overall, 33% were susceptible: 26% among those below and 44% among those above 35 years of age. These results suggest that about 35% of the population of the United Kingdom are susceptible to diphtheria.

Passive transfer of antibodies of maternal origin from blood to cerebrospinal fluid in infants.

The demonstration that specific IgM antibodies are present in the serum of infants is useful in the diagnosis of several congenital infections. However, it is less certain whether the detection of antibodies in cerebrospinal fluid (c.s.f.) of infants indicates congenital infection of the central nervous system, because the origins of such antibodies have not been established. In the present study diphtheria and tetanus antitoxins of maternal origin have been detected both in the serum and in the c.s.f. of infants. These observations suggest that an important source of immunoglobulins in c.s.f. is passive transfer of antibodies from serum which should be considered in interpreting serological studies with c.s.f.

Proteolytic degradation of exocrine and serum immunoglobulins.

The susceptibility of exocrine and serum immunoglobulins and antibodies to proteolytic degradation was assessed. Colostral and duodenal fluid exocrine 11S IgA, monomeric serum IgA, and IgG were digested with trypsin, chymotrypsin, or duodenal fluid. Exocrine IgA was more resistant to digestion than were the serum immunoglobulins. Under conditions of the experiments, most of colostral IgA retained its 11S quaternary structure, including the secretory piece; the portion degraded was reduced almost entirely to peptides. The superior resistance of exocrine IgA was also demonstrated by digestion of serum IgG and nasal exocrine IgA diphtheria antitoxins with trypsin or duodenal fluid. Selective precipitation of trypsin-digested antitoxins with antibodies to heavy chains, light chains, or secretory piece revealed that the differences in susceptibility to digestion were due to differences in lability of the Fc portions of the IgA and IgG antibody molecules. The Fc portions of IgG antibody molecules were degraded or cleaved from the Fab units of the molecules, whereas the Fc-like portions of IgA antibody molecules remained associated with their Fab-like units and the secretory piece. On the other hand, trypsin treatment did not affect the antigen binding ability of the Fab parts of either the exocrine IgA or IgG antibodies. The Fc-like portions of exocrine IgA may be protected from tryptic degradation by the quaternary structure of the 11S molecules, which includes a dimer of 7S IgA subunits and the secretory piece.