Human monoclonal antibody HCV1 effectively prevents and treats HCV infection in chimpanzees.

Hepatitis C virus (HCV) infection is a leading cause of liver transplantation and there is an urgent need to develop therapies to reduce rates of HCV infection of transplanted livers. Approved therapeutics for HCV are poorly tolerated and are of limited efficacy in this patient population. Human monoclonal antibody HCV1 recognizes a highly-conserved linear epitope of the HCV E2 envelope glycoprotein (amino acids 412-423) and neutralizes a broad range of HCV genotypes. In a chimpanzee model, a single dose of 250 mg/kg HCV1 delivered 30 minutes prior to infusion with genotype 1a H77 HCV provided complete protection from HCV infection, whereas a dose of 50 mg/kg HCV1 did not protect. In addition, an acutely-infected chimpanzee given 250 mg/kg HCV1 42 days following exposure to virus had a rapid reduction in viral load to below the limit of detection before rebounding 14 days later. The emergent virus displayed an E2 mutation (N415K/D) conferring resistance to HCV1 neutralization. Finally, three chronically HCV-infected chimpanzees were treated with a single dose of 40 mg/kg HCV1 and viral load was reduced to below the limit of detection for 21 days in one chimpanzee with rebounding virus displaying a resistance mutation (N417S). The other two chimpanzees had 0.5-1.0 log(10) reductions in viral load without evidence of viral resistance to HCV1. In vitro testing using HCV pseudovirus (HCVpp) demonstrated that the sera from the poorly-responding chimpanzees inhibited the ability of HCV1 to neutralize HCVpp. Measurement of antibody responses in the chronically-infected chimpanzees implicated endogenous antibody to E2 and interference with HCV1 neutralization although other factors may also be responsible. These data suggest that human monoclonal antibody HCV1 may be an effective therapeutic for the prevention of graft infection in HCV-infected patients undergoing liver transplantation.

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.

Treatment with monoclonal antibodies against Clostridium difficile toxins.

BACKGROUND: New therapies are needed to manage the increasing incidence, severity, and high rate of recurrence of Clostridium difficile infection. METHODS: We performed a randomized, double-blind, placebo-controlled study of two neutralizing, fully human monoclonal antibodies against C. difficile toxins A (CDA1) and B (CDB1). The antibodies were administered together as a single infusion, each at a dose of 10 mg per kilogram of body weight, in patients with symptomatic C. difficile infection who were receiving either metronidazole or vancomycin. The primary outcome was laboratory-documented recurrence of infection during the 84 days after the administration of monoclonal antibodies or placebo. RESULTS: Among the 200 patients who were enrolled (101 in the antibody group and 99 in the placebo group), the rate of recurrence of C. difficile infection was lower among patients treated with monoclonal antibodies (7% vs. 25%; 95% confidence interval, 7 to 29; P<0.001). The recurrence rates among patients with the epidemic BI/NAP1/027 strain were 8% for the antibody group and 32% for the placebo group (P=0.06); among patients with more than one previous episode of C. difficile infection, recurrence rates were 7% and 38%, respectively (P=0.006). The mean duration of the initial hospitalization for inpatients did not differ significantly between the antibody and placebo groups (9.5 and 9.4 days, respectively). At least one serious adverse event was reported by 18 patients in the antibody group and by 28 patients in the placebo group (P=0.09). CONCLUSIONS: The addition of monoclonal antibodies against C. difficile toxins to antibiotic agents significantly reduced the recurrence of C. difficile infection. (ClinicalTrials.gov number, NCT00350298.)

Cryptosporidium species: new insights and old challenges.

Cryptosporidium species are protozoan parasites that cause mainly enteric illnesses in humans and other animals. The mode of transmission is most commonly waterborne, but other sources of infection, including foodborne and person-to-person spread, have been documented. The environmental form of the parasite is resistant to most water purification methods, including chlorination. Cryptosporidium infection usually causes a self-limited diarrheal illness but can be life-threatening in immunocompromised individuals. There is no effective therapy for cryptosporidiosis.

Serum anti-toxin B antibody correlates with protection from recurrent Clostridium difficile infection (CDI).

BACKGROUND: Previous studies have demonstrated a correlation between Clostridium difficile anti-toxin A serum antibodies and protection against symptomatic disease and recurrence. METHODS: A neutralizing monoclonal antibody to C. difficile toxin A (CDA1) developed by MBL and Medarex, Inc. was studied in a phase II, randomized, double-blind, placebo-controlled trial in patients receiving standard of care treatment for C. difficile infection (CDI). Twenty-nine subjects received a single intravenous infusion of 10mg/kg CDA1 and 17 subjects received placebo and were evaluated for recurrence of CDI during the 56-day study period. Serum antibodies against C. difficile toxin A and B were measured by ELISA and cytotoxicity assay at various time points before and after infusion. FINDINGS: CDI recurrence occurred in 5 of 29 (17%) in the CDA1 group and 3 of 17 (18%) (p=NS) in the placebo group with a trend toward delay in time to recurrence in the group treated with CDA1. The geometric mean concentration of antibody to an epitope of the receptor-binding domain of toxin B (0.300 and 1.20microg/ml, respectively; p=0.02) and geometric mean titer of neutralizing B antibody (8.00 and 100, respectively; p=0.02) at study day 28 were lower for those subjects with recurrence compared to those who did not recur. In addition, a significantly greater proportion of subjects who recurred were infected with the epidemic BI/NAP1/027 strain compared with those that did not recur (88% vs. 22%; p=0.002). Finally, in a multiple logistic regression analysis neutralizing anti-toxin B at day 14 (p<0.001), anti-toxin A at day 28 (p<0.001) and infection with the BI/NAP1/027 strain at enrollment (p=0.002) were all predictive of CDI recurrence. INTERPRETATION: In this prospective study, lower concentrations of neutralizing anti-toxin B and anti-toxin A antibody and infection with the BI/NAP1/027 strain of C. difficile were significantly associated with recurrence of CDI.

Quantification of in vitro and in vivo Cryptosporidium parvum infection by using real-time PCR.

Established methods for quantifying experimental Cryptosporidium infection are highly variable and subjective. We describe a new technique using quantitative real-time PCR (qPCR) that can be used to measure in vitro and in vivo laboratory infections with Cryptosporidium. We show for the first time that qPCR permits absolute quantification of the parasite while simultaneously controlling for the amount of host tissue and correlates significantly with established methods of quantification in in vitro and in vivo laboratory models of infection.

An early intestinal mucosal source of gamma interferon is associated with resistance to and control of Cryptosporidium parvum infection in mice.

Resistance to and control of Cryptosporidium parvum infection in mice in the absence of adaptive immunity appears to be gamma interferon (IFN-gamma) dependent. Using an IFN-gamma-neutralizing antibody in a murine model, we demonstrated increased susceptibility to infection within 24 h. We correlated this early resistance and control with increased mucosal expression of IFN-gamma and demonstrate that CD8+ T-cell receptor alphabeta intestinal intraepithelial lymphocytes express and secrete this cytokine shortly after infection. The rapid kinetics of IFN-gamma expression and secretion by naive CD8+ T cells in response to a protozoan pathogen have not previously been demonstrated.

Analysis of sequence diversity at the highly polymorphic Cpgp40/15 locus among Cryptosporidium isolates from human immunodeficiency virus-infected children in South Africa.

Cryptosporidium sp. is a significant cause of diarrheal disease, particularly in human immunodeficiency virus (HIV)-infected patients in developing countries. We recently cloned and sequenced several alleles of the highly polymorphic single-copy Cryptosporidium parvum gene Cpgp40/15. This gene encodes a precursor protein that is proteolytically cleaved to yield mature cell surface glycoproteins gp40 and gp15, which are implicated in zoite attachment to and invasion of enterocytes. The most-striking feature of the Cpgp40/15 alleles and proteins is their unprecedented degree of sequence polymorphism, which is far greater than that observed for any other gene or protein studied in C. parvum to date. In this study we analyzed nucleic acid and amino acid sequence polymorphism at the Cpgp40/15 locus of 20 C. parvum isolates from HIV-infected South African children. Fifteen isolates exhibited one of four previously identified genotype I alleles at the Cpgp40/15 locus (Ia, Ib, Ic, and Id), while five displayed a novel set of polymorphisms that defined a new Cpgp40/15 genotype I allele, designated genotype Ie. Surprisingly, only 15 of these isolates exhibited concordant type I alleles at the thrombospondin-related adhesive protein of Cryptosporidium and Cryptosporidium oocyst wall protein loci, while five isolates (all of which displayed Cpgp40/15 genotype Ic alleles) displayed genotype II alleles at these loci. Furthermore, the last five isolates also manifested chimeric genotype Ic/Ib or Ic/II alleles at the Cpgp40/15 locus, raising the possibility of sexual recombination within and between prototypical parasite genotypes. Lastly, children infected with isolates having genotype Ic alleles were significantly older than those infected with isolates displaying other genotype I alleles.