Analgesia by Deletion of Spinal Neurokinin 1 Receptor Expressing Neurons Using a Bioengineered Substance P-Pseudomonas Exotoxin Conjugate.

Abstract: Cell deletion approaches to pain directed at either the primary nociceptive afferents or second-order neurons are highly effective analgesic manipulations. Second-order spinal neurons expressing the neurokinin 1 (NK1) receptor are required for the perception of many types of pain. To delete NK1+ neurons for the purpose of pain control, we generated a toxin­peptide conjugate using DTNB-derivatized (Cys0) substance P (SP) and a N-terminally truncated Pseudomonas exotoxin (PE35) that retains the endosome-release and ADP-ribosylation enzymatic domains but with only one free sulfhydryl side chain for conjugation. This allowed generation of a one-to-one product linked by a disulfide bond (SP-PE35). In vitro, Chinese hamster ovary cells stably transfected with the NK1 receptor exhibited specific cytotoxicity when exposed to SP-PE35 (IC50 = 5 × 10−11 M), whereas the conjugate was nontoxic to NK2 and NK3 receptor-bearing cell lines. In vivo studies showed that, after infusion into the spinal subarachnoid space, the toxin was extremely effective in deleting NK1 receptor-expressing cells from the dorsal horn of the spinal cord. The specific cell deletion robustly attenuated thermal and mechanical pain sensations and inflammatory hyperalgesia but did not affect motoric capabilities. NK1 receptor cell deletion and antinociception occurred without obvious lesion of non­receptor-expressing cells or apparent reorganization of primary afferent innervation. These data demonstrate the extraordinary selectivity and broad-spectrum antinociceptive efficacy of this ligand-directed protein therapeutic acting via receptor-mediated endocytosis. The loss of multiple pain modalities including heat and mechanical pinch, transduced by different populations of primary afferents, shows that spinal NK1 receptor-expressing neurons are critical points of convergence in the nociceptive transmission circuit. We further suggest that therapeutic end points can be effectively and safely achieved when SP-PE35 is locally infused, thereby producing a regionally defined analgesia.

A view of the E2-CD81 interface at the binding site of a neutralizing antibody against hepatitis C virus.

UNLABELLED: Hepatitis C virus (HCV) glycoprotein E2 is considered a major target for generating neutralizing antibodies against HCV, primarily due to its role of engaging host entry factors, such as CD81, a key cell surface protein associated with HCV entry. Based on a series of biochemical analyses in combination with molecular docking, we present a description of a potential binding interface formed between the E2 protein and CD81. The virus side of this interface includes a hydrophobic helix motif comprised of residues W(437)LAGLF(442), which encompasses the binding site of a neutralizing monoclonal antibody, mAb41. The helical conformation of this motif provides a structural framework for the positioning of residues F442 and Y443, serving as contact points for the interaction with CD81. The cell side of this interface likewise involves a surface-exposed hydrophobic helix, namely, the D-helix of CD81, which coincides with the binding site of 1D6, a monoclonal anti-CD81 antibody known to block HCV entry. Our illustration of this virus-host interface suggests an important role played by the W(437)LAGLF(442) helix of the E2 protein in the hydrophobic interaction with the D-helix of CD81, thereby facilitating our understanding of the mechanism for antibody-mediated neutralization of HCV. IMPORTANCE: Characterization of the interface established between a virus and host cells can provide important information that may be used for the control of virus infections. The interface that enables hepatitis C virus (HCV) to infect human liver cells has not been well understood because of the number of cell surface proteins, factors, and conditions found to be associated with the infection process. Based on a series of biochemical analyses in combination with molecular docking, we present such an interface, consisting of two hydrophobic helical structures, from the HCV E2 surface glycoprotein and the CD81 protein, a major host cell receptor recognized by all HCV strains. Our study reveals the critical role played by hydrophobic interactions in the formation of this virus-host interface, thereby contributing to our understanding of the mechanism for antibody-mediated neutralization of HCV.

Amino acid residue-specific neutralization and nonneutralization of hepatitis C virus by monoclonal antibodies to the E2 protein.

Antibodies to epitopes in the E2 protein of hepatitis C virus (HCV) reduce the viral infectivity in vivo and in vitro. However, the virus can persist in patients in the presence of neutralizing antibodies. In this study, we generated a panel of monoclonal antibodies that bound specifically to the region between residues 427 and 446 of the E2 protein of HCV genotype 1a, and we examined their capacity to neutralize HCV in a cell culture system. Of the four monoclonal antibodies described here, two were able to neutralize the virus in a genotype 1a-specific manner. The other two failed to neutralize the virus. Moreover, one of the nonneutralizing antibodies could interfere with the neutralizing activity of a chimpanzee polyclonal antibody at E2 residues 412 to 426, as it did with an HCV-specific immune globulin preparation, which was derived from the pooled plasma of chronic hepatitis C patients. Mapping the epitope-paratope contact interfaces revealed that these functionally distinct antibodies shared binding specificity for key amino acid residues, including W(437), L(438), L(441), and F(442), within the same epitope of the E2 protein. These data suggest that the effectiveness of antibody-mediated neutralization of HCV could be deduced from the interplay between an antibody and a specific set of amino acid residues. Further understanding of the molecular mechanisms of antibody-mediated neutralization and nonneutralization should provide insights for designing a vaccine to control HCV infection in vivo.

Parvovirus B19 infection transmitted by transfusion of red blood cells confirmed by molecular analysis of linked donor and recipient samples.

BACKGROUND: Extremely high viremic levels of parvovirus B19 (B19V) can be found in acutely infected, but asymptomatic donors. However, reports of transmission by single-donor blood components are rare. In this prospective study, paired donor-recipient samples were used to investigate the transfusion risk. STUDY DESIGN AND METHODS: Posttransfusion plasma or blood samples from recipients were tested for B19V DNA by polymerase chain reaction, generally at 4 and 8 weeks, and for anti-B19V immunoglobulin (Ig)G by enzyme immunoassay, at 12 and 24 weeks. To rule out infection unrelated to transfusion, pretransfusion samples and linked donor's samples for each B19V DNA-positive recipient were assayed for B19V DNA and anti-B19V IgG and IgM. To confirm transmission, sequencing and phylogenetic analysis were performed. RESULTS: A total of 14 of 869 (1.6%) recipients were B19V DNA positive, but only 1 of 869 (0.12%; 95% confidence interval, 0.0029%-0.6409%) was negative for B19V DNA and anti-B19V IgG before transfusion and seroconverted posttransfusion. This newly infected patient received 5 × 10(10) IU B19V DNA in one red blood cell (RBC) unit from an acutely infected anti-B19V-negative donor in addition to RBCs from three other donors that cumulatively contained 1320 IU of anti-B19V IgG. DNA sequencing and phylogenetic analysis showed that sequences from the linked donor and recipient were identical (Genotype 1), thus establishing transfusion transmission. CONCLUSIONS: The 0.12% transmission rate documented here, although low, could nonetheless result in hundreds or thousands of infections annually in the United States based on calculated confidence limits. Although most would be asymptomatic, some could have severe clinical outcomes, especially in neonates and those with immunocompromised or hemolytic states.

Antibody-mediated synergy and interference in the neutralization of SARS-CoV at an epitope cluster on the spike protein.

Incomplete neutralization of virus, especially when it occurs in the presence of excess neutralizing antibody, represents a biological phenomenon that impacts greatly on antibody-mediated immune prophylaxis of viral infection and on successful vaccine design. To understand the mechanism by which a virus escapes from antibody-mediated neutralization, we have investigated the interactions of non-neutralizing and neutralizing antibodies at an epitope cluster on the spike protein of severe acute respiratory syndrome coronavirus (SARS-CoV). The epitope cluster was mapped at the C-terminus of the spike protein; it consists of structurally intertwined epitopes recognized by two neutralizing monoclonal antibodies (mAbs), 341C and 540C, and a non-neutralizing mAb, 240C. While mAb 341C binds to a mostly linear epitope composed of residues (507)PAT(509) and V(349), mAb 240C binds to an epitope that partially overlaps the former by at least two residues (P(507) and A(508)). The epitope corresponding to mAb 540C is a conformational one, involving residues L(504) and N(505). In neutralization assays, non-neutralizing 240C disrupted virus neutralization by mAb 341C and/or mAb 540C, whereas a combination of mAbs 341C and 540C blocked virus infectivity synergistically. These findings indicate that the epitope cluster on the spike protein may serve as an evolutionarily conserved platform at which a dynamic interplay between neutralizing and non-neutralizing antibodies occurs, thereby determining the outcome of SARS-CoV infection.

A simple and rapid Hepatitis A Virus (HAV) titration assay based on antibiotic resistance of infected cells: evaluation of the HAV neutralization potency of human immune globulin preparations.

BACKGROUND: Hepatitis A virus (HAV), the causative agent of acute hepatitis in humans, is an atypical Picornaviridae that grows poorly in cell culture. HAV titrations are laborious and time-consuming because the virus in general does not cause cytopathic effect and is detected by immunochemical or molecular probes. Simple HAV titration assays could be developed using currently available viral construct containing selectable markers. RESULTS: We developed an antibiotic resistance titration assay (ARTA) based on the infection of human hepatoma cells with a wild type HAV construct containing a blasticidin (Bsd) resistance gene. Human hepatoma cells infected with the HAV-Bsd construct survived selection with 2 microg/ml of blasticidin whereas uninfected cells died within a few days. At 8 days postinfection, the color of the pH indicator phenol red in cell culture media correlated with the presence of HAV-Bsd-infected blasticidin-resistant cells: an orange-to-yellow color indicated the presence of growing cells whereas a pink-to-purple color indicated that the cells were dead. HAV-Bsd titers were determined by an endpoint dilution assay based on the color of the cell culture medium scoring orange-to-yellow wells as positive and pink-to-purple wells as negative for HAV. As a proof-of-concept, we used the ARTA to evaluate the HAV neutralization potency of two commercially available human immune globulin (IG) preparations and a WHO International Standard for anti-HAV. The three IG preparations contained comparable levels of anti-HAV antibodies that neutralized approximately 1.5 log of HAV-Bsd. Similar neutralization results were obtained in the absence of blasticidin by an endpoint dilution ELISA at 2 weeks postinfection. CONCLUSION: The ARTA is a simple and rapid method to determine HAV titers without using HAV-specific probes. We determined the HAV neutralization potency of human IG preparations in 8 days by ARTA compared to the 14 days required by the endpoint dilution ELISA. The ARTA reduced the labour, time, and cost of HAV titrations making it suitable for high throughput screening of sera and antivirals, determination of anti-HAV antibodies in human immune globulin preparations, and research applications that involve the routine evaluation of HAV titers.

A neutralization epitope in the hepatitis C virus E2 glycoprotein interacts with host entry factor CD81.

The identification of a specific immunogenic candidate that will effectively activate the appropriate pathway for neutralizing antibody production is fundamental for vaccine design. By using a monoclonal antibody (1H8) that neutralizes HCV in vitro, we have demonstrated here that 1H8 recognized an epitope mapped between residues A524 and W529 of the E2 protein. We also found that the epitope residues A524, P525, Y527 and W529 were crucial for antibody binding, while the residues T526, Y527 and W529 within the same epitope engaged in the interaction with the host entry factor CD81. Furthermore, we detected "1H8-like" antibodies, defined as those with amino acid-specificity similar to 1H8, in the plasma of patients with chronic HCV infection. The time course study of plasma samples from Patient H, a well-characterized case of post-transfusion hepatitis C, showed that "1H8-like" antibodies could be detected in a sample collected almost two years after the initial infection, thus confirming the immunogenicity of this epitope in vivo. The characterization of this neutralization epitope with a function in host entry factor CD81 interaction should enhance our understanding of antibody-mediated neutralization of HCV infections.

Measles-virus-neutralizing antibodies in intravenous immunoglobulins.

Measles infection induces lifelong immunity; however, wild-type infection stimulates higher levels of measles-virus-neutralizing antibodies (mnAbs) than does vaccination. Because the proportion of the donor population with vaccine-induced measles immunity is increasing, this study was conducted to determine whether this shift in demographic characteristics affects mnAb levels in contemporary lots of Immune Globulin Intravenous (Human) (IGIV). When 166 lots of 7 IGIV products manufactured between 1998 and 2003 were assayed by plaque-reduction neutralization test, there was a progressive decrease in geometric mean titers in lots manufactured between 1999 and 2002. IGIV products manufactured from recovered plasma had significantly higher titers than did those manufactured from Source Plasma, which could reflect a change in donor demographic characteristics, because Source Plasma donors tend to be much younger. A reduction in mnAbs also correlated with the loss of either IgG1 and IgG3, possibly because of certain manufacturing procedures, or bivalent antibodies (i.e., intact IgG and F(ab')2), because of fragmentation.