Gregory J. Babcock

EBV Persistence in Memory B Cells In Vivo

Epstein-Barr virus establishes latency in vitro by activating human B cells to become proliferating blasts, but in vivo it is benign. In the peripheral blood, the virus resides latently in resting B cells that we now show are restricted to the sIgD memory subset. However, in tonsils the virus shows no such restriction. We propose that EBV indiscriminately infects B cells in mucosal lymphoid tissue and that these cells differentiate to become resting memory B cells that then enter the circulation. Activation to the blastoid stage of latency is an essential intermediate step in this process. Thus, EBV may persist by exploiting the mechanisms that produce and maintain long-term B cell memory.

The Expression Pattern of Epstein-Barr Virus Latent Genes In Vivo Is Dependent upon the Differentiation Stage of the Infected B Cell

Epstein-Barr virus-infected B cells in vivo demonstrate three distinct patterns of latent gene expression, depending on the differentiation stage of the cell. Tonsillar naive B cells express the EBNA2-dependent lymphoblastoid phenotype, characteristic of direct infection. Germinal center centroblasts and centrocytes as well as tonsillar memory B cells express a more restricted pattern of latent genes (EBNA1(Q-K)+, LMP1+, LMP2+, EBNA2-) that has only been seen previously in EBV-positive tumors. Peripheral memory cells express an even more restricted pattern where no latent genes are expressed, with the possible exception of LMP2. These results are consistent with a model where EBV uses the normal biology of B lymphocytes to gain access to and persist within the long-lived memory B cell compartment.

Human Monoclonal Antibodies Directed against Toxins A and B Prevent Clostridium difficile-Induced Mortality in Hamsters

ABSTRACT Clostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea, and recent outbreaks of strains with increased virulence underscore the importance of identifying novel approaches to treat and prevent relapse of Clostridium difficile-associated diarrhea (CDAD). CDAD pathology is induced by two exotoxins, toxin A and toxin B, which have been shown to be cytotoxic and, in the case of toxin A, enterotoxic. In this report we describe fully human monoclonal antibodies (HuMAbs) that neutralize these toxins and prevent disease in hamsters. Transgenic mice carrying human immunoglobulin genes were used to isolate HuMAbs that neutralize the cytotoxic effects of either toxin A or toxin B in cell-based in vitro neutralization assays. Three anti-toxin A HuMAbs (3H2, CDA1, and 1B11) could all inhibit the enterotoxicity of toxin A in mouse intestinal loops and the in vivo toxicity in a systemic mouse model. Four anti-toxin B HuMAbs (MDX-1388, 103-174, 1G10, and 2A11) could neutralize cytotoxicity in vitro, although systemic toxicity in the mouse could not be neutralized. Anti-toxin A HuMAb CDA1 and anti-toxin B HuMAb MDX-1388 were tested in the well-established hamster model of C. difficile disease. CDA1 alone resulted in a statistically significant reduction of mortality in hamsters; however, the combination treatment offered enhanced protection. Compared to controls, combination therapy reduced mortality from 100% to 45% (P < 0.0001) in the primary disease hamster model and from 78% to 32% (P < 0.0001) in the less stringent relapse model.

Ligand-independent Dimerization of CXCR4, a Principal HIV-1 Coreceptor

CXCR4, a member of the G protein-coupled receptor family of proteins, is the receptor for stromal cell-derived factor (SDF-1α) and is a principal coreceptor for human immunodeficiency virus type 1 (HIV-1). CXCR4 has also been implicated in breast cancer metastasis. We examined the ability of CXCR4 to homomultimerize in detergent-solubilized cell lysates and in the membranes of intact cells. CXCR4 was found to multimerize in cell lysates containing the detergents CHAPSO or Cymal-7 but not other detergents that have been shown to disrupt the native conformation of CXCR4. CXCR4 expression levels did not affect the observed multimerization and differentially tagged CXCR4 molecules associated only when coexpressed in the same cell. CXCR4 did not interact with CCR5, the other principal HIV-1 coreceptor, when the two proteins were coexpressed. Using bioluminescence resonance energy transfer (BRET2), we demonstrated that CXCR4 multimers are found naturally in the intact cell membrane, in both the presence and absence of multiple CXCR4 ligands. Ligand binding did not significantly alter the observed BRET2 signal, suggesting that CXCR4 exists as a constitutive oligomer. In cell lysates prepared with non-denaturing detergents, CXCR4 sedimented in a manner consistent with a dimer, whereas CCR5 sedimented as a monomer under these conditions. The stable, constitutive dimerization of CXCR4 may contribute to its biological functions in chemokine binding, signaling, and HIV-1 entry.

The role of post-translational modifications of the CXCR4 amino terminus in stromal-derived factor 1α association and HIV-1 entry

The chemokine receptor CXCR4 plays critical roles in development, immune function, and human immunodeficiency virus type 1 (HIV-1) entry. Here we demonstrate that, like the CC-chemokine receptors CCR5 and CCR2b, CXCR4 is posttranslationally modified by sulfation of its amino-terminal tyrosines. The sulfate group at tyrosine 21 contributes substantially to the ability of CXCR4 to bind its ligand, stromal derived factor 1α. Tyrosine sulfation plays a less significant role in CXCR4-dependent HIV-1 entry than in CCR5-dependent HIV-1 entry. In some cell lines, CXCR4 is efficiently modified by a chondroitin sulfate chain at serine 18, but neither HIV-1 entry nor stromal derived factor 1α binding was affected by loss of this glycosaminoglycan. These data demonstrate a functional role for tyrosine sulfate in the CXC-chemokine receptor family and underscore a general difference in HIV-1 utilization of CCR5 and CXCR4.

Is EBV Persistence In Vivo a Model for B Cell Homeostasis

We have measured the absolute numbers of EBV-infected B cells in the peripheral blood of healthy persistently infected individuals. Single measurements on a panel of 15 healthy individuals demonstrate that the frequency varies over a wide range from 1-50 per 10(6) B cells. Repeat measurements over 1-3.5 years on several individuals whose frequencies varied over a 10-fold range showed that the variation does not represent the fluctuation in the frequency that can occur within an individual; rather, the frequencies are specific to the individual. The frequency within an individual measured over time is stable and contributes less than 10% to the variance seen in the whole population. These measurements suggest that the level of EBV-infected B cells is tightly regulated and we propose that the same homeostatic mechanisms that regulate the levels of normal B cells also regulate B cells latently infected with EBV.

Amino Acids 270 to 510 of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein Are Required for Interaction with Receptor

ABSTRACT A novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), has recently been identified as the causative agent of severe acute respiratory syndrome (SARS). SARS-CoV appears similar to other coronaviruses in both virion structure and genome organization. It is known for other coronaviruses that the spike (S) glycoprotein is required for both viral attachment to permissive cells and for fusion of the viral envelope with the host cell membrane. Here we describe the construction and expression of a soluble codon-optimized SARS-CoV S glycoprotein comprising the first 1,190 amino acids of the native S glycoprotein (S1190). The codon-optimized and native S glycoproteins exhibit similar molecular weight as determined by Western blot analysis, indicating that synthetic S glycoprotein is modified correctly in a mammalian expression system. S1190 binds to the surface of Vero E6 cells, a cell permissive to infection, as demonstrated by fluorescence-activated cell sorter analysis, suggesting that S1190 maintains the biologic activity present in native S glycoprotein. This interaction is blocked with serum obtained from recovering SARS patients, indicating that the binding is specific. In an effort to map the ligand-binding domain of the SARS-CoV S glycoprotein, carboxy- and amino-terminal truncations of the S1190 glycoprotein were constructed. Amino acids 270 to 510 were the minimal receptor-binding region of the SARS-CoV S glycoprotein as determined by flow cytometry. We speculate that amino acids 1 to 510 of the SARS-CoV S glycoprotein represent a unique domain containing the receptor-binding site (amino acids 270 to 510), analogous to the S1 subunit of other coronavirus S glycoproteins.

A model for persistent infection with Epstein-Barr virus: The stealth virus of human B cells☆

Most adult humans are infected benignly and for life with the herpesvirus Epstein-Barr virus. EBV has been a focus of research because of its status as a candidate tumor virus for a number of lymphomas and carcinomas. In vitro EBV has the ability to establish a latent infection in proliferating B lymphoblasts. This is the only system available for studying human herpesvirus latency in culture and has been extremely useful for elucidating how EBV promotes cellular growth. However, to understand how EBV survives in the healthy host and what goes awry, leading to disease, it is essential to know how EBV establishes and maintains a persistent infection in vivo. Early studies on the mechanism of EBV persistence produced inconclusive and often contradictory results because the techniques available were crude and insensitive. Recent advances in PCR technology and the application of sophisticated cell fractionation techniques have now provided new insights into the behavior of the virus. Most dramatically it has been shown that EBV in vivo does not establish latency in a proliferating lymphoblast, but in a resting memory B cell. The contrasting behaviors of being able to establish a latent infection in proliferating B blasts and resting memory B cells can be resolved in terms of a model where EBV performs its complete life cycle in B lymphocytes. The virus achieves this not by disrupting normal B cell biology but by using it.

The dispersal of mucosal memory B cells: Evidence from persistent EBV infection

We have used latent infection with the human herpesvirus Epstein-Barr virus to track the dispersal of memory B cells from the mucosal lymphoid tissue of Waldeyers ring (tonsils/adenoids). EBV is evenly distributed between the memory compartments of Waldeyers ring and the peripheral blood. However, it has an approximately 20-fold higher preference for Waldeyers ring over the spleen or mesenteric lymph nodes. These observations are consistent with a model whereby the virus preferentially establishes persistent infection within memory B cells from Waldeyers ring. The virus then colonizes the entire peripheral lymphoid system, at a low level, by trafficking with these memory B cells as they circulate through the body and back to Waldeyers ring. This pathway may reflect that of normal memory B cells derived from nasopharyngeal and other mucosal lymph nodes.

Identification and Characterization of Broadly Neutralizing Human Monoclonal Antibodies Directed against the E2 Envelope Glycoprotein of Hepatitis C Virus

ABSTRACT Nearly all livers transplanted into hepatitis C virus (HCV)-positive patients become infected with HCV, and 10 to 25% of reinfected livers develop cirrhosis within 5 years. Neutralizing monoclonal antibody could be an effective therapy for the prevention of infection in a transplant setting. To pursue this treatment modality, we developed human monoclonal antibodies (HuMAbs) directed against the HCV E2 envelope glycoprotein and assessed the capacity of these HuMAbs to neutralize a broad panel of HCV genotypes. HuMAb antibodies were generated by immunizing transgenic mice containing human antibody genes (HuMAb mice; Medarex Inc.) with soluble E2 envelope glycoprotein derived from a genotype 1a virus (H77). Two HuMAbs, HCV1 and 95-2, were selected for further study based on initial cross-reactivity with soluble E2 glycoproteins derived from genotypes 1a and 1b, as well as neutralization of lentivirus pseudotyped with HCV 1a and 1b envelope glycoproteins. Additionally, HuMAbs HCV1 and 95-2 potently neutralized pseudoviruses from all genotypes tested (1a, 1b, 2b, 3a, and 4a). Epitope mapping with mammalian and bacterially expressed proteins, as well as synthetic peptides, revealed that HuMAbs HCV1 and 95-2 recognize a highly conserved linear epitope spanning amino acids 412 to 423 of the E2 glycoprotein. The capacity to recognize and neutralize a broad range of genotypes, the highly conserved E2 epitope, and the fully human nature of the antibodies make HuMAbs HCV1 and 95-2 excellent candidates for treatment of HCV-positive individuals undergoing liver transplantation.