Publications

Infections with hepatitis C virus (HCV) represent a worldwide health burden and a prophylactic vaccine is still not available. Liver transplantation (LT) is often the only option for patients with HCV-induced end-stage liver disease. However, immediately after transplantation, the liver graft becomes infected by circulating virus, resulting in accelerated progression of liver disease. Although the efficacy of HCV treatment using direct-acting antivirals has improved significantly, immune compromised LT-patients and patients with advanced liver disease remain difficult to treat. As an alternative approach, interfering with viral entry could prevent infection of the donor liver. We generated a human monoclonal antibody (mAb), designated 2A5, which targets the HCV envelope. The neutralizing activity of mAb 2A5 was assessed using multiple prototype and patient-derived HCV pseudoparticles (HCVpp), cell culture produced HCV (HCVcc), and a human-liver chimeric mouse model. Neutralization levels observed for mAb 2A5 were generally high and mostly superior to those obtained with AP33, a well-characterized HCV-neutralizing monoclonal antibody. Using humanized mice, complete protection was observed after genotype 1a and 4a HCV challenge, while only partial protection was achieved using gt1b and 6a isolates. Epitope mapping revealed that mAb 2A5 binding is conformation-dependent and identified the E2-region spanning amino acids 434 to 446 (epitope II) as the predominant contact domain. Conclusion: mAb 2A5 shows potent anti-HCV neutralizing activity both in vitro and in vivo and could hence represent a valuable candidate to prevent HCV recurrence in LT-patients. In addition, the detailed identification of the neutralizing epitope can be applied for the design of prophylactic HCV vaccines.

Mice were immunized with Adenovirus expressing the H1-con, H2-con, H3-con and H5-con HA consensus genes in combination (multivalent) and compared to mice immunized with the traditional 2010–2011 FluZone and FluMist seasonal vaccines. Immunized mice were challenged with 10–100 MLD50 of H1N1, H3N1, H3N2 and H5N1 influenza viruses. The traditional vaccines induced robust levels of HA inhibition (HI) titers, but failed to protect against five different heterologous lethal influenza challenges. Conversely, the multivalent consensus vaccine (1 × 1010 virus particles (vp)/mouse) induced protective HI titers of ≥40 against 8 of 10 influenza viruses that represent a wide degree of divergence within the HA subtypes and protected 100% of mice from 8 of 9 lethal heterologous influenza virus challenges. The vaccine protection was dose dependent, in general, and a dose as low as 5 × 107 vp/mouse still provided 100% survival against 7 of 9 lethal heterologous influenza challenges. These data indicate that very low doses of Adenovirus-vectored consensus vaccines induce superior levels of immunity against a wide divergence of influenza subtypes as compared to traditional vaccines. These doses are scalable and translatable to humans and may provide the foundation for complete and long-lasting anti-influenza immunity.

Naturally acquired antibodies to Plasmodium falciparum schizont egress antigen 1 (PfSEA-1A) are associated with protection against severe malaria in children. Vaccination of mice with SEA-1A from Plasmodium berghei (PbSEA-1A) decreases parasitemia and prolongs survival following P. berghei ANKA challenge. To enhance the immunogenicity of PfSEA-1A, we identified five linear B-cell epitopes using peptide microarrays probed with antisera from nonhuman primates vaccinated with recombinant PfSEA-1A (rPfSEA-1A). We evaluated the relationship between epitope-specific antibody levels and protection from parasitemia in a longitudinal treatment-reinfection cohort in western Kenya. Antibodies to three epitopes were associated with 16 to 17% decreased parasitemia over an 18-week high transmission season. We are currently designing immunogens to enhance antibody responses to these three epitopes.

Cytomegalovirus (CMV) entry into fibroblasts differs from entry into epithelial cells. CMV also spreads cell to cell and can induce syncytia. To gain insights into these processes, 27 antibodies targeting epitopes in CMV virion glycoprotein complexes, including glycoprotein B (gB), gH/gL, and the pentamer, were evaluated for their effects on viral entry and spread. No antibodies inhibited CMV spread in fibroblasts, including those with potent neutralizing activity against fibroblast entry, while all antibodies that neutralized epithelial cell entry also inhibited spread in epithelial cells and a correlation existed between the potencies of these two activities. This suggests that exposure of virions to the cell culture medium is obligatory during spread in epithelial cells but not in fibroblasts. In fibroblasts, the formation of syncytiumlike structures was impaired not only by antibodies to gB or gH/gL but also by antibodies to the pentamer, suggesting a potential role for the pentamer in promoting fibroblast fusion. Four antibodies reacted with linear epitopes near the N terminus of gH, exhibited strain specificity, and neutralized both epithelial cell and fibroblast entry. Five other antibodies recognized conformational epitopes in gH/gL and neutralized both fibroblast and epithelial cell entry. That these antibodies were strain specific for neutralizing fibroblast but not epithelial cell entry suggests that polymorphisms external to certain gH/gL epitopes may influence antibody neutralization during fibroblast but not epithelial cell entry. These findings may have implications for elucidating the mechanisms of CMV entry, spread, and antibody evasion and may assist in determining which antibodies may be most efficacious following active immunization or passive administration. IMPORTANCE Cytomegalovirus (CMV) is a significant cause of birth defects among newborns infected in utero and morbidity and mortality in transplant and AIDS patients. Monoclonal antibodies and vaccines targeting humoral responses are under development for prophylactic or therapeutic use. The findings reported here (i) confirm that cell-to-cell spread of CMV is sensitive to antibody inhibition in epithelial cells but not fibroblasts, (ii) demonstrate that antibodies can restrict the formation in vitro of syncytiumlike structures that resemble syncytial cytomegalic cells that are associated with CMV disease in vivo , and (iii) reveal that neutralization of CMV by antibodies to certain epitopes in gH or gH/gL is both strain and cell type dependent and can be governed by polymorphisms in sequences external to the epitopes. These findings serve to elucidate the mechanisms of CMV entry, spread, and antibody evasion and may have important implications for the development of CMV vaccines and immunotherapeutics.

The prototypic β-herpesvirus human cytomegalovirus (CMV) establishes life-long persistence within its human host. The CMV envelope consists of various protein complexes that enable wide viral tropism. More specifically, the glycoprotein complex gH/gL/gO (gH-trimer) is required for infection of all cell types, while the gH/gL/UL128/130/131a (gH-pentamer) complex imparts specificity in infecting epithelial, endothelial and myeloid cells. Here we utilize state-of-the-art robotics and a high-throughput neutralization assay to screen and identify monoclonal antibodies (mAbs) targeting the gH glycoproteins that display broad-spectrum properties to inhibit virus infection and dissemination. Subsequent biochemical characterization reveals that the mAbs bind to gH-trimer and gH-pentamer complexes and identify the antibodies’ epitope as an ‘antigenic hot spot’ critical for virus entry. The mAbs inhibit CMV infection at a post-attachment step by interacting with a highly conserved central alpha helix-rich domain. The platform described here provides the framework for development of effective CMV biologics and vaccine design strategies.

Background Porcine reproductive and respiratory syndrome (PRRS) is an important pig endemic disease in pork-producing countries worldwide. The etiology, porcine reproductive and respiratory syndrome virus (PRRSV), is characterized by fast antigen variability. Glycosylated protein 4 (GP4) is a minor protein in PRRSV virion, but contributes to induce protective immune responses. However, the antigenic characterization of PRRSV GP4 and the role of the mutations in this protein in PRRSV evolution are not clear. Methods Peptides chip scanning and peptide based ELISA was used to analyze the antigenic characterization of HP-PRRSV GP4. A total of 142 peptides printed on a chip were used to reveal the antigen reaction characteristics of the HP-PRRSV. The reactions of these peptides with HP-PRRSV-specific pig serum were scanned and quantified using the software PepSlide® Analyzer by fluorescence intensity. The active reaction regions (AR) were identified based on the scanning results and then the amino acids (aa) sequences of AR(s) is aligned among PRRSV strains for further identify the key aa site(s) impact the antigenicity of the protein. Peptide based ELISA is then reacted with PRRSV positive sera derived from pig inoculated with different PRRSV strains for further analysis the role of specific amino acid in AR. Results The intensity plot was used to show the reactions of the peptides with PRRSV serum and it showed that enormously different response happened to various parts of GP4. The highest reaction intensity value reached 6401.5 against one peptide with the sequence DIKTNTTAASDFVVL. An AR from S29 to G56 was identified. Sequence alignment revealed various mutations in site 43 and possibly played an important role in this AR. Peptides ELISA reaction with sera from pigs inoculated with different PRRSV strain revealed that the change of aa in site 43 reduced the reaction of the peptide with PRRSV positive sera derived from pigs inoculated with the peptide related PRRSV strains. Conclusion In this study, one AR covering S29 to G56 was identified in GP4. The aa in site 43 play an important role in determining the antigenic character of GP4. The continual mutations (S → G → D → N) occurred in this site alter the antigenicity of PRRSV GP4.