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- M. Brown, H. Seitz, V. Towne, M. Müller, A.C. Finnefrock
Development of Neutralizing Monoclonal Antibodies for Oncogenic HPV Types 31, 33, 45, 52, and 58Clinical and Vaccine Immunology, 2014;21(4).Human Papillomavirus (HPV) is the etiological agent for all cervical cancers, a significant number of other anogenital cancers, and a growing number of head-and-neck cancers. Two licensed vaccines offer protection against the most prevalent oncogenic types, 16 and 18, responsible for approximately 70% of cervical cancer cases worldwide and one of these also offers protection against types 6 and 11, responsible for 90% of genital warts. The vaccines are comprised of recombinantly expressed major capsid proteins that self-assemble into virus-like particles (VLPs) and prevent infection by elicting neutralizing antibodies. Adding the other frequently identified oncogenic types 31, 33, 45, 52, and 58 to a vaccine would increase the coverage against HPV-induced cancers to approximately 90%. We describe the generation and characterization of panels of monoclonal antibodies to these five additional oncogenic HPV types, and the selection of antibody pairs that were high affinity, type-specific, and recognized conformation-dependent neutralizing epitopes. Such characteristics make these antibodies useful tools for monitoring the production and potency of a prototype vaccine as well as monitoring vaccine-induced immune response in the clinic.
- J.S. Armistead, I. Morlais, D.K. Mathias, J.G. Jardim, J. Joy, A. Fridman, A.C. Finnefrock, A. Bagchi, M. Plebanski, D.G. Scorpio, T.S. Churcher, N.A. Borg, J. Sattabongkot, R.R. Dinglasan
Antibodies to a single, conserved epitope in Anopheles APN1 inhibit universal transmission of falciparum and vivax malariaInfection and Immunity, 2014;82(2):818-29.Malaria transmission-blocking vaccines (TBVs) represent a promising approach for the elimination and eradication of this disease. AnAPN1 is a lead TBV candidate that targets a surface antigen on the midgut of the obligate vector of the Plasmodium parasite, the Anopheles mosquito. In this study, we demonstrated that antibodies targeting AnAPN1 block transmission of Plasmodium falciparum and Plasmodium vivax across distantly related anopheline species in countries to which malaria is endemic. Using a biochemical and immunological approach, we determined that the mechanism of action for this phenomenon stems from antibody recognition of a single protective epitope on AnAPN1, which we found to be immunogenic in murine and nonhuman primate models and highly conserved among anophelines. These data indicate that AnAPN1 meets the established target product profile for TBVs and suggest a potential key role for an AnAPN1-based panmalaria TBV in the effort to eradicate malaria.
- D.C. Freed, Q. Tang, A. Tang, F. Li, X. He, Z. Huang, W. Meng, L. Xia, A.C. Finnefrock, E. Durr, A.S. Espeseth, D.R. Casimiro, N. Zhang, J.W. Shiver, D. Wang, Z. An, T.M. Fu
Pentameric complex of viral glycoprotein H is the primary target for potent neutralization by a human cytomegalovirus vaccineProceedings of the National Academy of Sciences of the USA, 2013; 110(51): E4997-5005.Human cytomegalovirus (HCMV) can cause serious morbidity/mortality in transplant patients, and congenital HCMV infection can lead to birth defects. Developing an effective HCMV vaccine is a high medical priority. One of the challenges to the efforts has been our limited understanding of the viral antigens important for protective antibodies. Receptor-mediated viral entry to endothelial/epithelial cells requires a glycoprotein H (gH) complex comprising five viral proteins (gH, gL, UL128, UL130, and UL131). This gH complex is notably missing from HCMV laboratory strains as well as HCMV vaccines previously evaluated in the clinic. To support a unique vaccine concept based on the pentameric gH complex, we established a panel of 45 monoclonal antibodies (mAbs) from a rabbit immunized with an experimental vaccine virus in which the expression of the pentameric gH complex was restored. Over one-half (25 of 45) of the mAbs have neutralizing activity. Interestingly, affinity for an antibody to bind virions was not correlated with its ability to neutralize the virus. Genetic analysis of the 45 mAbs based on their heavy- and light-chain sequences identified at least 26 B-cell linage groups characterized by distinct binding or neutralizing properties. Moreover, neutralizing antibodies possessed longer complementarity-determining region 3 for both heavy and light chains than those with no neutralizing activity. Importantly, potent neutralizing mAbs reacted to the pentameric gH complex but not to gB. Thus, the pentameric gH complex is the primary target for antiviral antibodies by vaccination.
- V. Towne, Q. Zhao, M. Brown, A.C. Finnefrock
Pairwise antibody footprinting using surface plasmon resonance technology to characterize human papillomavirus type 16 virus-like particles with direct anti-HPV antibody immobilization.Journal of Immunological Methods, 2013, 388(1–2):1-7.This paper describes an approach to surface plasmon resonance (SPR) based epitope mapping, also referred to as pairwise antibody footprinting, involving the direct immobilization of an antigen-specific primary mAb to the surface of an SPR interface. This technique offers a more straightforward approach than indirect capture (e.g., via rabbit anti-mouse Fc) as it does not require additional steps to block the unoccupied immobilized anti-Fc to prevent non-specific antibody binding. This is also an alternative to the direct immobilization of an antigen of interest, which may cause conformational changes in the antigen or epitope degradation upon chemical immobilization, particularly in successive regeneration cycles. It is particularly suitable for highly multivalent targets such as virus-like particles (VLPs). Using this technique, we assessed a panel of eight monoclonal antibodies against HPV (human papilloma virus) L1 protein VLPs expressed by S. cerevisiae. In the antibody epitope screening studies, HPV16 L1-directed conformational mAbs were clearly distinguished from the linear mAbs and consistent with known epitope information. Additional studies using a linear mAb and a conformational mAb demonstrate the practical application of this technique for characterizing the result of process changes and the consistency of recombinant HPV16 VLPs. The method is readily extensible to other VLPs and VLP-based vaccines.
- A. Fridman, A.C. Finnefrock, D. Peruzzi, I. Pak, N. La Monica, A. Bagchi, D.R. Casimiro, G. Ciliberto, L. Aurisicchio
An efficient T-cell epitope discovery strategy using in silico prediction and the iTopia assay platform.OncoImmunology, 2012; 8(1).Functional T-cell epitope discovery is a key process for the development of novel immunotherapies, particularly for cancer immunology. In silico epitope prediction is a common strategy to try to achieve this objective. However, this approach suffers from a significant rate of false-negative results and epitope ranking lists that often are not validated by practical experience. A high-throughput platform for the identification and prioritization of potential T-cell epitopes is the iTopiaTM Epitope Discovery SystemTM, which allows measuring binding and stability of selected peptides to MHC Class I molecules. So far, the value of iTopia combined with in silico epitope prediction has not been investigated systematically. In this study, we have developed a novel in silico selection strategy based on three criteria: (1) predicted binding to one out of five common MHC Class I alleles; (2) uniqueness to the antigen of interest; and (3) increased likelihood of natural processing. We predicted in silico and characterized by iTopia 225 candidate T-cell epitopes and fixed-anchor analogs from three human tumor-associated antigens: CEA, HER2 and TERT. HLA-A2-restricted fragments were further screened for their ability to induce cell-mediated responses in HLA-A2 transgenic mice. The iTopia binding assay was only marginally informative while the stability assay proved to be a valuable experimental screening method complementary to in silico prediction. Thirteen novel T-cell epitopes and analogs were characterized and additional potential epitopes identified, providing the basis for novel anticancer immunotherapies. In conclusion, we show that combination of in silico prediction and an iTopia-based assay may be an accurate and efficient method for MHC Class I epitope discovery among tumor-associated antigens.
- T.M. Fu, D. Wang, D.C. Freed, A. Tang, F. Li, X. He, S. Cole, S. Dubey, A.C. Finnefrock, J.t. Meulen, J.W. Shiver, D.R. Casimiro
Restoration of viral epithelial tropism improves immunogenicity in rabbits and rhesus macaques for a whole virion vaccine of human cytomegalovirus.Vaccine, 30(52):7469–7474.Maternal immunity to human cytomegalovirus (HCMV) prior to conception is ∼70% protective against congenital transmission and in utero infection of HCMV. Both functional antibodies capable of neutralizing virus and effective T-cells are believed to be important for the protection. Previous HCMV vaccines have rarely been shown able to induce neutralizing antibody titers comparable to those seen in naturally infected HCMV seropositive subjects. Recent studies link a glycoprotein H (gH) complex to receptor-mediated viral entry of endothelial/epithelial cells and leukocytes. This pentameric gH complex, composed of five proteins (gH, gL, UL128, UL130 and UL131 proteins), is notably missing in all HCMV vaccine previously evaluated in clinic. Here we showed that a HCMV virus, with restored expression of the pentameric gH complex, can induce 10-fold higher neutralizing antibody titers than an attenuated AD169 virus or a recombinant glycoprotein B vaccine in multiple animal species in which viral replication is not expected. Encouragingly, the peak neutralizing titers post vaccination in rabbits and monkeys were within 2–4-fold of the levels determined in HCMV seropositive subjects. Functional antibodies by vaccination could further be improved when formulated with a novel adjuvant, and the titers of the antiviral antibodies were sustained in rabbits for over a year after vaccination. These results indicate that the pentameric gH complex is associated with greatly improved functional antibodies following vaccination, and support a vaccine concept based on a nonreplicating whole HCMV with the pentameric gH-associated epithelial tropism restored.
- D. Wang, F. Li, D.C. Freed, A.C. Finnefrock, A. Tang, S.N. Grimes, D.R. Casimiro, T.M. FuQuantitative Analysis of Neutralizing Antibody Response to Human Cytomegalovirus in Natural Infection.Vaccine, 2011 Nov 8;29(48):9075-80.
Naturally acquired immunity significantly reduces the risk of congenital cytomegalovirus (CMV) infection in future pregnancies. An immune response comparable to that of natural infection has been used as a benchmark for CMV vaccine efficacy; however, the magnitude and persistence of the neutralizing antibody responses in naturally infected women are not completely understood. In this study, we quantitatively analyzed a panel of 375 female CMV convalescent sera ranging in age from 18 to 84 years, for its ability to block virus entry into epithelial cells and fibroblasts, as well as its binding potential to CMV particles. The geometric mean titer of the sera in this panel to neutralize 50% of the virus entry into epithelial cells was 7491, compared to 802 for entry into fibroblasts. The epithelial neutralizing titers were statistically indistinguishable among different age groups, and conformed to a normal distribution. There was a weak correlation between the levels of neutralization and the binding activities to viral particles. Our data confirmed that natural CMV infection in healthy women induces potent neutralizing antibodies against infection of both fibroblasts and epithelial cells. The serum neutralizing activities were maintained at high levels throughout the child bearing age. The corresponding titers may serve as a biomarker for CMV vaccine efficacy.
- A. Tang, F. Li, D.C. Freed, A.C. Finnefrock, D.R. Casimiro, D. Wang, T.M. Fu A novel high-throughput neutralization assay for supporting clinical evaluations of human cytomegalovirus vaccines.Vaccine, 2011 Oct 26;29(46):8350-6.
Neutralizing antibodies are considered an important component of protective immunity against congenital infection of human cytomegalovirus (HCMV), a frequently cited cause of birth defects. An effective HCMV vaccine is desired to induce potent neutralizing antibodies in seronegative females, so that the viral transmission to fetus would be blocked if the women contracted HCMV infections during their pregnancies. We describe a novel microneutralization assay to measure antiviral activities against HCMV in serum samples. The assay is based on detection of a dominant HCMV antigen expressed in cells, using near infrared dye-labeled immune reagents. Since the detection is independent of viral cytopathic effects, this assay format has the appeal of a short turn-around time and a read-out that is not subject to operator experience and judgment, making it a promising platform to support large scale clinical studies. In a serological survey of a cohort of 200 healthy females, we showed that the neutralizing titers measured in this assay are highly comparable to those from a neutralization assay based on an enzyme-linked immunostaining method. Lastly, to demonstrate the utility of this assay to support HCMV vaccine study, we presented the results of neutralizing titers from a rhesus macaque vaccination study.
- F. Li, A.C. Finnefrock, S.A. Dubey, B.T.M. Korber, J. Szinger, S. Cole, M.J. McElrath, J.W. Shiver, D.R. Casimiro, L. Corey, S.G. SelfMapping HIV-1 Vaccine Induced T-Cell Responses: Bias towards Less-Conserved Regions and Potential Impact on Vaccine Efficacy in the Step Study.PLoS ONE, 2011; 6(6):e20479.
T cell directed HIV vaccines are based upon the induction of CD8+ T cell memory responses that would be effective in inhibiting infection and subsequent replication of an infecting HIV-1 strain, a process that requires a match or near-match between the epitope induced by vaccination and the infecting viral strain. We compared the frequency and specificity of the CTL epitope responses elicited by the replication-defective Ad5 gag/pol/nef vaccine used in the Step trial with the likelihood of encountering those epitopes among recently sequenced Clade B isolates of HIV-1. Among vaccinees with detectable 15-mer peptide pool ELISpot responses, there was a median of four (one Gag, one Nef and two Pol) CD8 epitopes per vaccinee detected by 9-mer peptide ELISpot assay. Importantly, frequency analysis of the mapped epitopes indicated that there was a significant skewing of the T cell response; variable epitopes were detected more frequently than would be expected from an unbiased sampling of the vaccine sequences. Correspondingly, the most highly conserved epitopes in Gag, Pol, and Nef (defined by presence in >80% of sequences currently in the Los Alamos database) were detected at a lower frequency than unbiased sampling, similar to the frequency reported for responses to natural infection, suggesting potential epitope masking of these responses. This may be a generic mechanism used by the virus in both contexts to escape effective T cell immune surveillance. The disappointing results of the Step trial raise the bar for future HIV vaccine candidates. This report highlights the bias towards less-conserved epitopes present in the same vaccine used in the Step trial. Development of vaccine strategies that can elicit a greater breadth of responses, and towards conserved regions of the genome in particular, are critical requirements for effective T-cell based vaccines against HIV-1.
- T. Ebert, S. Smith, G. Pancari, D. Clark, R. Hampton, S. Secore, V. Towne, H. Fan, X.M. Wang, X. Wu, R. Ernst, B.R. Harvey, A.C. Finnefrock, F. Wang, C. Tan, E. Durr, L. Cope, A. Anderson, Z. An, T. McNeelyA fully human monoclonal antibody to Staphylococcus aureus iron regulated surface determinant B (IsdB) with functional activity in vitro and in vivo.Human Antibodies, 2010; 19(4):113-28.
A fully human monoclonal antibody (CS-D7, IgG1) specific for the iron regulated surface determinant B (IsdB) of Staphylococcus aureus was isolated from the Cambridge Antibody Technology (CAT) scFv antibody library. As compared to previously described IsdB specific murine monoclonals, CS-D7 has a unique, non-overlapping binding site on IsdB, and exhibits increased in vivo activity. The antibody recognizes a conformational epitope spanning amino acids 50 to 285 and has a binding affinity of 340 (± 75) pM for IsdB. CS-D7 bound to a wide variety of S. aureus strains, but not to an isdB deletion mutant. The antibody mediated opsonophagocytic (OP) killing in vitro and mediated significant protection in vivo. In a murine lethal sepsis model, the antibody conferred protection from death when dosed prior to challenge, but not when dosed after challenge. Importantly, in a central venous catheter (CVC) model in rats, the antibody reduced bacteremia and prevented colonization of indwelling catheters. Protection was observed when rats were dosed with CS-D7 prior to challenge as well as post challenge. IsdB is currently being investigated for clinical efficacy against S. aureus infection, and the activity of this human IsdB specific antibody supplements the growing body of evidence to support targeting this antigen for vaccine development.
- L.A. Hirao, L. Wu, A. Satishchandran, A.S. Khan, R. Draghia-Akli, A.C. Finnefrock, A.J. Bett, M.R. Betts, D.R. Casimiro, N.Y. Sardesai, J.J. Kim, J.W. Shiver, D.B. WeinerComparative Analysis of Immune Responses Induced by Vaccination with SIV Antigens by Recombinant Ad5 Vector or Plasmid DNA in Rhesus Macaques.Molecular Therapy, 2010 Aug;18(8):1568-76.
DNA vaccines have undergone important enhancements in their design, formulation, and delivery process. Past literature supports that DNA vaccines are not as immunogenic in nonhuman primates as live vector systems. The most potent recombinant vector system for induction of cellular immune responses in macaques and humans is adenovirus serotype 5 (Ad5), an important benchmark for new vaccine development. Here, we performed a head-to-head evaluation of the Merck Ad5 SIV vaccine and an optimized electroporation (EP) delivered SIV DNA vaccine in macaques. Animals receiving the Ad5 vaccine were immunized three times, whereas the DNA-vaccinated animals were immunized up to four times based on optimized protocols. We observed significant differences in the quantity of IFN responses by enzyme-linked immunosorbent spot (ELISpot), greater proliferative capacity of CD8+ T cells, and increased polyfunctionality of both CD4+ and CD8+ T cells in the DNA-vaccinated group. Importantly, Ad5 immunizations failed to boost following the first immunization, whereas DNA responses were continually boosted with all four immunizations demonstrating a major advantage of these improved DNA vaccines. These optimized DNA vaccines induce very different immune phenotypes than traditional Ad5 vaccines, suggesting that they could play an important role in vaccine research and development.
- E. Bianchi, J.G. Joyce, M.D. Miller, A.C. Finnefrock, X. Liang, M. Finotto, P. Ingallinella, P. McKenna, M. Citron, E. Ottinger, R.W. Hepler, R. Hrin, D. Nahas, C. Wu, D. Montefiori, J.W. Shiver, A. Pessi, P.S. Kim.
Vaccination with peptide mimetics of the gp41 prehairpin fusion intermediate yields neutralizing antisera against HIV-1 isolates.Proceedings of the National Academy of Sciences of the USA, 2010; 107(23):10655-10660. (PDF from journal)Eliciting a broadly neutralizing polyclonal antibody response against HIV-1 remains a major challenge. One approach to vaccine development is prevention of HIV-1 entry into cells by blocking the fusion of viral and cell membranes. More specifically, our goal is to elicit neutralizing antibodies that target a transient viral entry intermediate (the prehairpin intermediate) formed by the HIV-1 gp41 protein. Because this intermediate is transient, a stable mimetic is required to elicit an immune response. Previously, a series of engineered peptides was used to select a mAb (denoted D5) that binds to the surface of the gp41 prehairpin intermediate, as demonstrated by x-ray crystallographic studies. D5 inhibits the replication of HIV-1 clinical isolates, providing proof-of-principle for this vaccine approach. Here, we describe a series of peptide mimetics of the gp41 prehairpin intermediate designed to permit a systematic analysis of the immune response generated in animals. To improve the chances of detecting weak neutralizing polyclonal responses, two strategies were employed in the initial screening: use of a neutralization-hypersensitive virus and concentration of the IgG fraction from immunized animal sera. This allowed incremental improvements through iterative cycles of design, which led to vaccine candidates capable of generating a polyclonal antibody response, detectable in unfractionated sera, that neutralize tier 1 HIV-1 and simian HIV primary isolates in vitro. Our findings serve as a starting point for the design of more potent immunogens to elicit a broadly neutralizing response against the gp41 prehairpin intermediate.
- D.R. Casimiro, K. Cox, A. Tang, K.J. Sykes, M. Feng, F. Wang, A. Bett, W.A. Schleif, X. Liang, J. Flynn, T.W. Tobery, K. Wilson, A. Finnefrock, L. Huang, S. Vitelli, J. Lin, D. Patel, M.E. Davies, G.J. Heidecker, D.C. Freed, S. Dubey, D.H. O’Connor, D.I. Watkins, Z.Q. Zhang, J.W. Shiver.
Efficacy of Multi-Valent Adenovirus-Based Vaccine against Simian Immunodeficiency Virus Challenge.Journal of Virology, 2010; 84(6):2996-3003. (PDF from journal)The prophylactic efficacies of several multi-valent replication-incompetent adenovirus serotype 5 (Ad5) vaccines were examined in rhesus macaques using an intrarectal high-dose SIVmac239 challenge model. Cohorts of Mamu-A*01(+)/B*17(-) Indian rhesus macaques were immunized with one of several combinations of Ad5 vectors expressing Gag, Pol, Nef and Env gp140; for comparison, a Mamu-A*01(+) cohort was immunized using the Ad5 vector alone. There was no sign of immunological interference between antigens in the immunized animals. In general, expansion of the antigen breadth resulted in more favorable virological outcomes. In particular, the order of efficacy trended as follows: Gag/Pol/Nef/Env ∼ Gag/Pol > Gag ∼ Gag/Pol/Nef > Nef. However, the precision in ranking the vaccines based on the study results may be limited by the cohort size and as such, warrant additional testing. Implications of these results in light of the recent discouraging results of Phase IIb study of the trivalent Ad5 HIV-1 vaccine will be discussed.
- D.L. Montgomery, Y.J. Wang, R. Hrin, M. Luftig, B. Su, M.D. Miller, F. Wang, P. Haytko, L. Huang, S. Vitelli, J. Condra, X. Liu, R. Hampton, A. Carfi, A. Pessi, E. Bianchi, J. Joyce, C. Lloyd, R. Geleziunas, D. Bramhill, V.M. King, A.C. Finnefrock, W. Strohl, Z. An.Affinity maturation and characterization of a human monoclonal antibody against HIV-1 gp41.MAbs, 2009 Sep;1(5):462-474.
The human D5 monoclonal antibody binds to the highly conserved hydrophobic pocket on the N-terminal heptad repeat (NHR) trimer of HIV-1 gp41 and exhibits modest yet relatively broad neutralization activity. Both binding and neutralization depend on residues in the complementarity determining regions (CDRs) of the D5 IgG variable domains on heavy chain (VH) and light chain (VL). In an effort to increase neutralization activity to a wider range of HIV-1 strains, we have affinity matured the parental D5 scFv by randomizing selected residues in 5 of its 6 CDRs. The resulting scFv variants derived from four different CDR changes showed enhanced binding affinities to gp41 NHR mimetic (5-helix) which correlated to improved neutralization potencies by up to 8-fold. However, when converted to IgG1s, these D5 variants had up to a 12-fold reduction in neutralization potency over their corresponding scFvs despite their slightly enhanced in vitro binding affinities. Remarkably, D5 variant IgG1s bearing residue changes in CDRs that interact with epitope residues N-terminal to the hydrophobic pocket (such as VH CDR3 and VL CDR3) retained more neutralization potency than those containing residue changes in pocket-interacting CDRs (such as VH CDR2). These results provide compelling evidence for the existence of a steric block to an IgG that extends to the gp41 NHR hydrophobic pocket region, and can be a useful guide for developing therapeutic antibodies and vaccines circumventing this block.
- M. Brown, R. Kowalski, J. Zorman, X.M. Wang, V. Towne, Q. Zhao, S. Secore, A.C. Finnefrock, T. Ebert, G. Pancari, K. Isett, Y. Zhang, A.S. Anderson, D. Montgomery, L. Cope, T. McNeely.Selection and characterization of murine monoclonal antibodies to Staphylococcus aureus iron-regulated surface determinant B with functional activity in vitro and in vivo.Clinical Vaccine Immunology, 2009 Aug;16(8):1095-1104.
In an effort to characterize important epitopes of Staphylococcus aureus iron-regulated surface determinant B (IsdB), murine IsdB-specific monoclonal antibodies (MAbs) were isolated and characterized. A panel of 12 MAbs was isolated. All 12 MAbs recognized IsdB in enzyme-linked immunosorbent assays and Western blots; 10 recognized native IsdB expressed by S. aureus. The antigen epitope binding of eight of the MAbs was examined further. Three methods were used to assess binding diversity: MAb binding to IsdB muteins, pairwise binding to recombinant IsdB, and pairwise binding to IsdB-expressing bacteria. Data from these analyses indicated that MAbs could be grouped based on distinct or nonoverlapping epitope recognition. Also, MAb binding to recombinant IsdB required a significant portion of intact antigen, implying conformational epitope recognition. Four MAbs with nonoverlapping epitopes were evaluated for in vitro opsonophagocytic killing (OPK) activity and efficacy in murine challenge models. These were isotype switched from immunoglobulin G1 (IgG1) to IgG2b to potentially enhance activity; however, this isotype switch did not appear to enhance functional activity. MAb 2H2 exhibited OPK activity (> or =50% killing in the in vitro OPK assay) and was protective in two lethal challenge models and a sublethal indwelling catheter model. MAb 13C7 did not exhibit OPK (<50% killing in the in vitro assay) and was protective in one lethal challenge model. Neither MAb 13G11 nor MAb 1G3 exhibited OPK activity in vitro or was active in a lethal challenge model. The data suggest that several nonoverlapping epitopes are recognized by the IsdB-specific MAbs, but not all of these epitopes induce protective antibodies.
- M. Wallace, B. Evans, S. Woods, R. Mogg, L. Zhang, A.C. Finnefrock, D. Rabussay, M. Fons, J. Mallee, D. Mehrotra, F. Schödel, L. Musey. Tolerability of two sequential electroporation treatments using MedPulser DNA delivery system (DDS) in healthy adults.Molecular Therapy, 2009 May;17(5):922-928.
Immunotherapy against infectious agents and malignant tumors requires efficient priming of effector cells through direct expression and/or efficient cross-presentation of antigens by antigen-presenting cells. Electroporation is a new procedure aimed at transiently increasing cell membrane permeability and direct delivery of antigen or antigen-encoding nucleic acids inside targeted cells. We evaluated the tolerability including compliance with repeated electroporation treatments using MedPulser DDS in 24 healthy adults. Pain severity was evaluated at time of electroporation treatment, and at 1, 5, 10, and 20 minutes, and 24 hours thereafter, using two clinically validated questionnaires: McGill Pain Questionnaire (MPQ) (Present Pain Intensity) and Brief Pain Inventory (BPI). Electroporation treatments were generally well tolerated. Twenty-two out of 24 subjects returned for the second electroporation treatment 14 days after first treatment. Only two subjects reported a treatment-related systemic adverse experience following either electroporation application. For both pain assessment tools, maximum pain and/or discomfort were mostly reported immediately (within 5 minutes) after electroporation; Furthermore, no difference was observed when comparing peak-pain scores after first and second electroporation treatments. This study supports the clinical application of MedPulser DDS for the improvement of antigen-induced immune responses for prophylactic or therapeutic vaccines, especially in gene-based therapies for cancer.
- A.C. Finnefrock, A. Tang, F. Li, D.C. Freed, M. Feng, K.S. Cox, K.J. Sykes, J.P. Guare, M.D. Miller, D.B. Olsen, D.J. Hazuda, J.W. Shiver, D.R. Casimiro, T.M. Fu. PD-1 blockade in rhesus macaques: impact on chronic infection and prophylactic vaccination.Journal of Immunology, 2009 Jan 15;182(2):980-987.
Programmed Cell Death 1 (PD-1) plays a crucial role in immunomodulation. Binding of PD-1 to its ligand receptors down-regulates immune responses, and published reports suggest that this immune modulation is exploited in cases of tumor progression or chronic viral infection to evade immune surveillance. Thus, blockade of this signal could restore or enhance host immune functions. To test this hypothesis, we generated a panel of mAbs specific to human PD-1 that block PD ligand 1 and tested them for in vitro binding, blocking, and functional T cell responses, and evaluated a lead candidate in two in vivo rhesus macaque (Macaca mulatta) models. In the first therapeutic model, chronically SIV-infected macaques were treated with a single infusion of anti-PD-1 mAb; viral loads increased transiently before returning to, or falling below, pretreatment baselines. In the second prophylactic model, naive macaques were immunized with an SIV-gag adenovirus vector vaccine. Induced PD-1 blockade caused a statistically significant (p<0.05) increase in the peak percentage of T cells specific for the CM9 Gag epitope. These new results on PD-1 blockade in nonhuman primates point to a broader role for PD-1 immunomodulation and to potential applications in humans.
- A.J. Simon, D.R. Casimiro, A.C. Finnefrock, M.E. Davies, A. Tang, M. Chen, M. Chastain, G.S. Kath, L. Chen, J.W. Shiver.Enhanced in vivo transgene expression and immunogenicity from plasmid vectors following electrostimulation in rodents and primatesVaccine, 2008 Sep 19;26(40):5202-5209.
Safe and efficient methods for in vivo delivery of transgenes of interest must be developed so that the promise of these therapies can be practically used in the clinic. In this work, we describe the use of electrostimulation to enhance the in vivo efficiency of plasmid DNA delivery. The method was optimized to work over a range of moderate frequencies, utilizing low field strengths and simple symmetrical waveforms. After studying several parameters of delivery in mice, we demonstrate how this methodology can be employed to significantly improve both gene expression (over 16-fold) and the immunogenicity of HIV-1 vaccines (over 28-fold) compared to naked DNA in non-human primates. Compared to an efficient viral Ad5 vector system, the gene expression levels of DNA+electrostimulation were surprisingly within a factor of four of the viral delivery system.
- J.E. Drummond, E.E. Shaw, J.M. Antonello, T. Green, G.J. Page, C.O. Motley, K.A. Wilson, A.C. Finnefrock, X. Liang, D.R. Casimiro.
Design and optimization of a multiplex anti-influenza peptide immunoassay.Journal of Immunological Methods, 2008 May 20;334(1-2):11-20.Current flu vaccines are based on killed or attenuated virus vaccines that must be altered each year to include the hemagglutinin and neuraminidase genes from a strain of virus predicted to predominate in the coming year. A vaccine that could protect against multiple strains of influenza A and B would be a major asset in the fight against flu-related mortality and morbidity. To support development of such a vaccine, we have developed a Flu Multiplex Assay based on a Luminex platform to assess serum antibody levels to two conserved peptides derived from influenza A (M2 protein) and influenza B (hemagglutinin protein). The peptides were synthesized with a biotin label and subsequently coupled to two different LumAvidin microspheres. We then tested various sera against both types of peptide in the multiplex assay format. The data show that sera from Rhesus macaques immunized with a single peptide react only with the homologous peptide while Rhesus macaques immunized with both peptides respond well to both peptides. Additionally, we were able to specifically compete reactivity to both peptides. We have tested serial bleeds from 100 pediatric patients at ages ranging from 16 to 56 weeks as well as single bleeds from over 100 healthy adults. No overall trend in titer relative to pediatric age was detected. Both demographics exhibited a minimal response to either the A/M2 or B/HA0 peptides. However, the average titer for the pediatric serum samples was significantly lower than that found in the adult population. The adult population exhibited a higher prevalence of low reactive samples. Assay reagents and parameters have been optimized and the assay is shown to be repeatable and robust. The assay will be used to support clinical vaccine trials of a bivalent peptide vaccine.
- G.E.S. Toombes, A.C. Finnefrock, M.W. Tate, R. Ulrich, U. Wiesner, S.M. Gruner. A Re-Evaluation of the Morphology of a Bicontinuous Block Copolymer-Ceramic Material.Macromolecules, 2007; 40: 8974-8982. (PDF)
The structures of a poly(isoprene-block-ethylene oxide) (PI-b-PEO) block copolymer-directed aluminosilicate mesostructure and the resulting ceramic material obtained from calcination were studied via small-angle X-ray scattering (SAXS) and transmission electron microscopy (TEM). The PI minority phase (volume fraction 0.36) formed a continuous network of channels, previously reported to be consistent with the plumber’s nightmare morphology. The solvent casting process used to form the material caused it to shrink uniaxially by ~30%, deforming the network structure within it. Calculated structure factors for constant-curvature and constant-thickness models of a distorted double gyroid structure are consistent with SAXS from the material, while  and  projections of the distorted double gyroid structure match the TEM data. Because the structural data from the material is most consistent with a distorted version of the double gyroid morphology, the previous assignment of the plumber’s nightmare morphology must be reconsidered. Approaches for structural assignment are also discussed.
- A.C. Finnefrock, X. Liu, D.W. Opalka, J.W. Shiver, D.R. Casimiro, and J.H. Condra.
HIV Type 1 Vaccines for Worldwide Use: Predicting In-Clade and Cross-Clade Breadth of Immune Responses.AIDS Research and Human Retroviruses, 2007 Oct;23(10):1283-1292. (PDF)One of the greatest challenges in HIV vaccine development is accommodating the worldwide sequence diversity of the HIV-1 virus. To understand how viral sequence diversity may affect the potential breadth of HIV-1 vaccines designed to elicit antiviral T cell immunity, we have developed novel approaches to assess sequence conservation at the amino acid level, where vaccine effects are exerted. Taking each sequence from the LANL 2004 amino acid alignments as a potential vaccine or as a challenge virus, all pairwise combinations of sequences were evaluated by two methods: first, a traditional comparison of aligned sequences, and second, by a new walking 9-mer algorithm chosen to emphasize the typical length of an MHC-I epitope. The rules for comparing mismatched 9-mer pairs between vaccine and challenge sequences were empirically deduced from an experiment on Nef-specific CD8 epitopes and the viral sequences from naturally HIV-1-infected patients. Results were weighted such that each clade contributed in proportion to its global prevalence. Cross-clade breadth of response is best maintained for vaccines encoding Pol and Gag, while commonly proposed Env- and Tat-based vaccines would be more clade sensitive. We evaluated the additional breadth that could be expected from multiclade vaccines including consensus and ancestral sequences. For more diverse proteins, adding a second strain can add a significant increase in breadth, although for three or more strains the intrinsic diversity of the protein leads to diminishing improvement.
- I.J. Krauss, J.G. Joyce, A.C. Finnefrock, H.C. Song, V.Y. Dudkin, X. Geng, J.D. Warren, M. Chastain, J.W. Shiver, and S.J. Danishefsky.
Fully synthetic carbohydrate HIV antigens designed on the logic of the 2G12 antibody.Journal of the American Chemical Society 2007 Sep 12;129(36):11042-11044. (PDF)Di- and trivalent glycopeptide mimics of the HIV 2G12 epitope have been synthesized and evaluated for their comparative 2G12 binding characteristics. The epitope mimics consist of a cyclic peptide scaffold (unrelated to gp120 peptide sequences) attached via aspartate linkages to two or three copies of the high-mannose glycan, Man9GlcNAc2. The synthesis has been achieved via high-yielding double and triple Lansbury aspartylations of Man9GlcNAc2-NH2 with peptides containing, respectively, two and three aspartate residues. Conjugation of such constructs with an immunogenic carrier protein, OMPC, has been accomplished through the peptide’s cysteine sulfhydryl function, and Biacore assays have shown that binding affinity for 2G12 increases with increasing valency.
- D.R. Casimiro, F. Wang, W.A. Schleif, X. Liang, Z.Q. Zhang, T.W. Tobery, M.E. Davies, A.B. McDermott, D.H. O’Connor, A. Fridman, A. Bagchi, L.G. Tussey, A.J. Bett, A.C. Finnefrock, T.M. Fu, A. Tang, K.A. Wilson, M. Chen, H.C. Perry, G.J. Heidecker, D.C. Freed, A. Carella, K.S. Punt, K.J. Sykes, L. Huang, V.I. Ausensi, M. Bachinsky, U. Sadasivan-Nair, D.I. Watkins, E.A. Emini, and J.W. Shiver.Attenuation of simian immunodeficiency virus SIVmac239 infection by prophylactic immunization with dna and recombinant adenoviral vaccine vectors expressing Gag.Journal of Virology. 2005 Dec;79(24):15547-15555. (PDF)
The prophylactic efficacy of DNA and replication-incompetent adenovirus serotype 5 (Ad5) vaccine vectors expressing simian immunodeficiency virus (SIV) Gag was examined in rhesus macaques using an SIVmac239 challenge. Cohorts of either Mamu-A*01(+) or Mamu-A*01(-) macaques were immunized with a DNA prime-Ad5 boost regimen; for comparison, a third cohort consisting of Mamu-A*01(+) monkeys was immunized using the Ad5 vector alone for both prime and boost. All animals, along with unvaccinated control cohorts of Mamu-A*01(+) and Mamu-A*01(-) macaques, were challenged intrarectally with SIVmac239. Viral loads were measured in both peripheral and lymphoid compartments. Only the DNA prime-Ad5-boosted Mamu-A*01(+) cohort exhibited a notable reduction in peak plasma viral load (sevenfold) as well as in early set-point viral burdens in both plasma and lymphoid tissues (10-fold) relative to those observed in the control monkeys sharing the same Mamu-A*01 allele. The degree of control in each animal correlated with the levels of Gag-specific immunity before virus challenge. However, virus control was short-lived, and indications of viral escape were evident as early as 6 months postinfection. The implications of these results in vaccine design and clinical testing are discussed.
- I. Zampaglione, A.J. Simon, S. Capone, A.C. Finnefrock, D.R. Casimiro, G.S. Kath, A. Tang, A. Folgori, N. La Monica, J. Shiver,A. Nicosia, G. Ciliberto, R. Cortese, and E. Fattori.
Genetic vaccination by gene electro-transfer in non-human primates.Journal of Drug Delivery Science and Technology, Jan-Feb 2006, 16(1): 85-89. (PDF)Muscle gene electro-transfer (GET) of plasmid DNA is a promising approach for gene therapy and genetic vaccination. Several protocols have been described which give good levels of gene transduction in small animals. However, to progress towards human applications, efficacy must be demonstrated in non-human primates. Here, we extensively explore several electrical and injection parameters in Rhesus monkeys and define a series of conditions for efficient vaccination.
- M.D. Miller, R. Geleziunas, E. Bianchi, S. Lennard, R. Hrin, H. Zhang, M. Lu, Z. An, P. Ingallinella, M. Finotto, M. Mattu, A.C. Finnefrock, D. Bramhill, J. Cook, D.M. Eckert, R. Hampton, M. Patel, S. Jarantow, J. Joyce, G. Ciliberto, R. Cortese, P. Lu, W. Strohl, W. Schleif, M. McElhaugh, S. Lane, C. Lloyd, D. Lowe, J. Osbourn, T. Vaughan, E. Emini, G. Barbato, P.S. Kim, D.J. Hazuda, J.W. Shiver, and A. Pessi.A human monoclonal antibody neutralizes diverse HIV-1 isolates by binding a critical gp41 epitope.Proceedings of the National Academy of Sciences of the USA. 2005 Oct 11;102(41):14759-14764. (PDF)
HIV-1 entry into cells is mediated by the envelope glycoprotein receptor-binding (gp120) and membrane fusion-promoting (gp41) subunits. The gp41 heptad repeat 1 (HR1) domain is the molecular target of the fusion-inhibitor drug enfuvirtide (T20). The HR1 sequence is highly conserved and therefore considered an attractive target for vaccine development, but it is unknown whether antibodies can access HR1. Herein, we use gp41-based peptides to select a human antibody, 5H/I1-BMV-D5 (D5), that binds to HR1 and inhibits the assembly of fusion intermediates in vitro. D5 inhibits the replication of diverse HIV-1 clinical isolates and therefore represents a previously unknown example of a crossneutralizing IgG selected by binding to designed antigens. NMR studies and functional analyses map the D5-binding site to a previously identified hydrophobic pocket situated in the HR1 groove. This hydrophobic pocket was proposed as a drug target and subsequently identified as a common binding site for peptide and peptidomimetic fusion inhibitors. The finding that the D5 fusion-inhibitory antibody shares the same binding site suggests that the hydrophobic pocket is a “hot spot” for fusion inhibition and an ideal target on which to focus a vaccine-elicited antibody response. Our data provide a structural framework for the design of new immunogens and therapeutic antibodies with crossneutralizing potential.
- X. Liang, D.R. Casimiro, W.A. Schleif, F. Wang, M.E. Davies, Z.Q. Zhang, T.M. Fu, A.C. Finnefrock, L. Handt, M.P. Citron, G. Heidecker, A. Tang, M. Chen, K.A. Wilson, L. Gabryelski, M. McElhaugh, A. Carella, C. Moyer, L. Huang, S. Vitelli, D. Patel, J. Lin, E.A. Emini, and J.W. Shiver.Vectored Gag and Env but not Tat show efficacy against simian-human immunodeficiency virus 89.6P challenge in Mamu-A*01-negative rhesus monkeys.Journal of Virology 2005 Oct;79(19):12321-12331. (PDF)
Simian-human immunodeficiency virus (SHIV) challenge studies in rhesus macaques were conducted to evaluate the efficacy of adenovirus-based vaccines in the context of different major histocompatibility complex class I genetic backgrounds and different vaccine compositions. Mamu-A*01 allele-negative rhesus monkeys were immunized with one of the following vaccine constructs: (i) replication-defective recombinant adenovirus type 5 (Ad5) expressing human immunodeficiency virus type 1 (HIV-1) Tat (Ad5/HIVTat); (ii) Ad5 vector expressing simian immunodeficiency virus (SIV) Gag (Ad5/SIVGag); (iii) Ad5 vector expressing the truncated HIV-1(jrfl) Env, gp140 (Ad5/gp140jrfl); (iv) Ad5 vector expressing the SHIV-89.6P gp140 (Ad5/gp14089.6P); or (v) the combination of Ad5/SIVGag and Ad5/gp140jrfl. Following intravenous challenge with SHIV-89.6P, only those cohorts that received vaccines expressing Gag or Env exhibited an attenuation of the acute viremia and associated CD4-cell lymphopenia. While no prechallenge neutralizing antibody titers were detectable in either Ad5/gp140-vaccinated group, an accelerated neutralizing antibody response was observed in the Ad5/gp14089.6P-vaccinated group upon viral challenge. The set-point viral loads in the Ad5/SIVGag- and Ad5/gp140jrfl-vaccinated groups were associated with the overall strength of the induced cellular immune responses. To examine the contribution of Mamu-A*01 allele in vaccine efficacy against SHIV-89.6P challenge, Mamu-A*01-positive monkeys were immunized with Ad5/SIVGag. Vaccine-mediated protection was significantly more pronounced in the Mamu-A*01-positive monkeys than in Mamu-A*01-negative monkeys, suggesting the strong contributions of T-cell epitopes restricted by the Mamu-A*01 molecule. The implications of these results in the development of an HIV-1 vaccine will be discussed.
- A.C. Finnefrock, R. Ulrich, G.E. Toombes, S.M. Gruner, U. Wiesner.The plumber’s nightmare: a new morphology in block copolymer-ceramic nanocomposites and mesoporous aluminosilicates. Journal of the American Chemical Society 2003 Oct 29;125(43):13084-13093.
A novel cubic bicontinuous morphology is found in polymer-ceramic nanocomposites and mesoporous aluminosilicates that are derived by an amphiphilic diblock copolymer, poly(isoprene-b-ethylene oxide) (PI-b-PEO), used as a structure-directing agent for an inorganic aluminosilicate. Small-angle X-ray scattering (SAXS) was employed to unambiguously identify the Im3m crystallographic symmetry of the materials by fitting individual Bragg peak positions in the two-dimensional X-ray images. Structure factor calculations, in conjunction with results from transmission electron microscopy, were used to narrow the range of possible structures consistent with the symmetry and showed the plumber’s nightmare morphology to be consistent with the data. The samples are made by deposition onto a substrate that imposes a strain field, generating a lattice distortion. This distortion is quantitatively analyzed and shown to have resulted in shrinkage of the crystallites by approximately one-third in a direction perpendicular to the substrate, in both as-made composites and calcined ceramic materials. Finally, the observation of the bicontinuous block-copolymer-derived hybrid morphology is discussed in the context of a pseudo-ternary morphology diagram and compared to existing studies of ternary phase diagrams of amphiphiles in a mixture of two solvents. The calcined mesoporous materials have potential applications in the fields of catalysis, separation technology, and microelectronics.
- Rahul S. Deshpande, Stefanie L. Sharp-Goldman, Jennifer L. Willson, Andrew B. Bocarsly, Joachim Gross, Adam C. Finnefrock, and Sol M. Gruner.Morphology and Gas Adsorption Properties of Palladium-Cobalt-Based Cyanogels.Chemistry of Materials 2003 15:4239-4246.
The morphology of cyanide-bridged palladium-cobalt (Pd-Co) gels, synthesized from aqueous solutions of Na2PdCl4 and K3[Co(CN)6], is elucidated using nitrogen and carbon dioxide adsorption, permeability measurements, electron microscopy, and small-angle X-ray scattering. The aerogels formed from the Pd-Co hydrogels possess both micro- and mesoporosity. Electron microscopy indicates that the aerogels are constituted of spherical microporous particles; the interstices between these particles constitute the mesopores. The aerogel surfaces are found to be fractal as analyzed by gas adsorption and small-angle X-ray scattering. Both these techniques yield, within experimental error, the same surface fractal dimension of 2.6 ± 0.2 for the aerogels. The Pd-Co xerogels, unlike the aerogels, are predominantly microporous with a narrow microporosity. Transmission electron microscopy reveals that the xerogels are constituted of particles of an average size of ~10 nm. Unlike the aerogels, the xerogels do not possess surface fractality. The mechanism of adsorption of different gases on these gels is analyzed on the basis of the gel morphologies.
- Gilman E. S. Toombes, Adam C. Finnefrock, Mark W. Tate, and Sol M. Gruner.Determination of Lα:HII phase transition temperature for DOPE.Biophysical Journal, 82(5):2504–2510, May 2002. (PDF)
The thermodynamic properties of fully-hydrated lipids provide important information about the stability of membranes and the energetic interactions of lipid bilayers with membrane proteins. The Lamellar/Inverse Hexagonal (Lα–HII) phase transition of DOPE-water mixtures is a first order transition and, therefore, at constant pressure must have a thermodynamically well-defined equilibrium transition temperature. The observed transition temperature is known to be dependent upon the rate at which the temperature is changed, which accounts for the many different values in the literature. X-ray diffraction was used to study the phase transition of fully-hydrated DOPE in order to determine the rate-independent transition temperature, TLH . Samples were heated or cooled for a range of rates, 0.212 < r < 225°C/hr, and the rate-dependent apparent phase transition temperatures, TA(r) were determined from the x-ray data. By use of a model-free extrapolation method, the transition temperature was found to be TLH = 3.33 ± 0.16°C. The hysteresis, | TA(r) – TLH |, was identical for heating and cooling rates, ± r, and varied as |r|^β for β ≈ 1/4. This unexpected power-law relationship is consistent with a previous study but differs markedly from the exponential behavior of activation barrier kinetics. The methods used in this study are general and provide a simple way to determine the true mesomorphic phase transition temperatures of other lipid and lyotropic systems.
- L. Pollack, M.W. Tate, A.C. Finnefrock, S.M. Gruner, C. Kalidas, S. Trotter, C.A. Batt, N.C. Darnton, R.H. Austin, L. Lurio, and S.G.J. Mochrie.Time-resolved collapse of a folding protein observed with small angle X-ray scattering.Physical Review Letters, 86(21):4962–4965, May 2001. (PDF)
Using high-intensity, “pink” beam from an undulator in conjunction with microfabricated rapid-fluid mixing devices, we can monitor the early events in protein folding with time-resolved small angle x-ray scattering. This Letter describes recent work on the protein bovine β-lactoglobulin (BLG) where collapse from an expanded to compact set of states was directly observed on the millisecond time scale. The role of chain collapse, one of the initial stages of protein folding, is not currently understood. The characterization of transient, compact states is vital in assessing the validity of theories and models of the folding process.
- A. C. Finnefrock, R. Ulrich, A. Du Chesne, C. C. Honeker, K. Schumacher, K. K. Unger, S. M. Gruner, and U. Wiesner.Metal oxide-containing mesoporous silica with bicontinuous Plumber’s Nightmare morphology from a block copolymer-hybrid mesophase.Angewandte Chemie International Edition, 40(7):1207–1211, April 2001. (PDF)
- K. L. Ringland, A. C. Finnefrock, Y. Li, J. D. Brock, S. G. Lemay, and R. E. Thorne.Sliding charge-density waves as rough growth fronts.Physical Review B, 61(7):4405–4408, February 2000. (PDF)
Using high-resolution x-ray scattering techniques, we have measured the transverse static structure factor of the sliding charge-density wave (CDW) in NbSe3. For temperatures between 70 and 120 K and for applied currents up to 40× the threshold current for sliding, the scattering peak for the sliding CDW is significantly broader than that for the pinned CDW, indicating that the sliding state is less correlated than the pinned state. Using scaling analysis, we show that the CDW phase roughness exponent α rises from 0.60 ± 0.01 in the pinned state to 0.80 ± 0.01 in the sliding state, indicating that the phase fronts of the sliding CDW are significantly rougher than those of the pinned CDW.
- J. D. Brock, K. L. Ringland, A. C. Finnefrock, Y. Li, S. G. Lemay, and R. E. Thorne.Dynamic scaling during CDW relaxation from the sliding state.Journal de Physique IV, 9(P10):17–21, December 1999.
Using time-resolved high-resolution x-ray scattering techniques, we have measured the evolution of the structure of the Q1 charge-density wave in NbSe3 as it relaxes after an applied electric field is turned off. Measurements were made at temperatures between 70 and 120 K and at applied field strengths up to 10 times the threshold for sliding. These time- dependent structural data are accurately described by dynamic scaling theory. For threshold field strengths less than the threshold to sliding, the value of the dynamic scaling exponent μ is consistent with the value predicted by assuming that the CDW is an elastic medium. However, for field strengths greater than threshold, μ is significantly smaller, indicating that phase-slip (amplitude fluctuations) is (are) necessary for a correct physical description.
- J. D. Brock, K. L. Ringland, A. C. Finnefrock, Y. Li, S. G. Lemay, and R. E. Thorne.Sliding charge-density waves as rough growth surfaces.Journal de Physique IV, 9(P10):83–84, December 1999.
Using high-resolution x-ray scattering techniques we have measured the transverse structure of the sliding charge-density wave (CDW) in NbSe3. For temperatures between 70 K and 120 K and for applied currents up to 40 times the threshold current for sliding, the scattering peak for the sliding CDW is significantly broader than that for the pinned CDW, indicating that the sliding state is less correlated than the pinned state. Using scaling analysis, we show that the CDW phase roughness exponent α rises from 0.60 ± 0.01 in the pinned state to 0.80 ± 0.01 in the sliding state.
- K. L. Ringland, A. C. Finnefrock, Y. Li, J. D. Brock, S. G. Lemay, and R. E. Thorne.Scaling of charge-density-wave relaxation: Time-resolved x-ray scattering measurements.Physical Review Letters, 82(9):1923–1926, March 1999. (PDF)
Using time-resolved, high-resolution x-ray scattering techniques, we have measured the evolution of the transverse structure of the NbSe3 Q1 charge-density wave as it relaxes from the sliding state to the pinned state. Measurements were made at temperatures between 70 and 120 K and at electric field strengths between 2 times and 10 times the threshold for sliding. These time-dependent data are accurately described by dynamic scaling.
- A. C. Finnefrock, K. L. Ringland, J. D. Brock, L. J. Buller, and H. D. Abruña.Nucleation and ordering of an electrodeposited two-dimensional crystal: Real-time x-ray scattering and electronic measurements.Physical Review Letters, 81(16):3459–3462, October 1998. (PDF)
We have studied in situ the ordering of a two-dimensional Cu–Cl crystal electrodeposited on a Pt(111) surface. We simultaneously measured x-ray scattering and chronoamperometric transients during Cu desorption and subsequent ordering of the Cu–Cl crystal. In all cases, the current transient occurs on a shorter time-scale than the development of crystalline order. The ordering time diverges with applied potential, consistent with the nucleation and growth of two-dimensional islands. We see a time-dependent narrowing of the x-ray peak, corresponding to the growing islands.
- A. C. Finnefrock, L. J. Buller, K. L. Ringland, J. D. Brock, and H. D. Abruña.Time-resolved surface x-ray scattering study of surface ordering of electrodeposited layers.Journal of the American Chemical Society, 119:11703–11704, December 1997. (PDF)
- E. Herrero, L. J. Buller, J. Li, A. C. Finnefrock, A. B. Solomón, C. Alonso, J. D. Brock, and H.D. Abruña.Electrodeposition dynamics: Electrochemical and x-ray scattering studies.Electrochimica Acta, 44(6-7):983–992, 1998. (PDF)
Studies of the electrodeposition dynamics of underpotential deposition (UPD) processes based on electrochemical and in-situ surface X-ray scattering studies are presented. The studies focus on the UPD of Hg on Au(111) in sulfate media, and Cu UPD on Pt(111) in sulfate and chloride media. In the first case it is shown that Hg UPD on Au(111) in sulfuric acid follows a progressive nucleation mechanism which involves various surface structures. In the case of Cu UPD on Pt(111), the process appears to follow an instantaneous nucleation mechanism in both sulfate and chloride media. Time-resolved surface X-ray scattering studies of Cu UPD on Pt(111) in the presence of chloride demonstrate that the electrochemical relaxation and the achievement of long range order can take place on significantly different time scales. Analysis of time-resolved surface X-ray scattering data allows for the study of the dynamics of island growth.
- H. D. Abruña, J. M. Feliu, J. D. Brock, L. J. Buller, E. Herrero, J. Li, R. Gómez, and A. Finnefrock.Anion and electrode surface structure effects on the deposition of metal monolayers: Electrochemical and time-resolved surface diffraction studies.Electrochimica Acta, 43(19-20):2899–2909, 1998. (PDF)
The effects of anions and electrode surface structure on the UPD of metal monolayers are illustrated with three different examples. In the first, we show that for Cu UPD on Pt[n(111) x (110)] (n = 2, 3, 5, 9 and 19) stepped surfaces in sulfuric acid medium, submonolayer amounts of underpotentially deposited copper induce the adsorption of (bi)sulfate in the vicinity of copper adatoms deposited on (110) step sites. The induced anion adsorption increases with terrace width up to a three Pt atoms wide terrace, suggesting that this is the minimum width to accommodate the copper adatom and the coadsorbed anion. In the second case we present data from simultaneous time-resolved surface X-ray scattering and chronoamperometric measurements of Cu UPD on Pt(111) electrodes in the presence of chloride anions. These studies demonstrate that the kinetics of formation of the incommensurate CuCl adlayer from the commensurate (1 x 1) Cu layer takes place in a much longer time scale than the current response. This is a clear indication of the temporal separation (resolution) between electrochemical events and processes associated with surface reorganization to achieve long-range periodic ordering. Finally, we consider the UPD of Hg on Au(111) electrodes with emphasis on the relationship between the partial charge retained by the mercury and anion adsorption. At the early stages of Hg UPD, when mercury is still partially charged, an ordered mercurous-sulfate bilayer structure is formed on the electrode surface. At more negative potentials, where mercury is almost fully discharged, two additional ordered hexagonal mercury adlayers are formed with little, if any, interactions with the anions, suggesting that the interactions between them are largely electrostatic in nature.
- A. C. Finnefrock, L. J. Buller, K. L. Ringland, P. D. Ting, H. D. Abruña, and J. D. Brock.Time-resolved measurements of overlayer ordering in electrodeposition. In P. C. Andricacos, S. G. Corcoran, J.-L. Delplancke, T. P. Moffat, and P. S. Searson, editors, Electrochemical Synthesis and Modification of Materials. Symposium, volume 451, pages 49–55. Materials Research Society, 1997. (PDF)
We report in situ time-resolved surface x-ray scattering measurements of the underpotential deposition of Cu2+ on Pt(111) in the presence of Cl– in HClO4 solution. Chronoamperometric (current vs. time) measurements indicate that after a potential step, the electrodeposition current decays to 1/e of its initial value in at most 0.12 seconds. In contrast, our simultaneous time-resolved surface x-ray scattering data reveal that the overlayer requires on the order of two seconds to develop long-range periodic order. These results demonstrate that the kinetics of surface ordering can be significantly different from the kinetics of charge-transfer and illustrate the power of time-resolved surface x-ray scattering for in situ studies of electrodeposition.
- J. D. Brock, A. C. Finnefrock, K. L. Ringland, and E. Sweetland.Detailed structure of a charge-density wave in a quenched random field.Physical Review Letters, 73(26):3588–91, December 1994. (PDF, preprint)
Using high resolution x-ray scattering, we have measured the structure of the Q1 charge-density wave in Ta-doped NbSe3. Detailed line shape analysis of the data demonstrates that two length scales are required to describe the phase-phase correlation function. Phase fluctuations with wavelengths less than a new length scale a are suppressed, and this a is identified with the amplitude coherence length. We find that ξa=34.4 ± 10.3 Å. Implications for the physical mechanisms responsible for pinning are discussed.
- E. Sweetland, A. C. Finnefrock, W. J. Podulka, M. Sutton, J. D. Brock, D. DiCarlo, and R. E. Thorne.X-ray-scattering measurements of the transient structure of a driven charge-density wave.Physical Review B (Condensed Matter), 50(12):8157–65, September 1994. (PDF, preprint)
We report time-resolved X-ray-scattering measurements of the transient structural response of the sliding Q1 charge-density wave (CDW) in NbSe3 to a reversal of the driving electric field. The observed time scale characterizing this response at 70 K varies from approximately 15 msec for driving fields near threshold to approximately 2 msec for fields well above threshold. The position and time-dependent strain of the CDW is analyzed in terms of a phenomenological equation of motion for the phase of the CDW order parameter. The value of the damping constant, γ =(3.2 ± 0.7) times 10-19 eV sec Å-3, is in excellent agreement with the value determined from transport measurements. As the driving field approaches threshold from above, the line shape becomes bimodal, suggesting that the CDW does not depin throughout the entire sample at one well-defined voltage.
- I. M. Vitomirov, A. Raisanen, A. C. Finnefrock, R. E. Viturro, L. J. Brillson, P. D. Kirchner, G. D. Pettit, and J. M. Woodall.Geometric ordering, surface chemistry, band bending and work function at decapped GaAs(100) surfaces.Physical Review B (Condensed Matter), 46(20):13293–302, November 1992. (PDF)
The authors present a comprehensive study of epitaxially grown and As-coated GaAs (100) surfaces as a function of As desorption temperature and background pressure. They have used low-energy electron diffraction to determine surface reconstruction, and core-level and valence-band soft-X-ray photoemission spectroscopy to perform chemical and electronic characterization of these surfaces. They find gradual changes in surface geometry and composition, and a limited ( approximately 120 meV) Fermi-level movement over numerous reconstructions in the 250-650 degrees C annealing temperature range. The surface ionization potential and work function exhibit large changes between different surface reconstructions. In conjunction with other techniques, work-function measurements present evidence of surface inhomogeneity for many of the desorption temperatures and surface reconstructions. This inhomogeneity appears related to the existence of differently reconstructed patches on the surface. The authors’ results emphasize the complexity of reconstructed GaAs(100) surfaces and the advantages of a multiple- technique approach for their characterization.
- I. M. Vitomirov, A. D. Raisanen, A. C. Finnefrock, R. E. Viturro, L. J. Brillson, P. D. Kirchner, G.D. Pettit, and J. M. Woodall.
Temperature-dependent chemical and electronic structure of reconstructed GaAs (100) surfaces.Journal of Vacuum Science and Technology B, 10(4):1898–903, July–August 1992. (PDF)Low-energy electron diffraction, soft X-ray photoemission, cathodoluminescence (CL), and Auger electron spectroscopies have been performed to investigate the geometric, chemical, and electronic properties of GaAs (100) surfaces as a function of annealing temperature and surface reconstruction. These measurements indicate gradual changes in surface geometry, composition, deep level CL features, and Fermi-level (EF) position with increasing temperature of surface preparation. In contrast, it was observed that pronounced changes in the surface ionization potential and work function between different surface reconstructions. For most of the desorption temperatures and surface reconstructions, the secondary electron emission exhibits characteristic double onsets, possibly due to the existence of differently reconstructed patches on the surface. The implications of these variations in the surface chemical and electronic structure of GaAs (100) surfaces on their metal contact properties. It was concluded that (a) unique characterization of these surfaces requires measurements of geometric ordering, chemical composition and bonding, and deep level emission in the band gap, and (b) the correlation of the surface geometry with chemical and electronic surface and interface structure points to the central role of surface preparation in achieving controlled Schottky barrier behavior.
- I. M. Vitomirov, A. D. Raisanen, A. C. Finnefrock, R. E. Viturro, S. Chang, L. J. Brillson, P. D. Kirchner, G. D. Pettit, and J. M. Woodall.
Surface and interface states for GaAs(100) (1*1) and (4*2)-c(8*2) reconstructions.Journal of Vacuum Science and Technology A, 10(4):749–753, July–August 1992. (PDF)Low energy cathodoluminescence spectroscopy measurements of GaAs (100) surfaces prepared by thermal desorption of an As passivation layer reveal deep level transitions localized at the clean surfaces and metallized interfaces. These surface and interface state features extend from 0.7 to 1.3 eV and exhibit subtle differences between the As- rich (1*1) and Ga-rich (4*2)-c(8*2) reconstructions. Both Au deposition and subsequent annealing induce additional deep level emissions which appear relatively unchanged between these two reconstructions. In contrast, Al deposition introduces new features which depend significantly upon starting surface stoichiometry and reconstruction. The authors discuss the formation and energies of these states in relation to reported variations in Fermi level stabilization. They conclude that surface stoichiometry and atomic bond configuration are a significant factor in formation and evolution of electrically active, deep level metal-GaAs (100) interface states.