L R Budgeon

Pennsylvania State University, State College, PA, United States

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Publications (64)240.67 Total impact

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    ABSTRACT: NOD.B10 Idd9.3 mice are congenic for the insulin-dependent diabetes (Idd) Idd9.3 locus, which confers significant type 1 diabetes (T1D) protection and encodes 19 genes, including microRNA (miR)-34a, from T1D-resistant C57BL/10 mice. B cells have been shown to play a critical role in the priming of autoantigen-specific CD4+ T cells in T1D pathogenesis in non-obese diabetic (NOD) mice. We show that early B-cell development is impaired in NOD.B10 Idd9.3 mice, resulting in the profound reduction of transitional and mature splenic B cells as compared with NOD mice. Molecular analysis revealed that miR-34a expression was significantly higher in B cell progenitors and marginal zone B cells from NOD.B10 Idd9.3 mice than in NOD mice. Furthermore, miR-34a expression in these cell populations inversely correlated with levels of Foxp1, an essential regulator of B-cell lymphopoiesis, which is directly repressed by miR-34a. We further show that islet-specific CD4+ T cells proliferated inefficiently when primed by NOD.B10 Idd9.3 B cells in vitro or in response to endogenous autoantigen in NOD.B10 Idd9.3 mice. Thus, Idd9.3-encoded miR-34a is a likely candidate in negatively regulating B-cell lymphopoiesis, which may contribute to inefficient expansion of islet-specific CD4+ T cells and to T1D protection in NOD.B10 Idd9.3 mice.This article is protected by copyright. All rights reserved
    European Journal of Immunology 03/2014; · 4.97 Impact Factor
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    ABSTRACT: Long peptide immunization is a promising strategy to clear established tumors. In the current study, we investigated the therapeutic effect of a naturally existing long peptide that contained two HLA-A2.1 restricted epitopes (CRPVE1/149–157 and CRPVE1/161–169) from cottontail rabbit papillomavirus (CRPV) E1 using our CRPV/HLA-A2.1 transgenic rabbit model. A universal Tetanus Toxin helper motif (TT helper) was tagged at either the N-terminus or the carboxyl-terminus of this long peptide and designated as TT-E1 peptide and E1 peptide-TT, respectively. Four groups of HLA-A2.1 transgenic rabbits were infected with wild type CRPV DNA. Three weeks post-infection, the rabbits were immunized four times with TT-E1 peptide, E1 peptide only, E1 peptide-TT or TT-control peptide with two-week intervals between immunizations. Tumor outgrowth was monitored and recorded weekly. After the third booster immunization, tumors on two of the four E1 peptide-TT immunized rabbits began to shrink. One animal from this group was free of tumors at the termination of the study. The mean papilloma size of E1 peptide-TT immunized rabbits was significantly smaller when compared with that of the three other groups (P < 0.05, one way ANOVA analysis). It is interesting that E1 peptide-TT vaccination not only stimulated stronger T cell mediated immune responses but also stronger antibody generations. We conclude that the location of a TT helper motif tagged at the long peptide vaccine is critical for the outcome of therapeutic responses to persistent tumors in our HLA-A2.1 transgenic rabbit model.
    Trials in Vaccinology. 01/2014; 3:134–142.
  • Neil D Christensen, Lynn R Budgeon
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    ABSTRACT: Prophylactic and therapeutic immunization strategies are an effective method to control human papillomavirus (HPV)-associated diseases and cancers. Current protective virus-like particle and capsid-based vaccines are highly protective against vaccine-matched HPV types, and continued improvements in second-generation vaccines will lead to broader protection and cross-protection against the cancer-associated types. Increasing the effectiveness of broadly cross-protective L2-based immunogens will require adjuvants that activate innate immunity to thus enhance adaptive immunity. Therapeutic immunization strategies are needed to control and cure clinical disease and HPV-associated cancers. Significant advances in strategies to improve induction of cell-mediated immunity to HPV early (and capsid) proteins have been pretested in preclinical animal papillomavirus models. Several of these effective protocols have translated into successful therapeutic immune-mediated clearance of clinical lesions. Nevertheless, there are significant challenges in activating immunity to cancer-associated lesions due to various immune downregulatory events that are triggered by persistent HPV infections. A better understanding of immune responses to HPV lesions in situ is needed to optimize immune effector T cells that efficiently locate to sites of infection and which should lead to an effective immunotherapeutic management of this important human viral pathogen. The most effective immunization strategy may well require combination antiviral and immunotherapeutic treatments to achieve complete clearance of HPV infections and associated cancers. © 2014 S. Karger AG, Basel.
    Current problems in dermatology 01/2014; 45:252-264.
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    ABSTRACT: Papillomavirus disease poses a special challenge to people with compromised immune systems. Appropriate models to study infections in these individuals are lacking. We report here the development of a model that will help to address these deficiencies. The MmuPV1 genome was synthesized and used successfully to produce virus from DNA infections in immune-compromised mice. In these early studies, we have demonstrated both primary and secondary infections, expanded tissue tropism and extensive dysplasia.
    Journal of Virology 06/2013; · 5.08 Impact Factor
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    ABSTRACT: Papillomaviruses use rare codons with respect to the host. The reasons for this are incompletely understood but among the hypotheses is the concept that rare codons result in low protein production and this allows the virus to escape immune surveillance. We changed rare codons in the oncogenes E6 and E7 of the cottontail rabbit papillomavirus to make them more mammalian-like and tested the mutant genomes in our in vivo animal model. While the amino acid sequences of the proteins remained unchanged, the oncogenic potential of some of the altered genomes increased dramatically. In addition, increased immunogenicity, as measured by spontaneous regression, was observed as the numbers of codon changes increased. This work suggests that codon usage may modify protein production in ways that influence disease outcome and that evaluation of synonymous codons should be included in the analysis of genetic variants of infectious agents and their association with disease.
    Virology 02/2013; · 3.35 Impact Factor
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    ABSTRACT: Multiple sclerosis (MS) is an autoimmune disease that is mediated by myelin-reactive T cells resulting in CNS demyelination, however the mechanisms that control their activation are unclear. Mice that are transgenic for a myelin proteolipid protein (PLP)-specific TCR spontaneously develop experimental autoimmune encephalomyelitis (EAE), the animal model of MS. They mimic the spontaneous onset of MS and thus offer the unique opportunity to investigate the mechanisms that may contribute to the development of spontaneous CNS autoimmunity. MyD88 is an adaptor protein that mediates signal transduction by TLRs, IL-1R and IL-18R, resulting in the activation of innate immune cells, including DCs. We investigated the requirement of MyD88 in the pathogenesis of spontaneous EAE in PLP TCR transgenic SJL mice. We show that genetic loss of MyD88 does not intrinsically preclude development of spontaneous EAE and autoimmune demyelination in these mice. EAE was associated with functionally mature peripheral DCs that promoted superior PLP-specific Th1 and Th17 responses compared to those from disease-free mice. Together, our data suggest that MyD88-independent innate immune signaling critically contributes to priming of myelin-reactive T cells and development of spontaneous EAE in MyD88-deficient PLP TCR transgenic mice.
    Journal of neuroimmunology 12/2012; · 2.84 Impact Factor
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    ABSTRACT: Adoptive T-cell immunotherapy has garnered wide attention, but its effective use is limited by the need of multiple ex vivo manipulations and infusions that are complex and expensive. In this study, we show how highly reactive antigen (Ag)-specific CTLs can be generated from induced pluripotent stem (iPS) cells to provide an unlimited source of functional CTLs for adoptive immunotherapy. iPS cell-derived T cells can offer the advantages of avoiding possible immune rejection and circumventing ethical and practical issues associated with other stem cell types. iPS cells can be differentiated into progenitor T cells in vitro by stimulation with the Notch ligand Delta-like 1 (DL1) overexpressed on bone marrow stromal cells, with complete maturation occurring upon adoptive transfer into Rag1-deficient mice. Here, we report that these iPS cells can be differentiated in vivo into functional CTLs after overexpression of MHC I-restricted Ag-specific T-cell receptors (TCR). In this study, we generated murine iPS cells genetically modified with ovalbumin (OVA)-specific and MHC-I restricted TCR (OT-I) by retrovirus-mediated transduction. After their adoptive transfer into recipient mice, the majority of OT-I/iPS cells underwent differentiation into CD8+ CTLs. TCR-transduced iPS cells developed in vivo responded in vitro to peptide stimulation by secreting interleukin 2 and IFN-γ. Most importantly, adoptive transfer of TCR-transduced iPS cells triggered infiltration of OVA-reactive CTLs into tumor tissues and protected animals from tumor challenge. Taken together, our findings offer proof of concept for a potentially more efficient approach to generate Ag-specific T lymphocytes for adoptive immunotherapy.
    Cancer Research 05/2011; 71(14):4742-7. · 9.28 Impact Factor
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    ABSTRACT: Recent phylogenic studies indicate that DNA recombination could have occurred in ancient papillomavirus types. However, no experimental data are available to demonstrate this event because of the lack of human papillomavirus infection models. We have used the cottontail rabbit papillomavirus (CRPV)/rabbit model to study pathogenesis and immunogenicity of different mutant genomes in vivo. Although the domestic rabbit is not a natural host for CRPV infection, it is possible to initiate infection with naked CRPV DNA cloned into a plasmid and monitor papilloma outgrowth on these animals. Taking advantage of a large panel of mutants based on a CRPV strain (Hershey CRPV), we tested the hypothesis that two non-viable mutant genomes could induce papillomas by either recombination or complementation. We found that co-infection with a dysfunctional mutant with an E2 transactivation domain mutation and another mutant with an E7 ATG knock out generated papillomas in rabbits. DNA extracted from these papillomas contained genotypes from both parental genomes. Three additional pairs of dysfunctional mutants also showed similar results. Individual wild type genes were also shown to rescue the function of corresponding dysfunctional mutants. Therefore, we suggest that complementation occurred between these two non-viable mutant PV genomes in vivo.
    Virus Research 05/2009; 144(1-2):117-22. · 2.75 Impact Factor
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    ABSTRACT: Shope papillomavirus or cottontail rabbit papillomavirus (CRPV) is one of the first small DNA tumour viruses to be characterized. Although the natural host for CRPV is the cottontail rabbit (Sylvilagus floridanus), CRPV can infect domestic laboratory rabbits (Oryctolagus cuniculus) and induce tumour outgrowth and cancer development. In previous studies, investigators attempted to passage CRPV in domestic rabbits, but achieved very limited success, leading to the suggestion that CRPV infection in domestic rabbits was abortive. The persistence of specific anti-L1 antibody in sera from rabbits infected with either virus or viral DNA led us to revisit the questions as to whether L1 and infectious CRPV can be produced in domestic rabbit tissues. We detected various levels of L1 protein in most papillomas from CRPV-infected rabbits using recently developed monoclonal antibodies. Sensitive in vitro infectivity assays additionally confirmed that extracts from these papillomas were infectious. These studies demonstrated that the CRPV/New Zealand White rabbit model could be used as an in vivo model to study natural virus infection and viral life cycle of CRPV and not be limited to studies on abortive infections.
    Journal of General Virology 01/2008; 88(Pt 12):3286-93. · 3.13 Impact Factor
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    ABSTRACT: Three transgenic rabbit lines that express a well-characterized human major histocompatibility complex class I (MHC-I) gene (HLA-A2.1) have been established. All three lines carry the HLA-A2.1 heavy chain and are able to pass the transgene to their offspring with both the outbred and the inbred EIII/JC genetic background. HLA-A2.1 colocalizes exclusively with rabbit MHC-I on the cell surfaces. These HLA-A2.1 transgenic rabbits demonstrated infection patterns similar to those found after cottontail rabbit papillomavirus (CRPV) challenge when compared with results in normal rabbits, although higher regression rates were found in HLA-A2.1 transgenic rabbits. Because the CRPV genome can accommodate significant modifications, the CRPV/HLA-A2.1 rabbit model has the potential to be used to screen HLA-A2.1-restricted immunogenic epitopes from human papillomaviruses in the context of in vivo papillomavirus infection.
    Journal of Virology 08/2007; 81(13):7171-7. · 5.08 Impact Factor
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    ABSTRACT: The cottontail rabbit papillomavirus (CRPV)/rabbit model has been used to study oncogenicity and immunogenicity of different antigens from the papillomavirus genome and has therefore served as a preclinical model for the development of preventive and therapeutic vaccines against papillomavirus infections. One unique property of the CRPV model is that infection can be initiated using viral DNA. This property allows for the functional testing of viral mutants in vivo. We have introduced point mutations, insertions and deletions into all of the different coding and non-coding regions of the CRPV genome and have tested their infectivity in this model. We found that the majority of the mutant genomes retained viability and could induce papillomas in domestic rabbits. These data indicated that the CRPV genome is tolerant of many modifications without compromising its ability to initiate skin papillomas. In combination with our recently established HLA-A2.1 transgenic rabbit model, this plasticity allows us to extend the utility of the CRPV/rabbit model to the screening of HLA-A2.1 restricted epitopes from other human viral and tumor antigens.
    Virology 03/2007; 358(2):384-90. · 3.37 Impact Factor
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    ABSTRACT: A human papillomavirus (HPV) vaccine consisting of virus-like particles (VLPs) was recently approved for human use. It is generally assumed that VLP vaccines protect by inducing type-specific neutralizing antibodies. Preclinical animal models cannot be used to test for protection against HPV infections due to species restriction. We developed a model using chimeric HPV capsid/cottontail rabbit papillomavirus (CRPV) genome particles to permit the direct testing of HPV VLP vaccines in rabbits. Animals vaccinated with CRPV, HPV type 16 (HPV-16), or HPV-11 VLPs were challenged with both homologous (CRPV capsid) and chimeric (HPV-16 capsid) particles. Strong type-specific protection was observed, demonstrating the potential application of this approach.
    Journal of Virology 01/2007; 80(24):12393-7. · 5.08 Impact Factor
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    ABSTRACT: We have established several HLA-A2.1-transgenic rabbit lines to provide a host to study CD8(+) T cell responses during virus infections. HLA-A2.1 protein expression was detected on cell surfaces within various organ tissues. Continuous cultured cells from these transgenic rabbits were capable of presenting both endogenous and exogenous HLA-A2.1-restricted epitopes to an HLA-A2.1-restricted epitope-specific CTL clone. A DNA vaccine containing an HLA-A2.1-restricted human papillomavirus type 16 E7 epitope (amino acid residues 82-90) stimulated epitope-specific CTLs in both PBLs and spleen cells of transgenic rabbits. In addition, vaccinated transgenic rabbits were protected against infection with a mutant cottontail rabbit papillomavirus DNA containing an embedded human papillomavirus type 16 E7/82-90 epitope. Complete protection was achieved using a multivalent epitope DNA vaccine based on epitope selection from cottontail rabbit papillomavirus E1 using MHC class I epitope prediction software. HLA-A2.1-transgenic rabbits will be an important preclinical animal model system to study virus-host interactions and to assess specific targets for immunotherapy.
    The Journal of Immunology 01/2007; 177(11):8037-45. · 5.52 Impact Factor
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    ABSTRACT: Papillomaviruses (PVs) demonstrate both tissue and species tropisms. Because PVs replicate only in terminally differentiating epithelium, the recent production of infectious PV particles in 293 cells marks an important breakthrough. In this article, we demonstrate that infectious PV particles produced in 293TT cells can cause papillomatous growths in the natural host animal. Moreover, we show that species-matched PV genomes can be successfully delivered in vivo by a heterologous, species-mismatched PV capsid. Additionally, our results indicate that the addition of the simian virus 40 origin of replication to the papillomavirus genome increases the production of infectious papillomavirus particles by increasing genome amplification in the transfected 293TT cells.
    Journal of Virology 12/2006; 80(22):11381-4. · 5.08 Impact Factor
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    ABSTRACT: Human papillomaviruses (HPVs) replicate only in the terminally differentiating epithelium of the skin and mucosa. While infection of basal keratinocytes is considered a requirement for permissive infection, it remains unclear whether virions can specifically target basal cells for adsorption and uptake following epithelial wounding. We present evidence that HPV binds specifically to laminin 5 (LN5), a component of the extracellular matrix (ECM) secreted by migrating and basal keratinocytes. HPV type 11 capsids colocalized with LN5 in the ECM secreted by vaginal keratinocytes. Binding of both virions and virus-like particles to purified LN5 and to the LN5-rich ECM secreted by cultured keratinocytes was effectively blocked by pretreatment with anti-LN5 antibodies. HPV capsid binding to human cervical mucosa sections included the basement membrane which contains LN5. Cultured keratinocytes expressing alpha6 integrin, a transmembrane protein known to bind LN5, were readily infected by virions preadsorbed to LN5-containing substrates, whereas mutant keratinocytes lacking alpha6 integrin were relatively resistant to infection via this route. These findings suggest a model of natural HPV infection in which proliferating keratinocytes expressing alpha6 integrin at the site of epithelial wounding might be targeted by virions adsorbed transiently to LN5 secreted by migrating keratinocytes.
    Journal of Virology 10/2006; 80(18):8940-50. · 5.08 Impact Factor
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    ABSTRACT: An abundant human papillomavirus (HPV) protein E1/\E4 is expressed late in the virus life cycle in the terminally differentiated layers of epithelia. The expression of E1/\E4 usually coincides with the onset of viral DNA amplification. However, the function of E1/\E4 in viral life cycle is not completely understood. To examine the role of E1/\E4 in the virus life cycle, we introduced a single nucleotide change in the HPV-11 genome to result in a truncation of E1/\E4 protein without affecting the E2 amino acid sequence. This mutated HPV-11 genome was introduced into a human foreskin keratinocyte cell line immortalized by the catalytic subunit of human telomerase, deficient in p16(INK4a) expression, and previously shown to support the HPV-11 life cycle when grown in organotypic raft culture. We have demonstrated that E1/\E4 is dispensable for HPV-11 viral DNA amplification in the late stages of the viral life cycle.
    Virology 09/2006; 351(2):271-9. · 3.37 Impact Factor
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    ABSTRACT: The morphological effects of mutation and disease are often critical to our understanding of normal and abnormal function. The power and popularity of zebrafish as a forward and reverse genetic vertebrate model system, combined with its small size, have made it an ideal model in which to study the genetics of histologically scorable phenotypes. The presence of multiple tissue types in this organism's small larvae also makes it a potentially important model for toxicological analysis. Studying histological phenotypes is greatly enhanced by high-throughput methods of histology. Here, we describe details of high-throughput histology of the zebrafish using larval arrays, along with recent advances in mold design and discussion of work in progress that will lead to easier ways for people in the field to more rapidly score phenotypes in arrays. These detailed descriptions, together with the troubleshooting guide, should enable any laboratory with ties to a histology facility to perform high-throughput histology of zebrafish.
    Methods 08/2006; 39(3):246-54. · 3.64 Impact Factor
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    L Fang, C Meyers, L R Budgeon, M K Howett
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    ABSTRACT: The study of the human papillomavirus (HPV) life cycle was hampered for more than 50 years by the lack of a conventional cell culture system for propagating HPV. Considerable progress has been made in the production of several HPV types using either organotypic rafts or human epithelial xenografts in immunocompromised mice. In this study, we demonstrated episomal maintenance of HPV-11 DNA in N-Tert cells. HPV-11 episomal DNA containing cell populations grown in raft culture showed induction of the productive viral life cycle. HPV-11 DNA amplification and viral capsid antigen synthesis were detected in differentiated layers of epithelia. The viruses generated were able to infect keratinocytes in vitro, which indicate that viruses generated were infectious. The demonstration of the productive HPV-11 life cycle in raft culture from cloned HPV-11 DNA will facilitate genetic analyses of viral gene functions that was not possible using the human xenograft athymic mouse model.
    Virology 04/2006; 347(1):28-35. · 3.37 Impact Factor
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    ABSTRACT: Human papillomaviruses (HPVs) have previously been shown to adsorb to cultured cells via membrane-associated heparan sulfate (HS) and alpha6 integrin. We demonstrate that cultured keratinocytes uniquely secrete a component into the basal extracellular matrix (ECM) which can function to adsorb HPV particles which can then be internalized by adherent cells. This uncharacterized basal ECM adsorption receptor was secreted by normal human epidermal keratinocytes (NHEK) and by each of the four keratinocyte-derived cell lines we examined, but not by non-keratinocyte cell lines. Multiple HPV types bound preferentially to this keratinocyte-specific receptor over the membrane-associated receptor, and binding to the basal ECM adsorption receptor was refractory to inhibition by heparin. Like the membrane-associated receptor, this basal ECM component was functional as an adsorption receptor in our in vitro infection model using HPV-11. Unlike particle adsorption, however, successful infection with HPV-11 virions remained sensitive to the pretreatment of virions with heparin. The secreted basal ECM receptor did not colocalize with antibodies against HS, perlecan, or alpha6 integrin, but colocalized with antibody against laminin-5, a marker of keratinocyte ECM and an abundant component of the basement membrane in mucosa and skin. These findings suggest a model for natural infections in which HPV virions, nonspecifically adsorbed to HS on suprabasal keratinocytes throughout an epithelial wound, might be transferred to mitotically active migrating keratinocytes via an intermediate association with the ECM secreted by these cells as they reestablish the basement membrane.
    Virology 04/2006; 347(1):147-59. · 3.37 Impact Factor
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    ABSTRACT: Papillomavirus major capsid protein L1 has successfully stimulated protective immunity against virus infection by induction of neutralizing antibodies in animal models and in clinical trials. However, the potential impact of L1-induced protective cell-mediated immune (CMI) responses is difficult to measure in vivo because of the coincidence of anti-L1 antibody. In this study, we tested the hypothesis that L1 could activate CMI, using the Cottontail Rabbit Papillomavirus (CRPV)-rabbit model. A unique property of this model is that infections can be initiated with viral DNA, thus bypassing all contributions to protection via neutralizing anti-L1 antibody. DNA vaccines containing either CRPV L1, or subfragments of L1 (amino-terminal two-thirds of L1 [L1N] and the carboxylterminal two-thirds of L1 [L1C]), were delivered intracutaneously into rabbits, using a gene gun. After three booster immunizations, the rabbits were challenged with several viral DNA constructs: wild-type CRPV, CRPV L1ATGko (an L1 ATG knockout mutation), and CRPV-ROPV hybrid (CRPV with a replacement L1 from Rabbit Oral Papillomavirus). Challenge of L1 DNA-vaccinated rabbits with wild-type CRPV resulted in significantly fewer papillomas when compared with challenge with CRPV L1ATGko DNA. Significantly smaller papillomas were found in CRPV L1-, L1N-, and L1C-vaccinated rabbits. In addition, rabbits vaccinated with either L1 or L1N grew significantly fewer and smaller papillomas when challenged with CRPV-ROPV hybrid DNA. Therefore, CRPV L1 DNA vaccination induced CMI responses to CRPV DNA infections that can contribute to protective immunity. Cross-protective immunity against CRPV L1 and ROPV L1 was elicited in these CRPV L1- and subfragment-vaccinated rabbits.
    Viral Immunology 02/2006; 19(3):492-507. · 1.75 Impact Factor

Publication Stats

1k Citations
240.67 Total Impact Points

Institutions

  • 1982–2013
    • Pennsylvania State University
      • • Department of Pathology
      • • Department of Microbiology and Immunology
      State College, PA, United States
  • 2004–2006
    • Drexel University
      Philadelphia, Pennsylvania, United States
  • 2005
    • University of Pennsylvania
      Philadelphia, Pennsylvania, United States
  • 1994–2005
    • Penn State Hershey Medical Center and Penn State College of Medicine
      • • Comparative Medicine
      • • Microbiology and Immunology
      • • Department of Pathology
      Hershey, Pennsylvania, United States
  • 2002
    • Cancer Research Institute
      New York City, New York, United States
  • 1985
    • University of Arkansas at Little Rock
      Little Rock, Arkansas, United States