Publications by authors named "Timothy Lockey"

21 Publications

  • Page 1 of 1

Development and Optimization of a Hydrophobic Interaction Chromatography-Based Method of AAV Harvest, Capture, and Recovery.

Mol Ther Methods Clin Dev 2020 Dec 28;19:275-284. Epub 2020 Sep 28.

Department of Therapeutics Production & Quality, St. Jude Children's Research Hospital, Memphis, TN 38105-3678, USA.

With many ongoing clinical trials utilizing adeno-associated virus (AAV) gene therapy, it is necessary to find scalable and serotype-independent primary capture and recovery methods to allow for efficient and robust manufacturing processes. Here, we demonstrate the ability of a hydrophobic interaction chromatography membrane to capture and recover AAV1, AAV5, AAV8, and AAV "Mutant C" (a novel serotype incorporating elements of AAV3B and AAV8) particles from cell culture media and cell lysate with recoveries of 76%-100% of loaded material, depending on serotype. A simple, novel technique that integrates release and recovery of cell-associated AAV capsids is demonstrated. We show that by the addition of lyotropic salts to AAV-containing cell suspensions, AAV is released at an equivalent efficiency to mechanical lysis. The addition of the lyotropic salt also promotes a phase separation, which allows physical removal of large amounts of DNA and insoluble cellular debris from the AAV-containing aqueous fraction. The AAV is then captured and eluted from a hydrophobic interaction chromatography membrane. This integrated lysis and primary capture and recovery technique facilitates substantial removal of host-cell DNA and host-cell protein impurities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.omtm.2020.09.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7569186PMC
December 2020

Lentiviral Vector Production from a Stable Packaging Cell Line Using a Packed Bed Bioreactor.

Mol Ther Methods Clin Dev 2020 Dec 14;19:1-13. Epub 2020 Aug 14.

Department of Therapeutics Production & Quality, St. Jude Children's Research Hospital, Memphis, TN 38105-3678, USA.

Self-inactivating lentiviral vectors (LVVs) are used regularly for genetic modification of cells, including T cells and hematopoietic stem cells for cellular gene therapy. As vector demand grows, scalable and controllable methods are needed for production. LVVs are typically produced in HEK293T cells in suspension bioreactors using serum-free media or adherent cultures with serum. The iCELLis® is a packed-bed bioreactor for adherent or entrained cells with surface areas from 0.53 to 500 m. Media are pumped through the fixed bed and overflows, creating a thin film that is replenished with oxygen and depleted of CO as media return to the reservoir. We describe the optimization and scale-up of the production of GPRTG-EF1α-hγ-OPT LVV using a stable packaging cell line in the iCELLis Nano 2-cm to the 10-cm bed height low compaction bioreactors (0.53 and 2.6 m surface area) and compare to the productivity and efficacy of GPRTG-EF1α-hγ-OPT LVV manufactured under current Good Manufacturing Practice (cGMP) using 10-layer cell factories for the treatment of X-linked severe combined immunodeficiency. By optimizing fetal bovine serum (FBS) concentration, pH post-induction, and day of induction, we attain viral yields of more than 2 × 10 transducing units/mL. We compared transduction efficiency between LVVs produced from the iCELLis Nano and cell factories on healthy, purified CD34 cells and found similar results.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.omtm.2020.08.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7490643PMC
December 2020

Lentiviral Gene Therapy Combined with Low-Dose Busulfan in Infants with SCID-X1.

N Engl J Med 2019 04;380(16):1525-1534

From the Departments of Bone Marrow Transplantation and Cellular Therapy (E.M., B.T., W.J., S.G.), Hematology (S.Z., Z.M., J.C., J.D., X.T., B.Y.R., M.J.W., B.P.S.), Therapeutics Production and Quality (T.L., M.M.M.), Immunology (H.A., B.Y.), Pharmaceutical Sciences (S.J.C.), Biostatistics (G.K., C.L.), and Infectious Diseases (G.M.), St. Jude Children's Research Hospital, Memphis, TN; the Allergy and Clinical Immunology Division, Hospital Nacional Edgardo Rebagliati Martins, Lima, Peru (J.C.A.B.); the Department of Pediatrics, Allergy-Immunology Division, Children's Hospital Los Angeles, Los Angeles (J.A.C.), and the Department of Pediatrics, Division of Pediatric Allergy-Immunology-Bone Marrow Transplantation, University of California, San Francisco (UCSF) Benioff Children's Hospital, San Francisco (J.R.L.-B., J.M.P., M.J.C.) - both in California; the Department of Pediatrics, Pediatric Allergy and Immunology, University of New Mexico, Albuquerque (E.D.); University of Oklahoma Health Sciences Center, Tulsa (J.T.L.); Departamento de Pediatria da Universidade de Taubaté, Conselho Nacional de Medicina, São Paulo (A.C.M.A.); Copperfield Childcare, Claremont, South Africa (H.W.); and the Genetic Immunotherapy Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (S.S.D.R., H.L.M.).

Background: Allogeneic hematopoietic stem-cell transplantation for X-linked severe combined immunodeficiency (SCID-X1) often fails to reconstitute immunity associated with T cells, B cells, and natural killer (NK) cells when matched sibling donors are unavailable unless high-dose chemotherapy is given. In previous studies, autologous gene therapy with γ-retroviral vectors failed to reconstitute B-cell and NK-cell immunity and was complicated by vector-related leukemia.

Methods: We performed a dual-center, phase 1-2 safety and efficacy study of a lentiviral vector to transfer complementary DNA to bone marrow stem cells after low-exposure, targeted busulfan conditioning in eight infants with newly diagnosed SCID-X1.

Results: Eight infants with SCID-X1 were followed for a median of 16.4 months. Bone marrow harvest, busulfan conditioning, and cell infusion had no unexpected side effects. In seven infants, the numbers of CD3+, CD4+, and naive CD4+ T cells and NK cells normalized by 3 to 4 months after infusion and were accompanied by vector marking in T cells, B cells, NK cells, myeloid cells, and bone marrow progenitors. The eighth infant had an insufficient T-cell count initially, but T cells developed in this infant after a boost of gene-corrected cells without busulfan conditioning. Previous infections cleared in all infants, and all continued to grow normally. IgM levels normalized in seven of the eight infants, of whom four discontinued intravenous immune globulin supplementation; three of these four infants had a response to vaccines. Vector insertion-site analysis was performed in seven infants and showed polyclonal patterns without clonal dominance in all seven.

Conclusions: Lentiviral vector gene therapy combined with low-exposure, targeted busulfan conditioning in infants with newly diagnosed SCID-X1 had low-grade acute toxic effects and resulted in multilineage engraftment of transduced cells, reconstitution of functional T cells and B cells, and normalization of NK-cell counts during a median follow-up of 16 months. (Funded by the American Lebanese Syrian Associated Charities and others; LVXSCID-ND ClinicalTrials.gov number, NCT01512888.).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1056/NEJMoa1815408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6636624PMC
April 2019

Development and Optimization of AAV hFIX Particles by Transient Transfection in an iCELLis(®) Fixed-Bed Bioreactor.

Hum Gene Ther Methods 2016 06;27(3):112-21

Department of Therapeutics Production & Quality, St. Jude Children's Research Hospital , Memphis, Tennessee.

Adeno-associated virus (AAV) vectors are increasingly popular in gene therapy because they are unassociated with human disease, replication dependent, and less immunogenic than other viral vectors and can infect a variety of cell types. These vectors have been used in over 130 clinical trials, and one AAV product has been approved for treatment of lipoprotein lipase deficiency in Europe. To meet the demand for the increasing quantities of AAV required for clinical trials and treatment, a scalable high-capacity technology is required. Bioreactors meet these requirements but limited options are available for adherent HEK 293T/17 cells. Here we optimize the transient transfection of HEK293T/17 cells for the production of AAV human factor IX in a disposable fixed-bed bioreactor, the iCELLis(®) Nano (PALL Corporation). A fixed bed in the center of the iCELLis bioreactor is surrounded by culture medium that is pumped through the bed from the bottom of the bioreactor so that a thin film of the medium overflows the bed and is replenished with oxygen and depleted of CO2 as it returns to the surrounding medium reservoir. We show that this fixed-bed bioreactor can support as many as 2.5 × 10(8) cells/ml of fixed bed (1.9 × 10(6) cells/cm(2)). By optimizing culture and transfection parameters such as the concentration of DNA for transfection, day of harvest, size of PEI/DNA particles, and transfection medium, and adding an additional medium change to the process, we increased our yield to as high as 9.0 × 10(14) viral particles per square meter of fixed bed. We also show an average GFP transfection of 97% of cells throughout the fixed bed. These yields make the iCELLis a promising scalable technology for the clinical production of AAV gene therapy products.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/hgtb.2016.021DOI Listing
June 2016

Distribution of AAV8 particles in cell lysates and culture media changes with time and is dependent on the recombinant vector.

Mol Ther Methods Clin Dev 2016 30;3:16015. Epub 2016 Mar 30.

Department of Therapeutics Production & Quality, St. Jude Children's Research Hospital , Memphis, Tennessee, USA.

With clinical trials ongoing, efficient clinical production of adeno-associated virus (AAV) to treat large numbers of patients remains a challenge. We compared distribution of AAV8 packaged with Factor VIII (FVIII) in cell culture media and lysates on days 3, 5, 6, and 7 post-transfection and found increasing viral production through day 6, with the proportion of viral particles in the media increasing from 76% at day 3 to 94% by day 7. Compared to FVIII, AAV8 packaged with Factor IX and Protective Protein/Cathepsin A vectors demonstrated a greater shift from lysate towards media from day 3 to 6, implying that particle distribution is dependent on recombinant vector. Larger-scale productions showed that the ratio of full-to-empty AAV particles is similar in media and lysate, and that AAV harvested on day 6 post-transfection provides equivalent function in mice compared to AAV harvested on day 3. This demonstrates that AAV8 production can be optimized by prolonging the duration of culture post-transfection, and simplified by allowing harvest of media only, with disposal of cells that contain 10% or less of total vector yield. Additionally, the difference in particle distribution with different expression cassettes implies a recombinant vector-dependent processing mechanism which should be taken into account during process development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/mtm.2016.15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4813606PMC
April 2016

Ex vivo activation of CD56(+) immune cells that eradicate neuroblastoma.

Cancer Res 2013 Apr 25;73(8):2608-18. Epub 2013 Feb 25.

Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.

Despite the use of intensive contemporary multimodal therapy, the overall survival of patients with high-risk neuroblastoma is still less than 50%. Therefore, immunotherapy without cross-resistance and overlapping toxicity has been proposed. In this study, we report the development of a novel strategy to specifically activate and expand human CD56(+) (NCAM1) natural killer (NK) immune cells from normal donors and patients with neuroblastoma. Enriched CD56(+) cells from peripheral blood were mixed with CD56(-) fraction at 1:1 ratio and cultured in the presence of OKT3, interleukin (IL)-2, and -15 for five days and then without OKT3 for 16 more days. The final products contained more than 90% CD56(+) cells and could kill neuroblastoma cells effectively that were originally highly resistant to nonprocessed NK cells. Mechanistically, cytolysis of neuroblastoma was mediated through natural cytotoxicity receptor (NCR), DNAX accessory molecule-1 (DNAM-1; CD226), perforin, and granzyme B. Successful clinical scale-up in a good manufacturing practices (GMP)-compliant bioreactor yielded effector cells that in a neuroblastoma xenograft model slowed tumor growth and extended survival without GVHD. Investigation of CD56(+) cells from patients with neuroblastoma revealed a similar postactivation phenotype and lytic activity. Our findings establish a novel and clinically expedient strategy to generate allogeneic or autologous CD56(+) cells that are highly cytotoxic against neuroblastoma with minimal risk of GVHD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-12-3322DOI Listing
April 2013

Transduction of human CD34+ repopulating cells with a self-inactivating lentiviral vector for SCID-X1 produced at clinical scale by a stable cell line.

Hum Gene Ther Methods 2012 Oct 7;23(5):297-308. Epub 2012 Nov 7.

Department of Hematology, St. Jude Children's Hospital, Memphis, TN 38105, USA.

Self-inactivating (SIN)-lentiviral vectors have safety and efficacy features that are well suited for transduction of hematopoietic stem cells (HSCs), but generation of vector at clinical scale has been challenging. Approximately 280 liters of an X-Linked Severe Combined Immunodeficiency Disorder (SCID-X1) SIN-lentiviral vector in two productions from a stable cell line were concentrated to final titers of 4.5 and 7.2×10(8) tu/ml. These two clinical preparations and three additional development-scale preparations were evaluated in human CD34(+) hematopoietic cells in vitro using colony forming cell (CFU-C) assay and in vivo using the NOD/Lt-scid/IL2Rγ(null) (NSG) mouse xenotransplant model. A 40-hour transduction protocol using a single vector exposure conferred a mean NSG repopulating cell transduction of 0.23 vector genomes/human genome with a mean myeloid vector copy number of 3.2 vector genomes/human genome. No adverse effects on engraftment were observed from vector treatment. Direct comparison between our SIN-lentiviral vector using a 40-hour protocol and an MFGγ(c) γ-retroviral vector using a five-day protocol demonstrated equivalent NSG repopulating cell transduction efficiency. Clonality survey by linear amplification-mediated polymerase chain reaction (LAM-PCR) with Illumina sequencing revealed common clones in sorted myeloid and lymphoid populations from engrafted mice demonstrating multipotent cell transduction. These vector preparations will be used in two clinical trials for SCID-X1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/hgtb.2012.150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3732136PMC
October 2012

A clinically adaptable method to enhance the cytotoxicity of natural killer cells against B-cell malignancies.

Cytotherapy 2012 Aug 29;14(7):830-40. Epub 2012 Mar 29.

Department of Oncology, St Jude Children's Research Hospital, Memphis, Tennessee, USA.

Background Aims: Retroviral transduction of anti-CD19 chimeric antigen receptors significantly enhances the cytotoxicity of natural killer (NK) cells against B-cell malignancies. We aimed to validate a more practical, affordable and safe method for this purpose.

Methods: We tested the expression of a receptor containing CD3ζ and 4-1BB signaling molecules (anti-CD19-BB-ζ) in human NK cells after electroporation with the corresponding mRNA using a clinical-grade electroporator. The cytotoxic capacity of the transfected NK cells was tested in vitro and in a mouse model of leukemia.

Results: Median anti-CD19-BB-ζ expression 24 h after electroporation was 40.3% in freshly purified (n =18) and 61.3% in expanded (n = 31) NK cells; median cell viability was 90%. NK cells expressing anti-CD19-BB-ζ secreted interferon (IFN)-γ in response to CD19-positive target cells and had increased cytotoxicity. Receptor expression was detectable 6 h after electroporation, reaching maximum levels at 24-48 h; specific anti-CD19 cytotoxicity was observed at 96 h. Levels of expression and cytotoxicities were comparable with those achieved by retroviral transduction. A large-scale protocol was developed and applied to expanded NK cells (median NK cell number 2.5 × 10(8), n = 12). Median receptor expression after 24 h was 82.0%; NK cells transfected under these conditions exerted considerable cytotoxicity in xenograft models of B-cell leukemia.

Conclusions: The method described here represents a practical way to augment the cytotoxicity of NK cells against B-cell malignancies. It has the potential to be extended to other targets beyond CD19 and should facilitate the clinical use of redirected NK cells for cancer therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3109/14653249.2012.671519DOI Listing
August 2012

Heterologous Prime-Boost HIV-1 Vaccination Regimens in Pre-Clinical and Clinical Trials.

Viruses 2010 Feb;2(2):435-467

Department of Immunology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN, USA;

Currently, there are more than 30 million people infected with HIV-1 and thousands more are infected each day. Vaccination is the single most effective mechanism for prevention of viral disease, and after more than 25 years of research, one vaccine has shown somewhat encouraging results in an advanced clinical efficacy trial. A modified intent-to-treat analysis of trial results showed that infection was approximately 30% lower in the vaccine group compared to the placebo group. The vaccine was administered using a heterologous prime-boost regimen in which both target antigens and delivery vehicles were changed during the course of inoculations. Here we examine the complexity of heterologous prime-boost immunizations. We show that the use of different delivery vehicles in prime and boost inoculations can help to avert the inhibitory effects caused by vector-specific immune responses. We also show that the introduction of new antigens into boost inoculations can be advantageous, demonstrating that the effect of `original antigenic sin' is not absolute. Pre-clinical and clinical studies are reviewed, including our own work with a three-vector vaccination regimen using recombinant DNA, virus (Sendai virus or vaccinia virus) and protein. Promising preliminary results suggest that the heterologous prime-boost strategy may possibly provide a foundation for the future prevention of HIV-1 infections in humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/v2020435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2855973PMC
February 2010

SHIV infection protects against heterologous pathogenic SHIV challenge in macaques: a gold-standard for HIV-1 vaccine development?

Curr HIV Res 2009 Sep;7(5):497-503

Department of Infectious Diseases, St Jude Children's Research Hospital, Memphis, TN 38105, USA.

A current debate in the HIV-1 vaccine field concerns the ability of an immunodeficiency virus to elicit a protective response. One argument is that HIV-1 superinfections are frequent in healthy individuals, because virus evades conventional immune surveillance, a serious obstacle to vaccine design. The opposing argument is that protection from superinfection is significant, reflecting a robust immune response that might be harnessed by vaccination to prevent disease. In an experiment designed to address the debate, two macaques received an I.V. inoculation with SHIV KU-1-d (a derivative of SHIV KU-1) and were rested for >10 months. Infection elicited diverse neutralizing antibody activities in both animals. Animals were then exposed to SHIV 89.6P (I.V.), a virus carrying a heterologous envelope protein relative to the vaccine strain. Infection was monitored by viral load and CD4+ T-cell measurements. All control animals were infected and most succumbed to disease. In contrast, protection from superinfection was statistically significant in test monkeys; one animal showed no evidence of superinfection at any time point and the second showed evidence of virus at only one time point over a 6-month observation period. Neither animal showed signs of disease. Perhaps this protective state may serve as a 'gold-standard' for HIV-1 vaccine development, as a similar degree of protection against immunodeficiency virus infections in humans would be much desired.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2782824PMC
http://dx.doi.org/10.2174/157016209789346255DOI Listing
September 2009

Expansion of highly cytotoxic human natural killer cells for cancer cell therapy.

Cancer Res 2009 May 21;69(9):4010-7. Epub 2009 Apr 21.

Department of Oncology, Hartwell Center for Bioinformatics and Biotechnology, St Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.

Infusions of natural killer (NK) cells are an emerging tool for cancer immunotherapy. The development of clinically applicable methods to produce large numbers of fully functional NK cells is a critical step to maximize the potential of this approach. We determined the capacity of the leukemia cell line K562 modified to express a membrane-bound form of interleukin (IL)-15 and 41BB ligand (K562-mb15-41BBL) to generate human NK cells with enhanced cytotoxicity. Seven-day coculture with irradiated K562-mb15-41BBL induced a median 21.6-fold expansion of CD56(+)CD3(-) NK cells from peripheral blood (range, 5.1- to 86.6-fold; n = 50), which was considerably superior to that produced by stimulation with IL-2, IL-12, IL-15, and/or IL-21 and caused no proliferation of CD3(+) lymphocytes. Similar expansions could also be obtained from the peripheral blood of patients with acute leukemia undergoing therapy (n = 11). Comparisons of the gene expression profiles of the expanded NK cells and their unstimulated or IL-2-stimulated counterparts showed marked differences. The expanded NK cells were significantly more potent than unstimulated or IL-2-stimulated NK cells against acute myeloid leukemia cells in vitro. They could be detected for >1 month when injected into immunodeficient mice and could eradicate leukemia in murine models of acute myeloid leukemia. We therefore adapted the K562-mb15-41BBL stimulation method to large-scale clinical-grade conditions, generating large numbers of highly cytotoxic NK cells. The results that we report here provide rationale and practical platform for clinical testing of expanded and activated NK cells for cell therapy of cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-08-3712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2716664PMC
May 2009

Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection.

Blood 2009 May 13;113(21):5104-10. Epub 2009 Mar 13.

Department of Hematology, St Jude Children's Research Hospital, Memphis, TN 38105, USA.

Retroviral vectors containing internal promoters, chromatin insulators, and self-inactivating (SIN) long terminal repeats (LTRs) may have significantly reduced genotoxicity relative to the conventional retroviral vectors used in recent, otherwise successful clinical trials. Large-scale production of such vectors is problematic, however, as the introduction of SIN vectors into packaging cells cannot be accomplished with the traditional method of viral transduction. We have derived a set of packaging cell lines for HIV-based lentiviral vectors and developed a novel concatemeric array transfection technique for the introduction of SIN vector genomes devoid of enhancer and promoter sequences in the LTR. We used this method to derive a producer cell clone for a SIN lentiviral vector expressing green fluorescent protein, which when grown in a bioreactor generated more than 20 L of supernatant with titers above 10(7) transducing units (TU) per milliliter. Further refinement of our technique enabled the rapid generation of whole populations of stably transformed cells that produced similar titers. Finally, we describe the construction of an insulated, SIN lentiviral vector encoding the human interleukin 2 receptor common gamma chain (IL2RG) gene and the efficient derivation of cloned producer cells that generate supernatants with titers greater than 5 x 10(7) TU/mL and that are suitable for use in a clinical trial for X-linked severe combined immunodeficiency (SCID-X1).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2008-11-191049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2686181PMC
May 2009

Preclinical and clinical development of a multi-envelope, DNA-virus-protein (D-V-P) HIV-1 vaccine.

Int Rev Immunol 2009 ;28(1):49-68

Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.

The human immune system has evolved to recognize antigenic diversity, a strength that has been harnessed by vaccine developers to combat numerous pathogens (e.g., pneumococcus, influenza virus, rotavirus). In each case, vaccine cocktails were formulated to include antigenic variants of the target. To combat HIV-1 diversity, we assembled a cocktail vaccine comprising dozens of envelopes, delivered as recombinant DNA, vaccinia virus, and protein for testing in a clinical trial. One vaccinee has now completed vaccinations with no serious adverse events. Preliminary analyses demonstrate early proof-of-principle that a multi-envelope vaccine can elicit neutralizing antibody responses toward heterologous HIV-1 in humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/08830180802495605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2649747PMC
April 2009

Epstein-Barr virus vaccine development: a lytic and latent protein cocktail.

Front Biosci 2008 May 1;13:5916-27. Epub 2008 May 1.

Department of Immunology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105, USA.

Epstein-Barr Virus (EBV) is the causative agent of acute infectious mononucleosis and associates with malignancies such as Burkitt lymphoma, nasopharyngeal carcinoma, and non-Hodgkin's lymphoma. Additionally, EBV is responsible for B-lymphoproliferative disease in the context of HIV-infection, genetic immunodeficiencies and organ/stem-cell transplantation. Here we discuss past and current efforts to design an EBV vaccine. We further describe preliminary studies of a novel cocktail vaccine expressing both lytic and latent EBV proteins. Specifically, a tetrameric vaccinia virus (VV) -based vaccine was formulated to express the EBV lytic proteins gp350 and gp110, and the latent proteins EBNA-2 and EBNA-3C. In a proof-of-concept study, mice were vaccinated with the individual or mixed VV. Each of the passenger genes was expressed in vivo at levels sufficient to elicit binding antibody responses. Neutralizing gp350-specific antibodies were also elicited, as were EBV-specific T-cell responses, following inoculation of mice with the single or mixed VV. Results encourage further development of the cocktail vaccine strategy as a potentially powerful weapon against EBV infection and disease in humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2741/3126DOI Listing
May 2008

First phase I clinical trial of an HIV-1 subtype D gp140 envelope protein vaccine: immune activity induced in all study participants.

AIDS 2008 Jan;22(1):149-51

Department of Immunology, St Jude Children's Research Hospital, Memphis, Tennessee, USA.

To combat HIV-1 diversity, we are developing a multienvelope vaccine (comprising DNA, vaccinia virus and protein vectors). Toward this goal, we conducted a phase I clinical trial of EnvPro, a gp140 protein formulated in alum. The vaccine was well tolerated and elicited an immune response in every trial participant.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/QAD.0b013e3282f174edDOI Listing
January 2008

HIV-1 vaccine development: tackling virus diversity with a multi-envelope cocktail.

Front Biosci 2008 Jan 1;13:609-20. Epub 2008 Jan 1.

Department of Immunology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105, USA.

A major obstacle to the design of a global HIV-1 vaccine is viral diversity. At present, data suggest that a vaccine comprising a single antigen will fail to generate broadly reactive B-cell and T-cell responses able to confer protection against the diverse isolates of HIV-1. While some B-cell and T-cell epitopes lie within the more conserved regions of HIV-1 proteins, many are localized to variable regions and differ from one virus to the next. Neutralizing B-cell responses may vary toward viruses with different i) antibody contact residues and/or ii) protein conformations while T-cell responses may vary toward viruses with different (i) T-cell receptor contact residues and/or (ii) amino acid sequences pertinent to antigen processing. Here we review previous and current strategies for HIV-1 vaccine development. We focus on studies at St. Jude Children's Research Hospital (SJCRH) dedicated to the development of an HIV-1 vaccine cocktail strategy. The SJCRH multi-vectored, multi-envelope vaccine has now been shown to elicit HIV-1-specific B- and T-cell functions with a diversity and durability that may be required to prevent HIV-1 infections in humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2741/2706DOI Listing
January 2008

A Multi-Vector, Multi-Envelope HIV-1 Vaccine.

J Pediatr Pharmacol Ther 2007 Apr;12(2):68-76

Departments of Immunology ; Infectious Diseases, St. Jude Children's Research Hospital ; Departments of Pathology.

The St. Jude Children's Research Hospital (St. Jude) HIV-1 vaccine program is based on the observation that multiple antigenically distinct HIV-1 envelope protein structures are capable of mediating HIV-1 infection. A cocktail vaccine comprising representatives of these diverse structures (immunotypes) is therefore considered necessary to elicit lymphocyte populations that prevent HIV-1 infection. This strategy is reminiscent of that used to design a currently licensed and successful 23-valent pneumococcus vaccine. Three recombinant vector systems are used for the delivery of envelope cocktails (DNA, vaccinia virus, and purified protein), and each of these has been tested individually in phase I safety trials. A fourth FDA-approved clinical trial, in which diverse envelopes and vectors are combined in a prime-boost vaccination regimen, has recently begun. This trial will continue to test the hypothesis that a multi-vector, multi-envelope vaccine can elicit diverse B- and T-cell populations that can prevent HIV-1 infections in humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.5863/1551-6776-12.2.68DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3462093PMC
April 2007

Multi-envelope HIV-1 vaccine devoid of SIV components controls disease in macaques challenged with heterologous pathogenic SHIV.

Vaccine 2005 Nov 20;23(46-47):5306-20. Epub 2005 Jul 20.

Department of Infectious Diseases, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105, USA.

A central obstacle to the design of a global HIV-1 vaccine is virus diversity. Pathogen diversity is not unique to HIV-1, and has been successfully conquered in other fields by the creation of vaccine cocktails. Here we describe the testing of an HIV-1 envelope cocktail vaccine. Six macaques received the vaccine, delivered by successive immunizations with recombinant DNA, recombinant vaccinia virus and recombinant envelope proteins. Following vaccination, animals developed a diversity of anti-envelope antibody binding and neutralizing activities toward proteins and viruses that were not represented by sequence in the vaccine. T-cells were also elicited, as measured by gamma-interferon production assays with envelope-derived peptide pools. Vaccinated and control animals were then challenged with the heterologous pathogenic SHIV, 89.6P. Vaccinated monkeys experienced significantly lower virus titers and better maintenance of CD4+ T-cells than unvaccinated controls. The B- and T-cell immune responses were far superior post-challenge in the vaccinated group. Four of six vaccinated animals and only one of six control animals survived a 44-week observation period post-challenge. The present report is the first to describe pathogenic SHIV disease control mediated by a heterologous HIV-1 vaccine, devoid of 89.6 or SIV derivatives.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.vaccine.2005.07.008DOI Listing
November 2005

HIV vaccine rationale, design and testing.

Curr HIV Res 2005 Apr;3(2):107-12

Department of Infectious Diseases, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105, USA.

A central obstacle to the design of a global HIV vaccine is viral diversity. Antigenic differences in envelope proteins result in distinct HIV serotypes, operationally defined such that antibodies raised against envelope molecules from one serotype will not bind envelope molecules from a different serotype. The existence of serotypes has presented a similar challenge to vaccine development against other pathogens. In such cases, antigenic diversity has been addressed by vaccine design. For example, the poliovirus vaccine includes three serotypes of poliovirus, and Pneumovax presents a cocktail of 23 pneumococcal variants to the immune system. It is likely that a successful vaccine for HIV must also comprise a cocktail of antigens. Here, data relevant to the development of cocktail vaccines, designed to harness diverse, envelope-specific B-cell and T-cell responses, are reviewed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/1570162053506928DOI Listing
April 2005

T cell epitope "hotspots" on the HIV Type 1 gp120 envelope protein overlap with tryptic fragments displayed by mass spectrometry.

AIDS Res Hum Retroviruses 2005 Feb;21(2):165-70

Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.

Our previous work has shown that immunodominant T-helper cell epitopes cluster within distinct fragments on a single face of the HIV envelope gp120 protein. We show in this report that the general positions of immunodominant epitopes are shared by T cells derived from BALB/c, C57BL/6, and CB6F1 mice, yet the precise peptides recognized by the responding T cell populations may differ. In addition, we find that gp120 peptides displayed by tryptic digestion and mass spectrometry of a purified HIV envelope protein share location with peptides defined as immunodominant T cell targets. Results are consistent with the suggestion that gp120 peptide location influences antigen processing, which, in turn, influences the specificity of immunodominant T cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/aid.2005.21.165DOI Listing
February 2005

A five-residue HIV envelope helper T cell determinant: does this peptide-MHC interaction leave the binding groove half empty?

AIDS Res Hum Retroviruses 2002 Oct;18(15):1141-4

Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.

High-resolution structures have revealed major pockets in the MHC class II peptide binding groove within a region designated Pl-P9. The region can accommodate 9-mer peptides, consistent with the observation that minimal core helper T (Th) cell determinants are usually eight or nine residues in size. Here we describe mouse Th cell hybridomas that are specific for a core peptide of only five residues (NPIIL) in the HIV envelope glycoprotein. Effective Th cell stimulation requires that these MHC class II Ia(b)-presented peptides contain amino acids flanking the minimal pentamer, but the flanking residues may be located on either the N or C terminus. To explain these findings, we suggest that mini-Th cell epitopes may effectively associate with MHC when only five (or possibly fewer) of the P1-P9 positions are filled. The remaining positions may be empty, or may be associated with a second, perhaps unrelated, peptide moiety.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/088922202320567888DOI Listing
October 2002