Biomedical subjects

Christopher P Locher

Publications and source records attributed to Christopher P Locher.

11 recordsLinked to original sources

PubMed2005

DNA shuffling and screening strategies for improving vaccine efficacy.

The efficacy of vaccines can be improved by increasing their immunogenicity, broadening their crossprotective range, as well as by developing immunomodulators that can be coadministered with the vaccine antigen. One technology that can be applied to each of these aspects of vaccine development is MolecularBreeding directed molecular evolution. Essentially, this technology is used to evolve genes in vitro through an iterative process consisting of recombinant generation followed by selection of the desired recombinants. We have used DNA shuffling and screening strategies to develop and improve vaccine candidates against several infectious pathogens including Plasmodium falciparum (a common cause of severe and fatal human malaria), dengue virus, encephalitic alphaviruses such as Venezuelan, western and eastern equine encephalitis viruses (VEEV, WEEV, and EEEV, respectively), human immunodeficiency virus-1 (HIV-1), and hepatitis B virus (HBV). By recombining antigen-encoding genes from different serovar isolates, new chimeras are selected for crossreactivity; these vaccine candidates are expected to provide broader crossprotection than vaccines based on a single serovar. Furthermore, the vaccine candidates can be selected for improved immunogenicity, which would also improve their efficacy. In addition to vaccine candidates, we have applied the technology to evolve several immunomodulators that when coadministered with vaccines can improve vaccine efficacy by fine-tuning the T cell response. Thus, DNA shuffling and screening technology is a promising strategy to facilitate vaccine efficacy.

Antigens↗

PubMed2005

Differential effects of R5 and X4 human immunodeficiency virus type 1 infection on CD4+ cell proliferation and activation.

Human immunodeficiency virus type 1 (HIV-1) isolates can be distinguished by their chemokine coreceptor usage. Non-syncytium-inducing (NSI), macrophage-tropic viruses utilize CCR5 and are called R5 viruses; syncytium-inducing (SI) isolates use CXCR4 and are known as X4 viruses. R5 and X4 HIV isolates are both transmitted but, in most cases, R5 viruses predominate in the blood prior to the development of AIDS-related pathogenesis. The reason for the selective growth of the R5 strain is not known, but could reflect a replication advantage of R5 viruses over X4 viruses in CD4+ cells. To explore this possibility, eight phenotypically distinct viruses were used to infect CD4+ cells and cellular proliferation and activation were evaluated. In unstimulated CD4+ cells, R5 virus isolates increased the level of cell activation compared with X4 virus isolates and uninfected control cells. In CD4+ cells that were stimulated with interleukin 2, both R5 and X4 viruses were found to decrease the level of cell proliferation and reduce the majority of the activation markers studied when compared with uninfected control CD4+ cells from the same donors. However, although equal amounts of CD4+ cells were infected, R5 virus-infected CD4+ cells showed a two- to fourfold increase in cellular proliferation over X4 viruses, as measured by [3H]thymidine incorporation (P=0.001) and nuclear expression of Ki67 (P=0.001). In addition, a larger proportion of CD4+ T cells infected with R5 viruses had significantly higher levels of activation-marker expression (e.g. CD25, CD71 and HLA-DR) than CD4+ T lymphocytes infected with X4 viruses (P<0.02). Taken together, these results indicate that CD4+ cells infected with R5 virus isolates may have a selective advantage over X4 virus-infected CD4+ T cells for survival and, hence, virus spread.

Amino Acid Sequence↗

PubMed2004

Human immunodeficiency virus type 2 DNA vaccine provides partial protection from acute baboon infection.

We determined if the genetic adjuvants, granulocyte-macrophage colony stimulating factor (GM-CSF) and B7-2, could improve the immunogenicity and efficacy of an HIV-2 DNA vaccine. The vaccine consisted of the HIV-2 tat, nef, gag, and env genes synthesized using optimized codons and formulated with cationic liposomes. Baboons (Papio cynocephalus hamadryas) were immunized by the intramuscular, intradermal, and intranasal routes with these expression constructs and challenged with HIV-2(UC2) by the intravaginal route. In the first month after HIV-2 vaginal challenge, the baboons receiving the HIV-2 DNA vaccine with or without the genetic adjuvants had significant reductions in the viral loads in the peripheral blood mononuclear cells (PBMC) (P = 0.028) while the reductions in their plasma viremia were suggestive of a protective effect (P = 0.1). These data demonstrate that partial protection against HIV-2 vaginal challenge, as measured by reduced viral load, can be achieved using only a DNA vaccine formulation.

Adjuvants, Immunologic↗

PubMed2004

Evaluation of genetic immunization adjuvants to improve the effectiveness of a human immunodeficiency virus type 2 (HIV-2) envelope DNA vaccine.

In an effort to develop a more effective genetic immunization strategy for HIV, we developed an HIV-2 env DNA vaccine and evaluated three adjuvant formulations. The gp140 gene from HIV-2(UC2 )was synthesized using mammalian codons and cloned into a plasmid vector that expresses eukaryotic genes at high levels. We found that after three immunizations in mice, a novel cationic liposome formulation (Vaxfectin) was superior at inducing systemic and mucosal antibody responses compared to a naked DNA, a controlled release device (an Alzet minipump) and polysaccharide microparticles made from chitosan (P = 0.027). Vaxfectin also induced higher levels of systemic antibodies for each isotype and IgG subclass as well as levels of HIV-2-specific mucosal IgA (P = 0.034). When different routes of immunization were used with the Vaxfectin formulation, gp140-specific systemic antibody responses were highest by the intradermal route, mucosal antibody responses were highest by the intramuscular route, while the intranasal route was the least effective. These results suggest that this cationic liposome formulation is an important adjuvant to improve the effectiveness of genetic immunization strategies for AIDS, and that multiple routes of immunization should be employed for optimal efficacy for HIV vaccine candidates.

Adjuvants, Immunologic↗

PubMed2004

Overcoming antigenic diversity and improving vaccines using DNA shuffling and screening technologies.

Viral, bacterial and parasitic pathogens have evolved multiple strategies to evade the immune response, facilitate transmission and establish chronic infections. One of the underlying strategies that pathogens have evolved is antigenic variation of immune response targets that reduce the affinity of antigen binding to antibodies and major histocompatability complex class I and II receptors. Vaccine candidates generally target a limited number of these antigen variants or combine antigens from several variants to include in multivalent vaccine formulations. DNA shuffling and screening technologies, also known as MolecularBreeding (Maxygen, Inc.) directed molecular evolution, have been successfully used to identify and develop novel and chimaeric vaccine candidates capable of inducing immune responses that recognise and control multiple antigenic variants. DNA shuffling and screening strategies also select vaccine candidates with improved immunogenicity, increased expression as recombinant polypeptides and improved growth of whole viruses in cell culture. As DNA shuffling and screening strategies can be applied to many pathogens, there remain numerous applications of DNA shuffling to solve challenging problems in vaccine process development and manufacture.

Animals↗

PubMed2004

Development of novel vaccines using DNA shuffling and screening strategies.

DNA shuffling and screening technologies recombine and evolve genes in vitro to rapidly obtain molecules with improved biological activity and fitness. In this way, genes from related strains are bred like plants or livestock and their successive progeny are selected. These technologies have also been called molecular breeding-directed molecular evolution. Recent developments in bioinformatics-assisted computer programs have facilitated the design, synthesis and analysis of DNA shuffled libraries of chimeric molecules. New applications in vaccine development are among the key features of DNA shuffling and screening technologies because genes from several strains or antigenic variants of pathogens can be recombined to create novel molecules capable of inducing immune responses that protect against infections by multiple strains of pathogens. In addition, molecules such as co-stimulatory molecules and cytokines have been evolved to have improved T-cell proliferation and cytokine production compared with the wild-type human molecules. These molecules can be used to immunomodulate vaccine responsiveness and have multiple applications in infectious diseases, cancer, allergy and autoimmunity. Moreover, DNA shuffling and screening technologies can facilitate process development of vaccine manufacturing through increased expression of recombinant polypeptides and viruses. Therefore, DNA shuffling and screening technologies can overcome some of the challenges that vaccine development currently faces.

Animals↗

PubMed2003

Enhancement of a human immunodeficiency virus env DNA vaccine using a novel polycationic nanoparticle formulation.

In an effort to develop a more effective DNA immunization strategy for HIV, we synthesized an HIV-2 env DNA vaccine and delivered it in a novel polycationic adjuvant formulation that forms nanoparticles in solution and enhances protein expression. The polycationic adjuvant contained imidazole moieties to facilitate endosomal escape. Nanoparticles containing the DNA vaccine plasmid were formed by electrostatic condensation with the polycationic adjuvant. We hypothesized that this formulation would improve immune responses to the gp140 env gene from HIV-2(UC2) by increasing the level of expressed antigen. We found that the nanoparticles were superior at inducing high levels of systemic antibody responses compared to naked DNA when delivered by the intradermal route in BALB/c mice. In addition, the nanoparticles induced higher levels of IgM, IgG, and IgA antibodies. These results suggest that nanoparticles may be an important adjuvant formulation to improve the effectiveness of genetic immunization and rationalize its use in the evaluation of vaccine candidates in non-human primate models for AIDS.

AIDS Vaccines↗

PubMed2003

Expression patterns of phenotypic markers on lymphocytes from human immunodeficiency virus type 2-infected baboons.

The development of AIDS in HIV-1-infected humans is associated with profound changes in the expression patterns of lymphocyte phenotypic markers associated with increased immune activation and with decreased recall immune responses. In assessing these immunologic changes in an animal model, we characterized the expression patterns of immune activation markers on lymphocyte subsets during the acute, chronic, and end stages of HIV-2 infection in baboons. Using flow cytometry, we identified 21 human-specific monoclonal antibodies that were cross-reactive with baboon lymphocytes; however, expression of only 2 of these markers was altered significantly after HIV-2 infection. We found an increase in baboon class II antigen (as measured by anti-HLA-DR) in the CD4(+) T cell subset within 8 weeks of infection (p = 0.045). Moreover, after 1 year of infection, CD11b was downregulated on CD8(+) T lymphocytes (p = 0.027). This downregulation of CD11b was consistently observed in all of the groups of baboons that were chronically infected with three different HIV-2 isolates. In addition, we found substantial downregulation of the interleukin 2 receptor (CD25) and upregulation of class II antigen on CD8(+) lymphocytes in a baboon with an AIDS-like disease. These and other phenotypic markers of immune activation may facilitate characterization of the immunopathogenesis of AIDS in nonhuman primate animal models.

Acute Disease↗

PubMed2003

5HT1A serotonin receptor agonists inhibit Plasmodium falciparum by blocking a membrane channel.

To identify new leads for the treatment of Plasmodium falciparum malaria, we screened a panel of serotonin (5-hydroxytryptamine [5HT]) receptor agonists and antagonists and determined their effects on parasite growth. The 5HT1A receptor agonists 8-hydroxy-N-(di-n-propyl)-aminotetralin (8-OH-DPAT), 2,5-dimethoxy-4-iodoamphetamine, and 2,5-dimethoxy-4-bromophenylethylamine inhibited the growth of P. falciparum in vitro (50% inhibitory concentrations, 0.4, 0.7, and 1.5 microM, respectively). In further characterizing the antiparasitic effects of 8-OH-DPAT, we found that this serotonin receptor agonist did not affect the growth of Leishmania infantum, Trypanosoma cruzi, Trypanosoma brucei brucei, or Trichostrongylus colubriformis in vitro and did not demonstrate cytotoxicity against the human lung fibroblast cell line MRC-5. 8-OH-DPAT had similar levels of growth inhibition against several different P. falciparum isolates having distinct chemotherapeutic resistance phenotypes, and its antimalarial effect was additive when it was used in combination with chloroquine against a chloroquine-resistant isolate. In a patch clamp assay, 8-OH-DPAT blocked a P. falciparum surface membrane channel, suggesting that serotonin receptor agonists are a novel class of antimalarials that target a nutrient transport pathway. Since there may be neurological involvement with the use of 8-OH-DPAT and other serotonin receptor agonists in the treatment of falciparum malaria, new lead compounds derived from 8-OH-DPAT will need to be modified to prevent potential neurological side effects. Nevertheless, these results suggest that 8-OH-DPAT is a new lead compound with which to derive novel antimalarial agents and is a useful tool with which to characterize P. falciparum membrane channels.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed2003

Increased virus replication and virulence after serial passage of human immunodeficiency virus type 2 in baboons.

Similar to human immunodeficiency virus type 1 (HIV-1) infection of humans, the natural history of HIV-2 infection in baboons (Papio cynocephalus) is a slow and chronic disease that generally takes several years before an AIDS-like condition develops. To shorten the amount of time to the development of disease, we performed five serial passages of HIV-2(UC2) in baboons by using blood and bone marrow samples during the acute phase of infection when viral loads were at high levels. After these serial passages, virus levels in plasma, peripheral blood mononuclear cells (PBMC) and lymphatic tissues in the acutely infected baboons were increased. Within 1 year of the HIV-2 infection, all of the inoculated baboons showed specific signs of AIDS-related disease progression within the lymphatic tissues, such as vascular proliferation and lymphoid depletion. The HIV-2(UC2) recovered after four serial passages showed increased kinetics of viral replication in baboon PBMC and cytopathicity. This study suggests that the HIV-2 isolate recovered after several serial passages in baboons will be useful in future studies of AIDS pathogenesis and vaccine development by using this animal model.

Adoptive Transfer↗

PubMed2002

Enhancement of antibody responses to an HIV-2 DNA envelope vaccine using an expression vector containing a constitutive transport element.

Because immune responses to DNA vaccines in humans remains suboptimal, strategies need to be devised to facilitate expression of the vaccine in vivo. One method to improve response to a DNA vaccine is to construct plasmid vectors with leader sequences and post-transcriptional elements that facilitate export of transcribed RNA. In this study, we sought to determine if a mammalian expression vector (pND-14) containing a tissue plasminogen activator (TPA) leader sequence and a constitutive transport element (CTE) from simian retrovirus was superior to other mammalian expression vectors containing a post-transcriptional regulatory element (PRE) from hepatitis B virus (pCMV-link) or a minimal mammalian expression vector (pVAX1). Toward this objective, we evaluated protein expression of the HIV-2 envelope gene (gp140) in vitro and immune responses in immunized mice. We found that pVAX1 produced three- to fourfold lower levels of gp140 in vitro (5 ng/ml) in contrast to the pCMV-link and pND-14 vectors. When we immunized groups of mice intradermally with two of the HIV-2 gp140 DNA vaccine constructs, we found that pND-14 induced higher levels of envelope-specific systemic and mucosal antibodies than pCMV-link. We conclude that expression vectors for DNA vaccines should contain TPA and CTE sequences to facilitate immune responses.

AIDS Vaccines↗