Herpes simplex virus as a model vector system for gene therapy in renal disease

Herpes simplex virus as a model vector system for gene therapy in renal disease. The past decade has been marked by significant advances in the application of gene transfer into living cells of animals and humans. These approaches have been tested in a few animal models of inherited and acquired renal diseases, including carbonic anhydrase II deficiency 1 and experimental glomerulonephritis2,3. Gene transfer into proximal tubular cells has been successfully accomplished by intrarenal arterial infusion of a liposomal complex4 or an adenoviral vector5. Tubular cells from the papilla and medulla have been selectively transduced by retrograde infusion into the pelvi-calyceal system of an adenoviral vector containing a reporter for β-galactosidase5. Although the results of these initial studies are promising, further studies to optimize viral vectors, maximize gene delivery, minimize side-effects, and develop cell-specific and long-term regulated gene expression are critical to the success of gene therapy targeted to specific compartments of the kidney. Our recent efforts have focused on defining the cellular pathways responsible for viral entry and infection into renal epithelial cells using herpes simplex virus (HSV) as a model vector. We anticipate that a solid understanding of the basic scientific principles underlying viral entry and gene expression into specific populations of renal cells will facilitate the design of successful therapeutic viral-based gene transfer strategies

A Contributing Role for Anti-Neuraminidase Antibodies on Immunity to Pandemic H1N1 2009 Influenza A Virus

Exposure to contemporary seasonal influenza A viruses affords partial immunity to pandemic H1N1 2009 influenza A virus (pH1N1) infection. The impact of antibodies to the neuraminidase (NA) of seasonal influenza A viruses to cross-immunity against pH1N1 infection is unknown.Antibodies to the NA of different seasonal H1N1 influenza strains were tested for cross-reactivity against A/California/04/09 (pH1N1). A panel of reverse genetic (rg) recombinant viruses was generated containing 7 genes of the H1N1 influenza strain A/Puerto Rico/08/34 (PR8) and the NA gene of either the pandemic H1N1 2009 strain (pH1N1) or one of the following contemporary seasonal H1N1 strains: A/Solomon/03/06 (rg Solomon) or A/Brisbane/59/07 (rg Brisbane). Convalescent sera collected from mice infected with recombinant viruses were measured for cross-reactive antibodies to pH1N1 via Hemagglutinin Inhibition (HI) or Enzyme-Linked Immunosorbent Assay (ELISA). The ectodomain of a recombinant NA protein from the pH1N1 strain (pNA-ecto) was expressed, purified and used in ELISA to measure cross-reactive antibodies. Analysis of sera from elderly humans immunized with trivalent split-inactivated influenza (TIV) seasonal vaccines prior to 2009 revealed considerable cross-reactivity to pNA-ecto. High titers of cross-reactive antibodies were detected in mice inoculated with either rg Solomon or rg Brisbane. Convalescent sera from mice inoculated with recombinant viruses were used to immunize naïve recipient Balb/c mice by passive transfer prior to challenge with pH1N1. Mice receiving rg California sera were better protected than animals receiving rg Solomon or rg Brisbane sera.The NA of contemporary seasonal H1N1 influenza strains induces a cross-reactive antibody response to pH1N1 that correlates with reduced lethality from pH1N1 challenge, albeit less efficiently than anti-pH1N1 NA antibodies. These findings demonstrate that seasonal NA antibodies contribute to but are not sufficient for cross-reactive immunity to pH1N1

Host Differences in Influenza-Specific CD4 T Cell and B Cell Responses Are Modulated by Viral Strain and Route of Immunization

The antibody response to influenza infection is largely dependent on CD4 T cell help for B cells. Cognate signals and secreted factors provided by CD4 T cells drive B cell activation and regulate antibody isotype switching for optimal antiviral activity. Recently, we analyzed HLA-DR1 transgenic (DR1) mice and C57BL/10 (B10) mice after infection with influenza virus A/New Caledonia/20/99 (NC) and defined epitopes recognized by virus-specific CD4 T cells. Using this information in the current study, we demonstrate that the pattern of secretion of IL-2, IFN-γ, and IL-4 by CD4 T cells activated by NC infection is largely independent of epitope specificity and the magnitude of the epitope-specific response. Interestingly, however, the characteristics of the virus-specific CD4 T cell and the B cell response to NC infection differed in DR1 and B10 mice. The response in B10 mice featured predominantly IFN-γ-secreting CD4 T cells and strong IgG2b/IgG2c production. In contrast, in DR1 mice most CD4 T cells secreted IL-2 and IgG production was IgG1-biased. Infection of DR1 mice with influenza PR8 generated a response that was comparable to that in B10 mice, with predominantly IFN-γ-secreting CD4 T cells and greater numbers of IgG2c than IgG1 antibody-secreting cells. The response to intramuscular vaccination with inactivated NC was similar in DR1 and B10 mice; the majority of CD4 T cells secreted IL-2 and most IgG antibody-secreting cells produced IgG2b or IgG2c. Our findings identify inherent host influences on characteristics of the virus-specific CD4 T cell and B cell responses that are restricted to the lung environment. Furthermore, we show that these host influences are substantially modulated by the type of infecting virus via the early induction of innate factors. Our findings emphasize the importance of immunization strategy for demonstrating inherent host differences in CD4 T cell and B cell responses

Herpes simplex virus as a model vector system for gene therapy in renal disease

Herpes simplex virus as a model vector system for gene therapy in renal disease. The past decade has been marked by significant advances in the application of gene transfer into living cells of animals and humans. These approaches have been tested in a few animal models of inherited and acquired renal diseases, including carbonic anhydrase II deficiency 1 and experimental glomerulonephritis2,3. Gene transfer into proximal tubular cells has been successfully accomplished by intrarenal arterial infusion of a liposomal complex4 or an adenoviral vector5. Tubular cells from the papilla and medulla have been selectively transduced by retrograde infusion into the pelvi-calyceal system of an adenoviral vector containing a reporter for β-galactosidase5. Although the results of these initial studies are promising, further studies to optimize viral vectors, maximize gene delivery, minimize side-effects, and develop cell-specific and long-term regulated gene expression are critical to the success of gene therapy targeted to specific compartments of the kidney. Our recent efforts have focused on defining the cellular pathways responsible for viral entry and infection into renal epithelial cells using herpes simplex virus (HSV) as a model vector. We anticipate that a solid understanding of the basic scientific principles underlying viral entry and gene expression into specific populations of renal cells will facilitate the design of successful therapeutic viral-based gene transfer strategies

The B cell response following infection with different viruses that replicate in the lung.

<p>(A–F) Virus-specific ASC frequencies. DR1 (A–C) and B10 (D–F) mice were infected intranasally with the influenza viruses PR8 (A and D) and X31 (B and E), and with the non-influenza virus MHV68 (C and F). Virus-specific ASC frequencies in the MedLN were determined by ELISPOT assay at intervals after infection. (G) PR8 replication in the lung. Titers are represented as log₁₀ TCID₅₀/0.2 ml of lung homogenate. (H, I) Flow cytometric analysis of ASCs and germinal center B cells in the MedLN on day 8 after PR8 infection. ASC frequencies (H) represent the proportion of B220^int CD138⁺ cells after gating on live CD4⁻ CD8⁻ CD19⁺ cells. Germinal center B cell frequencies (I) represent the proportion of PNA⁺ Fas⁺ cells among live CD4⁻ CD8⁻ B220⁺ cells. (J) Serum levels of virus-specific IgG in DR1 and B10 mice on day 8 after PR8 infection. Titers determined by ELISA are shown as the reciprocal of the highest serum dilution scored as positive relative to naïve control serum. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#pone-0034377-g006" target="_blank">Fig. 6</a> data sets depict the mean+SE for 3–10 individual mice per group. * P<0.05, *** P<0.001.</p

Cytokine and chemokine production in the lung after influenza infection.

<p>DR1 and B10 mice were infected intranasally with NC or PR8 or were mock-infected with PBS. Mice were sampled 60 h after inoculation. Cytokine and chemokine concentrations in clarified lung homogenates were determined by Multiplex assay or by sandwich ELISA (IFN-α only). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#pone-0034377-g009" target="_blank">Figure 9</a> shows the results for a selection of the 30 cytokine and chemokine determinations. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#s3" target="_blank">Results</a> for the remaining cytokines and chemokines are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#pone.0034377.s004" target="_blank">figure S4</a>. The mean+SE is shown for 5 individual mice per group. * P<0.05, ** P<0.01, *** P<0.001.</p

The virus-specific B cell and CD4 T cell response to intramuscular immunization.

<p>(A–D) Virus-specific ASC frequencies. DR1 (A and B) and B10 (C and D) mice were immunized intramuscularly with inactivated NC. Virus-specific ASC frequencies in the iliac lymph nodes and spleen were determined by ELISpot assay at the indicated times after immunization. The mean+SE is shown for 3–4 individual mice per group. (E, F) Cytokine production by peptide-specific CD4 T cells. Enriched CD4 T cells from the spleen were analyzed on day 8 after immunization. Frequencies of CD4 T cells secreting IL-2, IFN-γ, or IL-4 were determined by ELISpot assay after in vitro stimulation with antigen-presenting cells and sets of 1–3 17-mer peptides from different viral proteins (HA, NA, NP, and M1). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#s3" target="_blank">Results</a> are normalized to spot counts per 10⁶ CD4 T cells and are shown as the mean ± range for 2 independent experiments (the M1 protein was represented in only one experiment with B10 mice). Cells from at least three mice were pooled for each experiment. (G, H) Proportions of peptide-specific CD4 T cells secreting IL-2, IFN-γ, or IL-4. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#s3" target="_blank">Results</a> are compiled from the data shown in E and F. The mean ± range is shown for the two independent experiments.</p

Serum levels of virus-specific Ab in DR1 and B10 mice after NC infection.

<p>NC-specific IgM (A), IgG1 (B), IgG2b (C), IgG2c (D), IgG3 (E), IgA (F), IgG (G), and Ig (H) titers were determined by ELISA on plates coated with disrupted viral particles. Titers are shown as the reciprocal of the highest serum dilution scored as positive relative to naïve control serum. The mean+SE is shown for 4–8 individual mice per group. * P<0.05, ** P<0.01.</p

The CD4 T cell response to PR8 infection.

<p>(A, B) Cytokine production by peptide-specific CD4 T cells. DR1 and B10 mice were infected intranasally with PR8. Enriched CD4 T cells from the MedLN were analyzed on day 10 after infection. Frequencies of CD4 T cells secreting IL-2, IFN-γ, or IL-4 were determined by ELISpot assay after in vitro stimulation with antigen-presenting cells and individual 17-mer peptides. Peptide designations (x-axis) include the viral proteins of origin (NP, M1, and NS1). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#s3" target="_blank">Results</a> are normalized to spot counts per 10⁶ CD4 T cells and are shown as the mean ± range for 2 independent experiments. Cells from at least three mice were pooled for each experiment. (C, D) Proportions of peptide-specific CD4 T cells secreting IL-2, IFN-γ, or IL-4. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#s3" target="_blank">Results</a> are compiled from the data shown in A and B. The mean ± range is shown for the two independent experiments.</p