Qiu Zhong

CD4+ T cell–independent vaccination against Pneumocystis carinii in mice

Host defenses are profoundly compromised in HIV-infected hosts due to progressive depletion of CD4+ T lymphocytes. Moreover, deficient CD4+ T lymphocytes impair vaccination approaches to prevent opportunistic infection. Therefore, we investigated a CD4+ T cell-independent vaccine approach to a prototypic AIDS-defining infection, Pneumocystis carinii (PC) pneumonia. Here, we demonstrate that bone marrow-derived dendritic cells (DCs) expressing the murine CD40 ligand, when pulsed ex vivo by PC antigen, elicited significant titers of anti-PC IgG in CD4-deficient mice. Vaccinated animals demonstrated significant protection from PC infection, and this protection was the result of an effective humoral response, since adoptive transfer of CD4-depleted splenocytes or serum conferred this protection to CD4-deficient mice. Western blot analysis of PC antigen revealed that DC-vaccinated, CD4-deficient mice predominantly reacted to a 55-kDa PC antigen. These studies show promise for advances in CD4-independent vaccination against HIV-related pathogens.

CD4+ T cell–independent DNA vaccination against opportunistic infections

Depletion or dysfunction of CD4+ T lymphocytes profoundly perturbs host defenses and impairs immunogenicity of vaccines. Here, we show that plasmid DNA vaccination with a cassette encoding antigen (OVA) and a second cassette encoding full-length CD40 ligand (CD40L), a molecule expressed on activated CD4+ T lymphocytes and critical for T cell helper function, can elicit significant titers of antigen-specific immunoglobulins in serum and Tc1 CD8+ T cell responses in CD4-deficient mice. To investigate whether this approach leads to CD4+ T cell-independent vaccine protection against a prototypic AIDS-defining infection, Pneumocystis (PC) pneumonia, we used serum from mice vaccinated with PC-pulsed, CD40L-modified DCs to immunoprecipitate PC antigens. Kexin, a PC antigen identified by this approach, was used in a similar DNA vaccine strategy with or without CD40L. CD4-deficient mice receiving DNA vaccines encoding Kexin and CD40L showed significantly higher anti-PC IgG titers as well as opsonic killing of PC compared with those vaccinated with Kexin alone. Moreover, CD4-depleted, Kexin-vaccinated mice showed a 3-log greater protection in a PC challenge model. Adoptive transfer of CD19+ cells or IgG to SCID mice conferred protection against PC challenge, indicating a role of humoral immunity in the protection. The results of these studies show promise for CD4-independent vaccination against HIV-related or other opportunistic pathogens.

The Granulocyte Colony-Stimulating Factor Response after Intrapulmonary and Systemic Bacterial Challenges

In contrast to many cytokines such as tumor necrosis factor (TNF)-alpha, we hypothesized that, after an intrapulmonary bacterial challenge, lung-derived granulocyte colony-stimulating factor (G-CSF) would subsequently enter the systemic circulation. BALB/c mice were given Escherichia coli or saline, either intratracheally or intravenously. Four hours after intratracheal E. coli administration, bronchoalveolar lavage fluid (BALF) and plasma G-CSF concentrations increased, compared with concentrations in phosphate-buffered saline-treated controls. Lung G-CSF messenger RNA (mRNA) increased to 586+/-229 copies G-CSF mRNA/ng ribosomal RNA (rRNA) from the values in control animals (<0.5 copies/ng rRNA). In contrast, G-CSF mRNA was not increased in the extrapulmonary tissues examined (liver, spleen, and kidney) in mice challenged with intratracheal E. coli (<1 copy/ng rRNA). Intravenous E. coli increased plasma G-CSF and TNF-alpha, but neither cytokine was detected in BALF. These data show that, after an intrapulmonary infection, both lung and circulating G-CSF increase and that the lung is the likely source.

IL-17F/IL-17R interaction stimulates granulopoiesis in mice

OBJECTIVE IL-17F, a member of the interleukin (IL)-17 cytokine family, most closely resembles IL-17A structurally. IL-17A is a potent stimulator of granulopoiesis; its expression is induced in response to microbial challenge. Although IL-17F is considered to be a weak IL-17A analog that is also mediating its effect via IL-17R, its exact role and in vivo functions are unknown. Our goal was to determine the in vivo activity of IL-17F on granulopoiesis as well as on release of granulopoiesis-stimulating downstream cytokines in mice and directly compare its effect to IL-17A. MATERIALS AND METHODS Murine IL-17A (mIL-17A) or IL-17F (mIL-17F) was expressed in vivo in C57BL6 mice using adenoviral gene transfer technology. Peripheral cell counts were assessed as well as hematopoietic precursors using colony-forming assays at set time points. Downstream cytokines were measured using enzyme-linked immunosorbent assay and reverse transcriptase polymerase chain reaction. RESULTS We found mIL-17F to have similar expression kinetics as mIL-17A in splenocytes in vitro and in vivo, following challenge with microbial agents. Overexpression of mIL-17F in vivo resulted in similar neutrophilia and only in slightly reduced myeloid progenitor expansion when compared to mIL-17A. In vivo, there was no difference in releases for granulocyte-macrophage colony-stimulating factor; regulated on activation, normal T expressed and secreted; interferon-inducible protein-10; IL-6; and monocyte chemotactic protein-1 between either cytokine. IL-1A, macrophage inflammatory protein -2 (MIP), KC, and granulocyte colony-stimulating factor expression was approximately half of that seen with mIL-17A. CONCLUSION Both IL-17A and IL-17F are induced by similar stimuli, have similar expression kinetics and despite only minimal in vitro activity for IL-17F, surprisingly they exert similar in vivo bioactivity. IL-17F bioactivity appears to be augmented in vivo through mechanisms that require further investigation.

Retrovirus Molecular Conjugates A NOVEL, HIGH TRANSDUCTION EFFICIENCY, POTENTIALLY SAFETY-IMPROVED, GENE TRANSFER SYSTEM

Two significant barriers limit the use of amphotropic retrovirus for human gene transfer protocols: 1) low transduction efficiency in cells with low receptor expression and 2) safety concerns originating from the risk of formation and propagation of replication competent virus in vivo. In principle, if ecotropic retrovirus, which is incapable of infecting human cells, could be transiently modified to effectively transduce human cells, this safety risk could be alleviated. Here we demonstrate that formation of amphotropic retrovirus polylysine molecular conjugates (aMMLV-PL) enhanced gene transfer up to 10-fold in a variety of human cell lines over the equivalent of unconjugated vector (aMMLV). The polylysine modification and formation of ecotropic retrovirus molecular conjugates (eMMLV-PL) permitted effective and stable transduction of different human cell lines as well as primary human bone marrow stroma cells at frequencies of greater than 80%. It is conceivable that this novel ecotropic-based conjugate retrovirus vector could also potentially provide enhanced safety characteristics not only over amphotropic retrovirus vectors but also over genetically tropism-modified recombinant ecotropic vectors. In contrast to genetic modifications, physical or chemical modifications are not propagated. Thus, formation of replication competent eMMLV from conjugates would be self-limited and would not result in virus propagation in humans.

Efficient c-kit Receptor-Targeted Gene Transfer to Primary Human CD34-Selected Hematopoietic Stem Cells

ABSTRACT We have previously reported effective gene transfer with a targeted molecular conjugate adenovirus vector through the c-kit receptor in hematopoietic progenitor cell lines. However, a c-kit-targeted recombinant retroviral vector failed to transduce cells, indicating the existence of significant differences for c-kit target gene transfer between these two viruses. Here we demonstrate that conjugation of an adenovirus to a c-kit-retargeted retrovirus vector enables retroviral transduction. This finding suggests the requirement of endosomalysis for successful c-kit-targeted gene transfer. Furthermore, we show efficient gene transfer to, and high transgene expression (66%) in, CD34-selected, c-kit+ human peripheral blood stem cells using a c-kit-targeted adenovirus vector. These findings may have important implications for future vector development in c-kit-targeted stem cell gene transfer.

Retrovirus molecular conjugates.

Abstract. Retrovirus-derived vectors are currently the preferred vectors used for human gene therapy protocols. Serious safety concerns persist, however, which are specifically related to the formation of a replication-competent virus, and no synthesis method currently employed precludes its formation with certainty. For many cell types, a low transduction efficiency results in insufficient therapeutic benefit. We describe the development of a molecular conjugate system, which permits transient chemical modification of a retrovirus with polylysine. This modification not only introduces additional safety features over standard unmodified retrovirus vectors, but also provides enhanced transduction efficiency.

Corrigendum. CD4+ T cell-independent DNA vaccination against opportunistic infections.

Original citation: J Clin Invest. 2005;115(12):3536–3544. doi:10.1172/JCI26306. Citation for this corrigendum: J Clin Invest. 2015;125(3):1364. doi:10.1172/JCI81228. Karen A. Norris has requested to be removed from the author list, and the corresponding author has agreed. The revised author list is shown above.

Erratum: CD4+ T cell'independent DNA vaccination against opportunistic infections (Journal of Clinical Investigation (2005) 115: 12 (3536-3544) DOI: 10.1172/JCI26306)

Original citation: J Clin Invest. 2005;115(12):3536–3544. doi:10.1172/JCI26306. Citation for this corrigendum: J Clin Invest. 2015;125(3):1364. doi:10.1172/JCI81228. Karen A. Norris has requested to be removed from the author list, and the corresponding author has agreed. The revised author list is shown above.

CD4+ T cell–independent DNA vaccination against opportunisticinfections

Original citation: J Clin Invest. 2005;115(12):3536–3544. doi:10.1172/JCI26306. Citation for this corrigendum: J Clin Invest. 2015;125(3):1364. doi:10.1172/JCI81228. Karen A. Norris has requested to be removed from the author list, and the corresponding author has agreed. The revised author list is shown above.