An AAV Vector-Mediated Gene Delivery Approach Facilitates Reconstitution of Functional Human CD8⁺ T Cells in Mice

<div><p>In the present study, a novel adeno-associated virus (AAV) vector-mediated gene delivery approach was taken to improve the reconstitution of functional CD8⁺ T cells in humanized mice, thereby mimicking the human immune system (HIS). Human genes encoding HLA-A2 and selected human cytokines (A2/hucytokines) were introduced to an immune-deficient mouse model [NOD/SCID/IL2rγ^null (NSG) mice] using AAV serotype 9 (AAV9) vectors, followed by transplantation of human hematopoietic stem cells. NSG mice transduced with AAV9 encoding A2/hucytokines resulted in higher levels of reconstitution of human CD45⁺ cells compared to NSG mice transduced with AAV9 encoding HLA-A2 alone or HLA-A2-transgenic NSG mice. Furthermore, this group of HIS mice also mounted the highest level of antigen-specific A2-restricted human CD8⁺ T-cell response upon vaccination with recombinant adenoviruses expressing human malaria and HIV antigens. Finally, the human CD8⁺ T-cell response induced in human malaria vaccine-immunized HIS mice was shown to be functional by displaying cytotoxic activity against hepatocytes that express the human malaria antigen in the context of A2 molecules. Taken together, our data show that AAV vector-mediated gene delivery is a simple and efficient method to transfer multiple human genes to immune-deficient mice, thus facilitating successful reconstitution of HIS in mice. The HIS mice generated in this study should ultimately allow us to swiftly evaluate the T-cell immunogenicity of various human vaccine candidates in a pre-clinical setting.</p></div

<i>In vitro</i> expression of human cytokines and HLA-A2.1 in MC57G cells.

<p>(A) Maps of Zac2.1 plasmids modified to encode human cytokines and HLA-A2.1 (HHD) containing α1 and α2 domains of HLA-A2.1, α3, cytoplasmic and transmembrane domains of murine H-2D^b, and hβ2m, are shown. These plasmids were used to construct AAV9 viral particles. (B) MC57G cells were infected <i>in vitro</i> with AAV9 encoding the respective cytokine, and the production of each cytokine was determined using ELISA. (C) MC57G cells were infected <i>in vitro</i> with different doses (1×10⁹, 1×10¹⁰, or 1×10¹¹ GC/mL) of AAV9-encoding HLA-A2.1/hβ2m (AAV9-A2). Expression of HLA-A2.1 and hβ2m was evaluated using flow cytometric analyses.</p

Reconstitution of a human immune system in NSG mice transduced with AAV9-A2/hucytokines.

<p>(A) Flow cytometric analyses were performed to determine the level of human CD45⁺ cell reconstitution in the blood of various groups of mice: AAV9-A2/hucytokines-transduced NSG mice, AAV9-A2-transduced NSG mice, A2-Tg NSG mice, or AAV9-GFP-transduced NSG mice 6, 10, or 14 weeks after engraftment of human CD34⁺ cells. (B) The level of human CD45⁺ cell reconstitution in the blood of various groups of NSG mice was determined using flow cytometric analyses 6, 10, or 14 weeks after engrafting human CD34⁺ cells. (C) Percentages of human CD3⁺ T cells and CD19⁺ B cells within the human CD45⁺ cells in the blood of various groups of HIS mice were determined 6, 10, and 14 weeks after HSC engraftment. (D) Percentages of human CD8⁺ and CD4⁺ T cells within the human CD3⁺ T cells in the blood of various groups of HIS mice were determined 6, 10, and 14 weeks after HSC engraftment. (E) The pie charts show the percentage of various human lymphocyte subsets, including naïve, central memory (CM), effector memory (EM) and effector CD8+ T cells, within the human CD45⁺ cells in the blood of various groups of HIS mice determined 14 weeks after HSC engraftment, compared to those in PBMCs of a healthy human subject. * indicates p values <0.05, ** indicates p values <0.01, and *** indicates p values <0.001.</p

<i>In vitro</i> and <i>in vivo</i> A2-restricted, PfCS-specific cytotoxic T cell responses induced in AA-A2/hucytokines-transduced HIS mice immunized with AdPfCS.

<p>(A) Splenocytes were collected from AAV9-A2/hucytokines transduced HIS mice 2 weeks after AdPfCS immunization. After CD8⁺ T cell enrichment, cells were used as an effector cells. A2⁺ hepatocytes were isolated from AA-A2/hucytokines-transduced NSG mice, labeled with CFSE, and used as target cells. These two cell populations were co-cultured and <i>in vitro</i> CTL assays were performed by measuring the amount of caspase 3 within the hepatocytes in the presence or absence of the peptide corresponding to the A2-restricted, CD8⁺ epitope of PfCS antigen. (B) Two weeks after AdPfCS immunization to AAV9-A2/hucytokines-transduced HIS mice, immunized and naïve AAV9-A2/hucytokines-transduced HIS mice were injected with 50 µg of DNA-PfCS dissolved in 2 ml PBS by HTV delivery. Another group of immunized and naïve AAV9-A2/hucytokines-transduced HIS mice received DNA-PyCS via HTV delivery as a negative control. Three days after DNA-PfCS or DNA-PyCS challenge, liver was collected and the amount of PfCS or PyCS mRNA was determined using real-time qRT-PCR.</p

Human leukocyte reconstitution in the peripheral blood of NSG mice transduced with AAV9-hucytokines or AAV9-A2.

<p>(A) Schematic representation of the strategic methodology for engrafting human CD34⁺ cells in AAV9-transduced NSG mice. NSG mice were inoculated with AAV9-hucytokines or AAV9-A2 and 2 weeks later, mice were irradiated to myeloablate mouse immune cells. The next day, mice were transplanted i.v. with 1×10⁵ human CD34⁺ cells previously isolated from human fetal liver. (B) The level of human CD45⁺ cell reconstitution in the blood was determined using flow cytometric analyses 10 weeks after engrafting human CD34⁺ cells into NSG mice transduced with AAV9 encoding human IL-3, IL-4, IL-7, IL-15, or GM-CSF. (C) The level of human CD45⁺ cell reconstitution in the blood was determined using flow cytometric analyses 10 weeks after engrafting human CD34⁺ cells into NSG mice transduced with individual or combination of AAV9 encoding selected human cytokines, IL-3 (5×10⁹ GC), IL-15 (5×10⁹ GC), and GM-CSF (1×10⁹ GC). (D) The level of human CD45⁺ cell reconstitution in the blood was determined using flow cytometric analyses 10 weeks after engrafting human CD34⁺ cells into NSG mice transduced with AAV9-A2.</p

GC number specific for AAV9 and the transgenes present in selected organs of NSG mice at 6 weeks and 20 weeks after AAV9 injection.

<p>Lung, liver, spleen, kidney and bone marrow, were collected 6 and 20 weeks after NSG mice were given recombinant AAV9 vectors (5×10⁹ GC of AAV9-GM-CSF, AAV9-IL-3, and AAV9-IL-15 by i.v., or 1×10¹¹ GC of AAV9-A2 by i.v. and intrathoracically), and DNA was isolated from the respective organs. (A) The number of AAV9 vector GC was determined by using a set of primers specific to AAV9 vector. (B) The number of GC specific for the transgenes - HLA-A2, hGM-CSF, hIL-3 and hIL-15 - was determined by using a set of primers to the respective transgene. There was a low number (10–10³ GC; depending on the transgene) of GC detected in naïve NSG mice, as a non-specific background, and, therefore, this background GC number was subtracted from the GC number in experimental groups.</p

An AAV Vector-Mediated Gene Delivery Approach Facilitates Reconstitution of Functional Human CD8^{+ - Table 1} T Cells in Mice

<p>An AAV Vector-Mediated Gene Delivery Approach Facilitates Reconstitution of Functional Human CD8^{+ - Table 1} T Cells in Mice</p

rCSP Strain Selection and Identification.

<p>(<b>A</b>) Reduced SDS-PAGE analysis of lysate samples prior to purification. Lanes 1–3 represent samples from strain CSP-1, CSP-3, and CSP-2 fermentations, respectively. Lane 4 represents a sample from a culture of <i>P. fluorescens</i> transformed with plasmid vector containing no insert. Arrow denotes rCSP in lysate samples. (<b>B</b>) Reduced SDS-PAGE analysis of purified proteins from a 2-column purification process. Lanes 1–3 represent samples from CSP-1, CSP-2, and CSP-3 productions, respectively. (<b>C</b>) Western blot of non-reduced purified CSP-1, CSP-2, and CSP-3 proteins (lanes 2–4, respectively). Lane 1 represents a sample from a culture of <i>P. fluorescens</i> transformed with plasmid vector containing no insert.</p

Activity of Prime-Boost Regimens in Mice.

<p>C57BL/6 mice (5–7 per cohort) were immunized 3x with 20 µg of rCSP formulated in GLA-SE, 1–2×10¹⁰ v.p. of Ad35CS, or a combination of the two. Spleens and livers were collected 10 days after the last immunization; sera were collected 10 or 14 days after the last immunization. To assess reduction in parasite load in the liver or sterile protection, mice were challenged with <i>Pb</i>-CS(<i>Pf</i>) parasites 3 weeks after the last immunization. (<b>A</b>) Percent inhibition of LS parasite development in immunized mice challenged with <i>Pb</i>-CS(<i>Pf</i>) sporozoites normalized to naïve mice received the challenge, and percent sterile protection in immunized BALB/c mice upon challenge with a fewer number of Pb-CS(Pf) sporozoites are shown. A statistically significant reduction of LS parasites in the livers of mice immunized with all regimens, except Ad35CS 3x and Ad35CS (1x), GLA-SE (2x), was seen upon sporozoites challenge, compared to the level of LS parasites in the livers of negative control mice following sporozoites challenge, based on the mean parasite's 18s rRNA copy number (as noted by asterisks). Three regimens were assessed in two independent studies (GLA-SE 3x; GLA-SE 1x, Ad35CS 2x; and Ad35CS 1x, GLA-SE 2x); for these regimens, average percent inhibition is shown with error bars represented as standard error. Statistical significance with the GLA-SE 3x; GLA-SE 1x, Ad35CS 2x was seen in both studies. (<b>B</b>) ELISA was used to evaluate the level of humoral response in immunized mice to the repeat region of CSP with pooled mouse sera and to whole rCSP with individual mouse sera. Titer was assessed at OD = 1.0 based on 4-parameter logistic curve fits. Average titers are shown with error bars representing standard error of the mean. (<b>C</b>) The level of T-cell response was determined via ELISpot using lymphocytes isolated from the spleens and livers of immunized mice (5 mice per cohort were used for this study) and overlapping peptides (15-mers encompassing the CSP repeat region) for in vitro restimulation. Average numbers of IFN-γ producing cells/10⁶ lymphocytes are shown with error bars representing standard error of the mean. No more than 5 IFN-γ producing cells/10⁶ lymphocytes were counted with cells harvested from naïve mice (data not shown).</p

Immunogenicity and Efficacy of rCSP Immunization in Mice.

<p>Five C57BL/6 mice per cohort were immunized 3x with 25 µg of rCSP from 3 production strains (CSP-1, CSP-2, and CSP-3) formulated in incomplete Freund's adjuvant. Sera were collected 2 weeks after the last immunization and pooled for analysis by ELISA, IFA, and ISI. Mice were challenged with <i>Pb</i>-CS(<i>Pf</i>) parasites 3 weeks after the last immunization. (<b>A</b>) Pooled sera from mice immunized with the 3 recombinant proteins showed similar ELISA and IFA titers. ELISA titers to a [NANP]₆ peptide were calculated at OD = 1.0 based on 4-parameter logistic curve fits, and IFA titers were determined based on the lowest serum dilution to give sporozoites-specific fluorescence above background level shown by sera from naïve mice. (<b>B</b>) Sera from rCSP-immunized mice reacted to <i>Pb</i>-CS(<i>Pf</i>) sporozoites demonstrated the expected pattern of surface fluorescence by IFA. (<b>C</b>) Sera from rCSP-immunized mice diluted 1∶100 and assayed for the ability to block <i>Pf</i> sporozoite infection of hepatocytes in an ISI assay demonstrated the highest ISI activity for anti-CSP-1 protein sera. The percent inhibition is shown relative to the number of sporozoites after incubating with the sera of naïve mice. Percent inhibition of LS parasite development in rCSP-immunized mice challenged with <i>Pb</i>-CS(<i>Pf</i>) sporozoites normalized to naïve control mice is also shown. The reduction of LS parasites in livers of mice immunized with CSP-1 or CSP-3 proteins was statistically significant based on the mean parasite-specific 18 s rRNA copy number compared the level of LS parasites in livers of naïve and adjuvant-only administered mice (as noted by asterisks).</p