T cells from mice immunized either intraperitoneally or orally with CVB4/p24(73₃) produce cytokines in response to gag p24 peptides.

<p>Splenic CD4 and CD8 T cells producing TNF-α, IL-2, or IFN-γ in response to p24(65–73) or p24(10–30) were identified by multiparameter flow cytometry. (A) Primary immune responses after immunization via the intraperitoneal route. (B) Secondary immune responses after immunization via the intraperitoneal route. (C) Secondary immune responses after immunization via the oral route. Each symbol represents the results from one experiment using splenocytes pooled from 3 mice. Experiments were done a total of three or four times and all of the data are shown here. Mean values are indicated by horizontal bars. Flow cytometric analysis was performed after gating on CD4 or CD8 T cells. The T cell frequency indicates the percentage of CD4 or CD8 T cells that is responding to the gag p24 peptides. T cell frequencies after IP immunization with one or two doses of virus were similar. The one exception is indicated by a closed arrowhead (P<0.05). T cell frequencies after IP or oral immunization with two doses of virus were also similar. The one exception is indicated by an open arrowhead (P<0.05). Statistical analysis was done using a one-way ANOVA. The functionality of gag p24-specific T cell responses was assessed using Boolean gating and FlowJo software, and the data is shown in pie charts. Polyfunctional T cells were detected after IP immunization but not after oral immunization.</p

Construction and biological properties of the CVB4/p24(73₃) recombinant.

<p>(A) Construction of the CVB4/p24(73₃) recombinant. The top line shows the two T cell epitopes of gag p24 that are contained within CVB4/p24(73₃). The middle line depicts the genetic organization of the CVB4 genome and shows the insertion site for the gag p24 sequence, between the 5′ untranslated region (UTR) and the VP4 sequence. The bottom line shows the 73 amino acids (residues 3 to 75) of gag p24 (HXB2) in the extended polyprotein sequence of CVB4/p24(73₃). The H-2^d restricted T cell epitopes are indicated along with the recognition sequence for the CVB4 3C protease. (B) The CVB4/p24(73₃) recombinant shares the thermostable phenotype of its parental virus, CVB4. Samples containing 10⁷ pfu of virus were heated at 44°C for different time intervals, and the residual infectivity was determined by plaque assay. Experiments were done twice and both inactivation profiles for each virus are shown. ▴, Δ: CVB4/p24(73₃); •, ○: CVB4. (C) The CVB4/p24(73₃) recombinant is replication-competent. The replication of CVB4/p24(73₃) and CVB4 in LLC-MK2(D) cells was assessed under single-step conditions. Each experiment was done twice and both growth curves for each virus are shown. ▴, Δ: CVB4/p24(73₃); •, ○: CVB4.</p

The CVB4/p24(73₃) recombinant induced a localized infection in the gut after oral delivery.

<p>*Peyer's patches (5–6 in each sample);</p><p># mesenteric lymph nodes (5–6 in each sample). Organ homogenates (0.2 ml) were used to infect HeLa cells and infectivity was evaluated by the development of CPE. ND, not done.</p

Replication of the CVB4/p24(73₃) recombinant in BALB/c mice.

<p>(A) Mice infected with CVB4/p24(73₃) gained weight. Mice (n = 3) were infected intraperitoneally with CVB4/p24(73₃) (▴) or CVB4 (□) or were mock-infected with PBS (•), and body weights were measured at various times after infection. (B) Intraperitoneal delivery of CVB4/p24(73₃) resulted in systemic infection, while oral delivery did not result in systemic infection. Mice (n = 3) were infected with 5×10⁴ pfu administered intraperitoneally, 5×10⁴ pfu administered orally, or 10⁶ pfu administered orally. Pancreas, spleen, and intestine were harvested from each mouse at 2 dpi, and organ homogenates were tested for viral infectivity by plaque assay. Dashed lines indicate the limit of detection of infectious virus. ND: not detected.</p

Mice immunized with either the CVB4 vector or the CVB4/p24(73₃) recombinant develop an anamnestic response to vector antigens.

<p>Mice (n = 3) were immunized via the intraperitoneal route with either CVB4 or CVB4/p24(73₃). Sera from mice immunized with one dose of virus (primary responses) or three doses of virus (secondary responses) were tested for anti-CVB4 antibodies by ELISA. Primary anti-CVB4 titers were similar in mice immunized with either CVB4 (open circles) or CVB4/p24(73₃) (open squares). Secondary anti-CVB4 titers were greater in mice immunized with CVB4/p24(73₃) (filled squares) than in mice immunized with CVB4 (filled circles). Statistical analysis was done using a one-way ANOVA.</p

Transient pancreatitis develops after intraperitoneal administration of CVB4/p24(73₃) but it does not develop after oral administration.

<p>Mice (n = 4) were infected with 5×10⁴ pfu administered intraperitoneally, 5×10⁴ pfu administered orally, or 10⁶ pfu administered orally. The pancreas was harvested from 2 mice at 4 dpi and from 2 mice at 7 dpi. Tissues were processed for routine histology and stained with hematoxylin and eosin. Representative sections are shown. (a) 4 dpi, IP infection with 5×10⁴ pfu, depicting pancreatitis; (b) 7 dpi, IP infection with 5×10⁴ pfu, depicting pancreatitis; (c) 4 dpi, oral infection with 5×10⁴ pfu, depicting a healthy, intact pancreas; (d) 7 dpi, oral infection with 5×10⁴ pfu, depicting a healthy, intact pancreas; (e) 4 dpi, oral infection with a higher dose,10⁶ pfu, depicting a healthy, intact pancreas; (f) 7 dpi, oral infection with a higher dose,10⁶ pfu, depicting a healthy, intact pancreas. A, acinus (core structure of the exocrine pancreas consisting of 8-12 acinar cells; IL, islet of Langerhans; in, inflammatory infiltrates. Original magnification, ×250. The scale bar is 25 µm.</p

Rapamycin alters the cytokine profile in the cerebrospinal fluid after gene delivery.

<p>Rapamycin alters the cytokine profile in the cerebrospinal fluid after gene delivery.</p

GFP-specific T cell responses as spot forming cells (SFC)/10⁶ PBMC after rapamycin treatment.

<p>T cell responses in PBMC were measured by ELISPOT at 14, 28, and 84 days after AAV9/GFP delivery in (A) AAV9/GFP only (controls); (B) AAV9/GFP + rapamycin. Arrow indicates undetectable (< 10 SFC/10⁶ PBMC) GFP-specific responses at necropsy (84 days).</p

GFP expression in lumbar spinal cords of NHPs monitored by IHC.

<p>GFP expression in lumbar spinal cords of NHPs monitored by IHC.</p

IHC was carried out to visualize GFP expression in the lumbar spinal cord.

<p>Shown are representative 40 micron lumbar spinal cord sections of all study macaques, stained for GFP. Magnified insets show examples of GFP-positive motor neurons. Macaque ID numbers are provided in each panel, along with a qualitative scoring of ventral horn expression. The (-) indicates no GFP-positive staining above that seen in uninjected NHPs; (+) indicates some positive cells observed, above that seen in uninjected macaques; (++) indicates relatively strong expression in a large number of neurons.</p