A double-blind randomized phase I clinical trial targeting ALVAC-HIV vaccine to human dendritic cells

Abstract

Background: We conducted a novel pilot study comparing different delivery routes of ALVAC-HIV (vCP205), a canarypox vaccine containing HIV gene inserts: env, gag and pol. We explored the concept that direct ex vivo targeting of human dendritic cells (DC) would enhance the immune response compared to either conventional intramuscular or intradermal injections of the vaccine alone.

Methodology/principal findings: Healthy HIV-1 uninfected volunteers were administered ALVAC-HIV or placebo by intramuscular injection (i.m.), intradermal injection (i.d.) or subcutaneous injection (s.q.) of autologous ex vivo transfected DC at months 0, 1, 3 and 6. All vaccine delivery routes were well tolerated. Binding antibodies were observed to both the ALVAC vector and HIV-1 gp160 proteins. Modest cellular responses were observed in 2/7 individuals in the DC arm and 1/8 in the i.m. arm as determined by IFN-γ ELISPOT. Proliferative responses were most frequent in the DC arm where 4/7 individuals had measurable responses to multiple HIV-1 antigens. Loading DC after maturation resulted in lower gene expression, but overall better responses to both HIV-1 and control antigens, and were associated with better IL-2, TNF-α and IFN-γ production.

Conclusions/significance: ALVAC-HIV delivered i.m., i.d. or s.q. with autologous ex vivo transfected DC proved to be safe. The DC arm was most immunogenic. Proliferative immune responses were readily detected with only modest cytotoxic CD8 T cell responses. Loading mature DC with the live viral vaccine induced stronger immune responses than loading immature DC, despite increased transgene expression with the latter approach. Volunteers who received the autologous vaccine loaded mature DC developed a broader and durable immune response compared to those vaccinated by conventional routes.

Trial registration: ClinicalTrials.gov NCT00013572.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Study Design.

(A) Consort diagram. (B) Study timeline. Vaccinations were administered at 0, 1, 3 and 6 months. All study subjects underwent apheresis prior to initial vaccination (visit 1) and 2 weeks following the final vaccination (visit 11).

Figure 2. Post-vaccination reactogenicity.

Post injection-site reactogenicity symptoms including induration (left panel) and erythema (right panel) after each vaccination for all study groups (autologous DC (DC), intradermal (ID) and intramuscular (IM) routes).

Figure 3. Antibodies develop after vaccination.

Humoral responses to the vaccine vector (ALVAC, left panel) and insert genes HIV-gp160 (middle panel) and HIV-p24 (right panel). Data points are the geometric mean titers (GMT)+SEM bars for all subjects within each study arm receiving vaccine. Visit numbers are indicated and an asterisk (*) denotes vaccination visits. Closed diamonds, autologous DC route; open circles, ID route; open squares, IM route.

Figure 4. HIV specific lymphocyte proliferation responses.

LPA responses of PBMC from (A) pre-vaccination (v.1, white bars) and two weeks post final vaccination (v.11, black bars) from all vaccinees. Responses to whole inactivated HIV (AT-2 HIV), HIV-gp160 and HIV-p24 were measured and reported as a lymphocyte stimulation index (LSI, antigen response/unstimulated control). An LSI of >5 (dashed line) is considered positive. (B) Durability of proliferative responses up to 18 months post final vaccination for the 4 responders in the DC vaccination arm. Responses to HIV antigens, keyhole limpet hemocyanin (KLH, positive control for DC injection) and aldrithiol-2 microvessicles (MV, negative control for AT-2 HIV) are expressed as mean LSI±SEM from LPAs performed on fresh PBMC. (C) Proliferative responses pre-vaccination (grey histograms) and 2 weeks post final vaccination (black lines) to HIV antigens were measured by flow cytometry using CFDA-SE. CD4+ and CD8+ T cell responses are shown for two donors from the DC arm stimulated with either AT-2 HIV (left panels) or HIV-p24 (right panels). Proliferative response is measured as the percent of cells that have undergone division and show reduced intensity of CFSE. The ratio of proliferative response post-vaccination to pre-vaccination is shown.

Figure 5. DC vaccine characterization.

(A) Schematic representation of the DC maturation sequence. Autologous DC were loaded with vaccine either after maturation (black bars) or before maturation (grey bars). (B) Characteristics of vaccine-loaded DC, segregated by loading/maturation sequence. Expression of HIV-p24 and the maturation marker CD83 were assessed by flow cytometry. The number of DC per injection and cell viability at the time of injection were assessed by visual counts using Trypan Blue exclusion. P-values represent the statistical difference as determined by an unpaired t-test. (C) Proliferative responses of DC arm vaccinees to KLH (white bars) and AT-2 HIV (black bars). Data is shown as mean LSI±SEM from LPAs performed on fresh PBMC isolated between visits 1–16. (D) Cytokine secretion following AT-2 HIV stimulation of PBMC collected pre-vaccination (v.1, white bars) and two weeks following the final vaccination (v.11, black bars). IFN-γ, TNF-α, IL-2 and IL-10 secretion from representative responder and non-responder vaccinees from the DC arm are shown.

Figure 6. Antigen-specific differential gene expression.

(A) Principal components analysis of differentially expressed genes for the group of responders in the DC arm. Red spheres denote pre-vaccination state (Visit 1) and blue spheres denote post-vaccination status (Visit 11). The X-axis is the first principal component, the Y-axis is the second principal component and the Z-axis is the third principal component. (B) Functional groups of gene families most significantly down-regulated in response to in vitro AT-2 HIV stimulation of PMBC from the DC arm responders.