Vector-mediated gene transfer engenders long-lived neutralizing activity and protection against SIV infection in monkeys

Abstract

The key to an effective HIV vaccine is development of an immunogen that elicits persisting antibodies with broad neutralizing activity against field strains of the virus. Unfortunately, very little progress has been made in finding or designing such immunogens. Using the simian immunodeficiency virus (SIV) model, we have taken a markedly different approach: delivery to muscle of an adeno-associated virus gene transfer vector expressing antibodies or antibody-like immunoadhesins having predetermined SIV specificity. With this approach, SIV-specific molecules are endogenously synthesized in myofibers and passively distributed to the circulatory system. Using such an approach in monkeys, we have now generated long-lasting neutralizing activity in serum and have observed complete protection against intravenous challenge with virulent SIV. In essence, this strategy bypasses the adaptive immune system and holds considerable promise as a unique approach to an effective HIV vaccine.

Figure 1

Schematic representation of immunoadhesin constructs. For molecules derived from macaque Fabs (4L6, 8S, 5L7, and 3V), V_H and V_L domains were joined by a synthetic linker. Rhesus CD4 (domains 1 and 2) was cloned as described in the Methods section. Antigen-binding domains were attached to the Fc fragment of a rhesus IgG2 molecule.

Figure 2

Neutralization of SIV in vitro. The five immunoadhesins shown in Fig. 1 were tested for in vitro neutralizing activity against SVmac316. All five showed significant 50% neutralization at < 1 μg ml⁻¹. The IC₅₀ (ug ml⁻¹) for each was 4L6 (0.01), 8S (0.01), 5L7 (0.02), 3V (0.20), and N4 (0.25).

Figure 3

Serum concentration of immunoadhesins or antibodies. Sera from unimmunized and immunized animals were tested over time by a SIV gp120 ELISA for immunoadhesins (or antibodies in infected animals) after immunization and challenge infection (see Methods). The pink shaded areas in panels (a) – (c) represent the time period after immunization and before challenge. Day of challenge was one month after immunization. (a) 4L6 immunized animals. (b) 5L7 immunized animals. Levels of reactivity to gp120 for monkeys 05C004 and 05C053 were off scale (due to SIV infection), and peaked at 5,023 and 1,191 μg ml⁻¹, respectively. (c) N4 immunized animals. Note scale of the y-axis. The level of reactivity to gp120 for monkey 05D043 was off scale (due to SIV infection), and peaked at 3,953 μg ml⁻¹. (d) Unimmunized controls were infected at 2 different times (at one month and 4.5 months). Note scale of the y-axis.

Figure 4

SIV viral loads after challenge infection. Plasma samples from unimmunized and immunized animals were tested after challenge infection for SIV RNA genomes (viral load). Day of challenge was 0 months (and 3.5 months for the second set of unimmunized control animals). (a) 4L6 immunized animals. (b) 5L7 immunized animals. (c) N4 immunized animals. (d) Unimmunized controls. Monkeys C009, D225, D059, and D152 from the unimmunized control group were euthanized between 57 and 60 weeks after infection due to AIDS-related complications.

Figure 5

Anti-immunoadhesin antibodies in immunized monkeys. (a) Serum (1:100 dilution) collected over time from each immunized animal was tested by ELISA for reactivity against the homologous transgene. For example, sera from 5L7 recipients 05C002, 05C004, and 05C053 were reacted against purified 5L7 protein (as indicated on the x-axis). The dashed line indicates the limit of sensitivity. Animals with positive homologous reactivity were 5L7 recipients 05C004 and 05C053, and N4 recipient 05D043. The inset shows sera from animal 05C004 at a 1:1000 dilution, demonstrating a peak of activity at eight weeks, and a significant waning of the response by week 32. (b) Sera from the three animals with homologous reactivity were tested against all three purified proteins (as indicated on the x-axis). As expected, each animal serum reacted with the homologous transgene. 05C004 reacted against 5L7 (homologous) and 4L6 (heterologous). 05C053 reacted against 5L7 (homologous), 4L6, and N4 (both heterologous). 05D043 reacted only with the homologous N4 protein. The dashed line indicates the limit of sensitivity.