Previous efforts to engineer humoral immunity using AAV-based vectors resulted in modest antibody production11
which was subsequently improved through the use of alternative capsids12
and self-complementary AAV (scAAV) vectors13
that increase expression at the expense of carrying capacity. Recently, scAAV vectors were employed to direct expression of SIV-neutralizing immunoadhesins consisting of small, artificially fused antibody fragments14
. However, the efficacy of this prophylaxis was limited by an endogenous immune response directed against the immunoadhesin proteins. To ask whether newer capsid serotypes and vector configurations might support long-lived expression of full-length human antibodies from muscle, we produced AAV vectors with the capsid from serotype 815
that expressed either luciferase or 4E10 HIV neutralizing antibody driven from CMV promoters and administered them through a single injection of the gastrocnemius muscle (). Within one week of vector administration, either luciferase or antibody gene expression was detectable (Supplementary Fig. 1a
, left and right respectively). Expression continued to rise, achieving maximum levels after 12–16 weeks and then decreasing two- to three-fold before stabilizing for the duration of the 64-week study. Given the long-lived nature of this expression, it seemed possible that these vectors could be used to engineer lifelong humoral immunity provided by full-length, fully human antibodies. Hence, we carried out a systematic process of vector and transgene optimization to improve the expression characteristics of this system (Supplementary Information
). The heavy and light chain variable regions of the HIV-neutralizing b12 antibody were cloned into the vector and AAV stock was produced for intramuscular administration of 1×1011
genome copies (GC) into the gastrocnemius muscle of two immunodeficient and two immunocompetent mouse strains: NOD/SCID/γc (NSG), Rag2/γc (RAG), B6, and Balb/C. Mice produced the encoded antibody at serum concentrations that were 100-fold higher than the levels achieved with the non-optimized vector and this level of expression persisted for at least 52 weeks ( compared to Supplementary Fig. 1a
, right). In agreement with previous studies of AAV-induced tolerance in mice16
, we detected very limited mouse antibodies raised against human b12-IgG in B6 mice while Balb/C animals generated detectable mouse antibodies against the transgene (data not shown) that did not appear to impact human IgG levels.
VIP protects against HIV-mediated CD4 cell depletion in humanized mice
To test the ability of VIP to protect mice from challenge in vivo
, we adapted a previously described humanized mouse model17
that exhibits CD4 cell depletion following challenge with replication-competent HIV (Supplementary Fig. 5
). We administered vector expressing either luciferase or b12 antibody to NSG mice, producing stable serum b12 antibody concentrations of approximately 100µg/mL within six weeks (). These mice were adoptively populated with expanded human peripheral blood mononuclear cells (huPBMCs), which engrafted over a period of two weeks. Mice were then challenged by intraperitoneal (IP) injection of the NL4-3 strain of HIV. Following HIV challenge, the majority of mice expressing luciferase showed dramatic loss of CD4 cells while mice expressing b12 antibody showed no CD4-cell depletion ().
To compare the protective abilities of the historically available broadly neutralizing antibodies, vectors expressing b12, 2G12, 4E10, and 2F5 were produced and administered to NSG mice. Seven weeks after administration, NSG mice produced 20–250µg/mL of the indicated antibodies (). Interestingly, in vivo
serum concentrations of 4E10 and 2F5 were somewhat lower than b12 and 2G12 despite comparable expression in vitro (Supplementary Figure 3b
), perhaps resulting from the previously described self-reactivity of these clones18
. Transduced mice were adoptively populated with huPBMCs, challenged by intravenous (IV) injection with HIV, and sampled weekly to quantify CD4 cell depletion over time (). Animals expressing b12 were completely protected from infection, while those expressing 2G12, 4E10 and 2F5 were partially protected. Groups demonstrating partial protection consisted of animals with delayed CD4 cell depletion as well as animals that maintained high CD4 cell levels throughout the course of the experiment. Interestingly, mice expressing 250µg/mL of the 2G12 antibody were only partially protected despite antibody levels being over 300-fold higher than previously established IC50
values for this antibody-strain combination in vitro19
. Eight weeks post-challenge, mice were sacrificed and paraffin-embedded spleen sections underwent immunohistochemical staining for the HIV-expressed p24 antigen to quantify the extent of infection (). Remarkably, mice expressing b12 had no detectable p24 expressing cells while those expressing other antibodies exhibited significant positive staining ().
Comparison of protection mediated by various broadly neutralizing HIV antibodies
To determine the robustness of protection mediated by VIP, a large cohort of mice expressing b12 antibody were adoptively populated with huPBMCs. Prior to challenge, all mice expressed high levels of human IgG, presumably due to engrafted human B-cells (Supplementary Fig. 6a
), but only those receiving the b12-expressing vector produced IgG specific for gp120, which reached 100µg/mL (Supplementary Fig. 6b
). Mock-infected mice expressing either luciferase or b12 demonstrated consistent high-level CD4 cell engraftment throughout the course of the experiment, showing that transgene toxicity was not contributing to CD4 cell loss (). In contrast, mice expressing luciferase that received 1ng of HIV experienced rapid and extensive CD4 cell depletion. At higher doses, infection in luciferase-expressing mice became more consistent and resulted in depletion of CD4 cells below the level of detection in some cases (25, 125ng doses). Remarkably, all mice expressing b12 demonstrated protection from CD4 cell loss, despite receiving HIV doses over 100-fold higher than necessary to deplete seven out of eight control animals ().
Robustness of CD4 cell protection mediated by b12 antibody
As newer anti-HIV antibodies have become available, we have compared the relative efficacy of b12 to VRC01 antibody. VRC01 neutralizes over 90% of circulating HIV strains in vitro1
, making it an excellent candidate for human trials. We administered decreasing doses of vector expressing either b12 or VRC01 to NSG mice and monitored expression of the antibodies over time. For both antibodies, we observed clear dose-dependent expression at all time points analyzed (Supplementary Fig. 7a
and (left)). Mice expressing luciferase or antibodies at various levels were adoptively populated with huPBMCs. Just prior to challenge, a gp120-specific ELISA confirmed the effective antibody concentration in each group (Supplementary Fig. 7b
and (middle)). Following IV challenge with 10ng of HIV, CD4 cells were monitored to determine the impact of antibody concentration. An average b12 concentration of 34µg/mL and VRC01 concentration of 8.3µg/mL protected mice from infection (Supplementary Fig. 7c
and (right)). Groups expressing lower concentrations of b12 and VRC01 were only partially protected, with several animals showing no detectable loss of CD4 cells and others exhibiting delayed CD4 cell depletion.
Determination of the minimum protective dose of VRC01 in vivo
Here we demonstrate that broadly neutralizing human antibodies expressed by VIP are capable of protecting animals from even high-dose HIV exposure in vivo
. Human-to-human HIV transmission rates vary with behavior but do not generally exceed one per hundred heterosexual exposures20
, and recent studies have demonstrated that infections are generally initiated by a single founder strain21
. Humanized mice with b12 serum concentrations of 100µg/mL were resistant to HIV infection at challenge doses 100-fold higher than necessary to infect the vast majority of animals, suggesting that this level of protection may far exceed what would be necessary to provide protection against HIV infection in humans. In contrast to previous approaches, VIP produces full-length antibodies that are identical in sequence to those produced by the immune system22
. Recent experiments have suggested that full-length antibody structures possess superior in vitro
neutralization activity as compared to modified architectures such as immunoadhesins23
. Utilization of such naturally occurring antibody architectures should also reduce immune responses against the transgene, which were previously shown to reduce the effectiveness of prophylaxis against SIV14
Our results demonstrate that VIP administration results in long-lived production of human antibodies at super-prophylactic levels in immunocompetent animals. Clinical trials involving AAV have demonstrated remarkable success when targeting immunoprivileged sites such as retinal tissue24
, but transduction of liver resulted in an adaptive immune response against vector capsid25
. Studies in non-human primates have shown that the elicitation of capsid-specific cytotoxic T-lymphocytes is limited to AAV capsids that exhibit heparin-binding activity26
. Interestingly, serotypes lacking heparin-binding activity, including AAV8, did not induce CTL responses, suggesting that AAV8-based vectors, like the one we have used, may circumvent previously observed immunological barriers to long-term transduction. Additionally, in contrast to liver transduction, administration of AAV via intramuscular injection has been shown to result in very long-lived, albeit low-level, Factor IX expression27
, suggesting that the route of administration can significantly impact the duration of expression. While the expression level attainable in humans remains to be determined, it is worth noting that the significantly longer serum half-life of a human antibody in humans28
, as opposed to mice29
, may result in higher steady-state levels than those observed in the present study. Regardless of this, our results suggest that even if VIP administration in humans results in serum antibody concentrations 100-fold lower than those observed in mice, it may still confer protection against HIV infection.
Given the urgency that exists in combating the ongoing global HIV pandemic and the incremental progress towards a vaccine, novel paradigms of prophylaxis must be explored towards solving this global health crisis. Our work demonstrates the feasibility of directly translating the existing repertoire of broadly neutralizing antibodies into functional immunoprophylaxis with robust protective abilities in vivo
. As more potent broadly neutralizing HIV antibodies are isolated30
, VIP can deliver these in concert with existing antibodies to provide increased potency, broader coverage, and greater resistance to escape mutations. This approach may find broad utility in the rapid development of effective prophylaxis against any existing or future infectious disease for which broadly neutralizing antibodies can be isolated. Beyond infectious diseases, VIP can be applied to therapeutic regimens in which continuous production of monoclonal antibodies in vivo
is desirable. Given the level of protection that VIP has demonstrated in vivo
, we believe that highly effective prophylaxis through expression of existing monoclonal antibodies against HIV in humans is achievable.