Wilson: Vector-Mediated Passive Immunization
Circulating adenovirus-specific central memory CD4 T cells are attractive targets for vaccine development since they could quickly expand into a population of HIV-targeting cells in response to vaccination with an adenoviral vector. Initially, Wilson-led research focused on clarifying the biology of adenovirus vaccines in the context of their application to HIV. The original project contained four objectives poised to define the mechanism by which pre-vaccination exposure to natural Ad may have contributed to the increased acquisition of HIV infection. Those objectives were completed under budget and one year before the grant term ended.
In 2010 funds were redirected to a new set of objectives that take advantage of the unique expertise of Dr. Wilson. Three new objectives explore the feasibility of using AAV vectors with different capsids (AAV1, 8, and 9) for delivery and expression of broad HIV antibodies as a vaccine-like, low cost, sustainable technology for HIV prevention. As a result of these studies, we selected rAAV8 as a candidate for sustainable Ab delivery.
Many of the studies conducted to date have been done in rhesus macaques, the species from which AAV2/8 was discovered. However, the biology of AAV8 in macaques may be different from the biology of AAV8 in other primate species, including humans, due to the level of pre-existing NAbs, which are much higher and more prevalent in rhesus macaques than in humans. Consequently, in 2011, the refined project was extended to include evaluation of multiple NHP species for use in the AAV8-mediated Abs expression studies.
Dr. Wilson and his team will continue to evaluate the feasibility of using AAV8 to deliver a transgene encoding a bNAb to muscle in order to achieve levels of the bNAb in blood, vagina and rectum which could prevent HIV infections. They will use concentrations of bNAbs in blood and mucosal surfaces as surrogates of efficacy. They plan to determine optimal conditions for injecting skeletal muscle by defining the best target muscle group, formulation, and promoter driving the Ab expression. Simultaneously, they will optimize the Ab gene construct for expression in muscle, and develop additional assays for structural and functional characterization of the
in vivo expressed HIV Abs.
1. Define the AAV capsid that confers the best expression; evaluate transduction and tropism in three NHP species; define impact of pre-existing vector NAbs on transgene expression; define optimal group of muscles for transduction; optimize ROA for muscle delivery; and define optimal promoter.
2. Establish optimal bNAb gene constructs and develop structure/function assays for in vivo expressed bNAb.
1. Evaluate the adenovirus ecology in the gastro-intestinal tracks of men who have sex with men.
2. Evaluate the natural history of adenovirus infections in macaque populations.
3. Create an adenovirus vector based on a macaque-derived virus.
4. Determine if vaccination with an adenoviral vector in macaques increases the number of activated CD4 T cells in gut lamina propria.
To identify the best AAV capsid for the expression of the therapeutic gene following IM injections, the team compared muscle transduction by AAV2/1, 2/8 and 2/9 in rhesus macaques, using alpha-fetoprotein (rhAFP) driven by the constitutive CMV promoter as a transgene. The primary evaluation criteria were transduction efficiency and stability of transgene expression. Based on this year-long study, they have chosen AAV2/8 as their preferred vector for the delivery of the NAbs into the muscle.
Comparative expression studies for AAV2/8-mediated transduction in rhesus macaques, African green monkeys and squirrel monkeys were initiated to help us to select preferred NHP species for gene expression studies. Rhesus bNAb in the immunoadhesin configuration was used to evaluate AAV2/8 efficacy for delivery of the transgene. Evaluation is based on the comparison of the peak and steady-state long-term expression levels between different NHP species. After six months, gene expression is similar in rhesus macaques and squirrel monkeys (both, peak and long-term), and is considerably lower in African green monkeys. This study is still ongoing.
To evaluate the effects of the pre-existing NAbs on transgene expression, rhesus macaques with different levels of pre-existing NAbs were monitored for expression of rhesus bNAb in immunoadhesin configuration after muscle transduction by AAV2/8. While peak expression was somewhat variable between different groups of NHP, the steady-state expression after one year remains very similar for pre-existing NAb levels with a titer of up to 1/320. This study is still ongoing.
The process of gene optimization is much like going from hit to a lead to a clinical candidate in drug development with the ultimate rate-limiting step being the cost of vector. Dr. Wilson and his research team plan to reduce the cost of vector production by combining additive/synergistic improvements that fall into 3 major categories: vector optimization (production methods that yield more potent vector), transgene optimization and improved transduction by optimization of injection protocols. The current focus of this set of studies is on the optimization of transgene format (i.e., two promoters vs. single promoter, IRES vs. 2A linker, codon bias) and improving transduction. These studies are still ongoing.
Through the analyses of human samples from various regions of GI tract, the researchers showed that human gut is colonized with a diverse flora of Ads primarily of the subtype E family with a high level of T cell responses in blood and variable T cell responses at gut mucosa. By analyzing samples from three animal facilities, they determined that macaques, like humans, are colonized with a diverse flora of Ad in the gut and have Ad-specific T cells in gut mucosa. While the structures and biologies of rhesus Ads are different from those found in humans and great apes, the only real difference in host responses to natural Ad infections is that humans have moderate to high levels of Ad-specific T cells in peripheral blood in comparison to macaques. The research team has created molecular clones from six of the novel Ad isolates and used SAdV‐7 to vaccinate macaques. Following vaccination, virtually all animals showed a global increase in activated CD4 and CD8 T cells, including T cells in the gut mucosa. This increase was independent of antigen, independent of pre-existing NAb and was not reflected in the peripheral blood since animals showed very little increase in Ad-specific T cells following vaccination. However, the pre-existing vector NAb did substantially diminish the transgene-specific responses as they were in the STEP trial. Their hypothesis regarding STEP is that both NAb+ and NAb‐ subjects realized an increase in T cells against HIV in the gut thus increasing the chances of acquiring HIV. The more robust response to HIV antigens in patients who were Ad NAb negative was able to offset the increased susceptibility to HIV infection leading to no real vaccine effects in terms of acquisition. However, NAb-positive vaccines mounted a less robust anti-HIV T cell response that was not able to compensate for the increased acquisition of HIV; the net effect would be increased acquisition. An alternative explanation is that there is a difference in the activation of CD4 T cells in the gut following an Ad-based vaccine as a function of pre‐existing NAb, although our study was not powered sufficiently to evaluate this question.