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. Later, in 2013 the scope of the project was further expanded to allow evaluation of the long-term consequences of transgene expression in three species of non-human primates.
1. Evaluate stability and safety of transgene expression
2. Evaluate potential for immune complex disease
3. Evaluate vector re-administration and antibody co-expression
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.
5. 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.
6. Establish optimal bNAb gene constructs and develop structure/function assays for in vivo expressed bNAb.
We are continuing the routine monitoring of the non-human primates, which express antibody delivered by intramuscular administration of AAV8. We also continue to monitor expression in the animals, which were re-administered with a second AAV vector expressing an additional antibody. At this time, most of the animals were expressing one or more antibodies for at least two years, and some for as long as four years with no visible complications. More data will become available as we near the completion of the studies.
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.
In order 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. With respect to primary evaluation criteria (transduction efficiency and stability of transgene expression), all vectors performed similarly. The AAV8 capsid was selected as a preferred candidate due to low impact of pre-existing neutralizing antibodies to the capsid on expression of transgene. Steady state expression after several years remains similar for pre-existing NAb levels of <1/320. Transduction and tropism were evaluated in three NHP species. After over a year post AAV2/8-mediated transduction in rhesus macaques, African green monkeys and squirrel monkeys expression in all species remains at 75, 175 and 50 μg/ml of serum, respectively. Our work on optimization of vector expression cassettes affords peak expression levels in rhesus macaques of up to 1056-1121 μg/ml of serum.