Modification of Ad5 Hexon Hypervariable Regions Circumvents Pre-Existing Ad5 Neutralizing Antibodies and Induces Protective Immune Responses

Abstract

The development of an effective malaria vaccine is a high global health priority. Vaccine vectors based on adenovirus type 5 are capable of generating robust and protective T cell and antibody responses in animal models and are currently being evaluated in clinical trials for HIV and malaria. They appear to be more effective in terms of inducing antigen-specific immune responses as compared with non-Ad5 serotype vectors. However, the high prevalence of neutralizing antibodies to Ad5 in the human population, particularly in the developing world, has the potential to limit the effectiveness of Ad5-based vaccines. We have generated novel Ad5-based vectors that precisely replace the hexon hypervariable regions with those derived from Ad43, a subgroup D serotype with low prevalence of neutralizing antibody in humans. We have demonstrated that these hexon-modified adenovectors are not neutralized efficiently by Ad5 neutralizing antibodies in vitro using sera from mice, rabbits and human volunteers. We have also generated hexon-modified adenovectors that express a rodent malaria parasite antigen, Py CSP, and demonstrated that they are as immunogenic as an unmodified vector. Furthermore, in contrast to the unmodified vector, the hexon-modified adenovectors induced robust T cell responses in mice with high levels of Ad5 neutralizing antibody. We also show that the hexon-modified vector can be combined with unmodified Ad5 vector in prime-boost regimens to induce protective responses in mice. Our data establish that these hexon-modified vectors are highly immunogenic even in the presence of pre-existing anti-adenovirus antibodies. These hexon-modified adenovectors may have advantages in sub-Saharan Africa where there is a high prevalence of Ad5 neutralizing antibody in the population.

Identification of a Suppressor Mutation That Improves the Yields of Hexon-Modified Adenovirus Vectors

“…were generated by using our plasmid-based adenovirus vector construction system as previously described (35). Briefly, the recombinant Ad5 genomes (containing a transgene expression cassette and hexon modification) were liberated from plasmids by digestion with PacI restriction endonuclease, transfected into monolayer 293 or 293-ORF6 cells, and cell lysates were serially passaged every 3 days until cytopathic effect (CPE) was observed.…”

“…Vector administrations to generate preexisting Ad5-neutralizing antibodies were delivered by IM injection into the left gastrocnemius muscle (1 ϫ 10 10 PU) followed by a booster immunization with the same vector (1 ϫ 10 10 PU) given 4 weeks later. Ad5 NAb were generated using an Ad5 vector expressing luciferase, Ad5L, and hexon-specific Ad5 NAb were generated using a chimeric Ad5 vector containing an Ad35 fiber, Ad5L.F (35).…”

“…These NAbs target the nine hypervariable regions that form the exposed surface of the hexon protein (31,33). We and others have shown that it is possible to replace the nine hypervariable regions of the Ad5 hexon with those derived from group C (31) or group D (34,35) serotypes. The Ad5-based hexon-modified vectors that contain subgroup D hypervariable regions (Ad43 or Ad48) induced robust transgene-specific immune responses that were unaffected by Ad5-specific NAb in murine and nonhuman primate vaccine models.…”

Adenovirus with DNA Packaging Gene Mutations Increased Virus Release

“…Each facet of the Ad capsid contains twelve hexon trimers with hexon pentamers positioned at each vertex of the icosahedral capsid [70,71]. in vivo, hexon is highly immunogenic [72] and contains many hypervariable regions (HVRs) which can be bound by antibodies to neutralize the infectivity of Ads [73]. Hexon is known to contain nine HVRs located in the DE1 and FG1 loops between the coded 126 and 461 amino acids [73].…”

“…in vivo, hexon is highly immunogenic [72] and contains many hypervariable regions (HVRs) which can be bound by antibodies to neutralize the infectivity of Ads [73]. Hexon is known to contain nine HVRs located in the DE1 and FG1 loops between the coded 126 and 461 amino acids [73]. The mutations located at 20378 and 21630 are not contained within one of these nine HVRs.…”