Combinatorial Approach to Hepadnavirus-Like Particle Vaccine Design

Abstract

The particulate hepatitis core protein (HBcAg) represents an efficient carrier platform with many of the characteristics uniquely required for the delivery of weak immunogens to the immune system. Although the HBcAg is highly immunogenic, the existing HBcAg-based platform technology has a number of theoretical and practical limitations, most notably the "preexisting immunity" and "assembly" problems. To address the assembly problem, we have developed the core protein from the woodchuck hepadnavirus (WHcAg) as a new particulate carrier platform system. WHcAg appears to tolerate insertions of foreign epitopes at a greater number of positions than HBcAg. For example, both within the external loop region and outside the loop region a total of 17 insertion sites were identified on WHcAg. Importantly, the identification of an expanded number of insertion sites was dependent on additional modifications to the C terminus that appear to stabilize the various internal insertions. Indeed, 21 separate C-terminal modifications have been generated that can be used in combination with the 17 insertion sites to ensure efficient hybrid WHcAg particle assembly. This combinatorial technology is also dependent on the sequence of the heterologous insert. Therefore, the three variables of insert position, C terminus, and epitope sequence are relevant in the design of hybrid WHcAg particles for vaccine purposes.

Immunogenic Display of Diverse Peptides on Virus-like Particles of RNA Phage MS2

“…Three different 5′-primers [called (NNY) 6 , (NNY) 8 and (NNY) 10 ] attach at codon 14 a Kpn I site and 6, 8 or 10 randomized codons of sequence NNY (where N=A,C,G, or T and Y=T or C). Each reaction employed a single 3′-primer that annealed downstream of a Bam HI site in the plasmid vector.…”

Advantages to the use of rodent hepadnavirus core proteins as vaccine platforms

“…In summary, the advantages of utilizing the rodent hepadnavirus core proteins as opposed to the HBcAg as a carrier moiety for vaccine purposes include: i) the rodent core proteins are equally or more immunogenic than the HBcAg at the T and B cell levels; ii) the rodent core proteins will not substantially compromise the use of the anti-HBc diagnostic assay because they are not significantly crossreactive with HBcAg at the antibody level as shown herein and previously [11] iii) pre-existing anti-HBc antibodies in HBV chronically infected patients or in previously infected and recovered persons may limit the efficacy of the HBcAg platform, whereas, hybrid-WHcAg and hybrid-GSHcAg (data not shown) platforms do not bind preexisting anti-HBc antibodies; (iv) the HBcAg-specific immune tolerance present in HBV chronic carriers can be circumvented by the use of the WHcAg and GSHcAg platforms because the HBcAg is only partially crossreactive at the T cell level with WHcAg and GSHcAg; and v) the WHcAg and GSHcAg combinatorial technologies are more versatile than the HBcAg in terms of accommodating the insertion of a greater variety of foreign epitopes [12]. The immunogenicity of hybrid-WHcAg particles.…”

“…Transformation of chemically-competent Top10 by heat shock was carried out according to the manufacturer's protocol (Invitrogen). For greater detail regarding constructs refer to a previous report [12].…”

“…The hybrid-WHcAg particles are designated by the C-terminus, the inserted epitope (e.g., M,malaria P. falciparum CS repeat; MV, malaria P. vivax CS repeat; HV4, HIV gp120 THGIRPVVSTQLLLNGSLAEEE; IM2(−), FLU-M2, SLLTEVETPIRNEWGARANDSSD; CE, cholesteryl ester transfer protein, FGFPEHLLVDFLQSL) and the position of the insert (e.g., 74). Full descriptions of the C-termini and the inserted epitopes have been previously described (12). ND, not determined.…”

“…The "assembly" problem has been addressed by developing a combinatorial approach to hepadnavirus core protein vaccine design, which consists of an expanded number of insertion sites, an expanded number of C-terminal modifications and insert-specific modifications that in combination allow for efficient hybrid core particle assembly [12]. In the current study we have examined in greater detail the ability of the rodent hepadnavirus core proteins, in particular the WHcAg, to function as vaccine platforms and explored possible advantages to their use as opposed to the HBcAg.…”