Efficient self-assembly of human papillomavirus type 16 L1 and L1-L2 into virus-like particles

Abstract

The L1 genes of two human papillomavirus type 16 (HPV16) isolates derived from condylomata acuminata were used to express the L1 major capsid protein in insect cells via recombinant baculoviruses. Both L1 major capsid proteins self-assembled into virus-like particles (VLP) with high efficiency and could be purified in preparative amounts on density gradients. The yield of VLP was 3 orders of magnitude higher than what has been obtained previously, using L1 derived from the prototype HPV16. DNA sequence comparison identified a single nonconserved amino acid change to be responsible for the inefficient self-assembly of the prototype L1. VLP were also obtained by expressing L1 of HPV6, HPV11, and cottontail rabbit papillomavirus, indicating that L1 from a variety of papillomaviruses has the intrinsic capacity to self-assemble into VLP. Coexpression of HPV16 L1 plus L2 by using a baculovirus double-expression vector also resulted in efficient self-assembly of VLP, and the average particle yield increased about fourfold in comparison to when L1 only was expressed. Coimmunoprecipitation of L1 and L2 and cosedimentation of the two proteins in a sucrose gradient demonstrated that L2 was incorporated into the particles. The ability to generate preparative amounts of HPV16 L1 and L1-L2 VLP may have implications for the development of a serological assay to detect anti-HPV16 virion immune responses to conformational epitopes and for immunoprophylaxis against HPV16 infection.

Identification of type-specific and cross-reactive neutralizing conformational epitopes on the major capsid protein of human papillomavirus type 31

“…In addition, it must be noted that no MAb directed against the L2 protein was produced. This could be due to the low proportion of L2 proteins in the capsid composition [20], which means that only a small proportion of the L2 epitopes are presented at the surface of VLPs [6,31], or to the fact that L1 epitopes are immunodominant. Further, it is possible that the test we used for anti-L2 screening is less sensitive than for L1.…”

“…The viral genome is approximately an 8-kb double-stranded DNA genome encapsidated in a structure consisting of 72 capsomers composed of L1. There is also a minor capsid protein involved in the capsid structure; its exact interaction and location in relation to L1 have not been fully characterised; however, it is proposed that the ratio of L1 to L2 is 30:1 [20]. Immunization with self-assembled VLPs made up of L1 or L1 and L2 induces neutralizing antibodies that confer type-specific and long-lasting protection in 3 different animal models of papillomavirus infection [2,21,34].…”

Virus-like particles as a highly efficient vaccine platform: Diversity of targets and production systems and advances in clinical development

“…Insect cells are equally effective in producing non-enveloped VLPs. Expression of HPV11 L1 or HPV16 L1 or L1 plus L2 proteins in baculovirus-infected Sf-9 insect cells has been demonstrated to result in the successful self-assembly of VLPs [174,175] that elicited high titers of serum neutralizing antibodies in mice and rabbits following IM or SC immunization with no adjuvant present [174,176]. Furthermore, HPV16 L1 VLPs have been shown to induce both systemic and cellular immunity in mice when administered IN or orally in the presence of cholera toxin or CpG, respectively, as mucosal adjuvants [177,178].…”

Bioengineering virus‐like particles as vaccines

“…Single layered non-enveloped VLPs can be assembled from (i) a single protein [e.g., hepatitis B core antigen (Clarke et al, 1987;Whitacre et al, 2009)]; or (ii) two proteins [e.g., cowpea mosaic virus VLPs (Saunders et al, 2009)]. Double layered non-enveloped VLPs can be assembled from (iii) two proteins [e.g., papillomavirus L1 and L2 VLPs (Kirnbauer et al, 1993)]; or (iv) four proteins [e.g., Foot-and-mouth disease virus (Porta et al, 2013)]. Triple layered VLPs (v) have been assembled from four coat proteins of bluetongue virus (Hewat et al, 1994) and rotavirus (Conner et al, 1996).…”