No abstract

At least 15 high-risk human papillomaviruses (HPVs) are linked to anogenital preneoplastic lesions and cancer. Currently, there are three licensed prophylactic HPV vaccines based on virus-like particles (VLPs) of the L1 major capsid protein from HPV-2, -4, or -9, including the AS04-adjuvanted HPV-16/18 L1 vaccine. The L2 minor capsid protein contains HPV-neutralizing epitopes that are well conserved across numerous high-risk HPVs. Therefore, the objective of our study was to assess the capacity to broaden vaccine-mediated protection using AS04-adjuvanted vaccines based on VLP chimeras of L1 with one or two L2 epitopes. Several chimeric VLPs were constructed by inserting L2 epitopes within the DE loop and/or C terminus of L1. Based on the shape, yield, size, and immunogenicity, one of seven chimeras was selected for further evaluation in mouse and rabbit challenge models. The chimeric VLP consisted of HPV-18 L1 with insertions of HPV-33 L2 (amino acid residues 17 to 36; L1 DE loop) and HPV-58 L2 (amino acid residues 56 to 75; L1 C terminus). This chimeric L1/L2 VLP vaccine induced persistent immune responses and protected against all of the different HPVs evaluated (HPV-6, -11, -16, -31, -35, -39, -45, -58, and -59 as pseudovirions or quasivirions) in both mouse and rabbit challenge models. The degree and breadth of protection in the rabbit were further enhanced when the chimeric L1/L2 VLP was formulated with the L1 VLPs from the HPV-16/18 L1 vaccine. Therefore, the novel HPV-18 L1/L2 chimeric VLP (alone or in combination with HPV-16 and HPV-18 L1 VLPs) formulated with AS04 has the potential to provide broad protective efficacy in human subjects. IMPORTANCEFrom evaluations in human papillomavirus (HPV) protection models in rabbits and mice, our study has identified a prophylactic vaccine with the potential to target a wide range of HPVs linked to anogenital cancer. The three currently licensed vaccines contain virus-like particles (VLPs) of the L1 major capsid protein from two, four, or nine different HPVs. Rather than increasing the diversity of L1 VLPs, this vaccine contains VLPs based on a recombinant chimera of two highly conserved neutralizing epitopes from the L2 capsid protein inserted into L1. Our study demonstrated that the chimeric L1/L2 VLP is an effective vehicle for displaying two different L2 epitopes and can be used in a quantity equivalent to what is used in the licensed vaccines. Hence, using the chimeric L1/L2 VLP may be a more cost-effective approach for vaccine formulation than adding different VLPs for each HPV. O ncogenic human papillomaviruses (HPVs) cause cervical cancer (1), and it is widely considered that all cervical cancers are linked to at least one HPV (2-5). The oncogenic HPV-16 and HPV-18 are detected in approximately 70% of HPV-positive cervical cancers and represent the two HPVs most frequently detected in these cancers (3,(6)(7)(8). Other high-risk oncogenic HPVs also detected in cervical cancer include 7,8). In addition to cervical cancer, other HPVs, such as HPV-5, ha...

Cervarix™ is a prophylactic human papillomavirus (HPV)-16 and -18 vaccine, developed for the prevention of cervical cancer. The vaccine antigens are HPV-16 and HPV-18 L1 virus-like particles (VLPs) made from recombinant HPV-16 and HPV-18 L1 proteins, respectively. HPV-16 and HPV-18 L1 proteins are expressed at high levels in insect cells using the baculovirus expression vector system (BEVS). Under natural conditions, L1 migrates to the nucleus where virus assembly occurs. The L1 antigen used for the HPV-16/18 VLP vaccine is truncated at the Cterminus by 34 amino acids for HPV-16 and 35 amino acids for HPV-18. This truncation removes the nuclear targeting signal as well as the DNA binding domain. The distribution of HPV-16 and HPV-18 L1 in insect cells expressing the proteins was investigated by immunogold EM. Both HPV-16 and HPV-18 L1 proteins were found exclusively in the cytoplasm and no VLPs were detected. This confirms that the truncation prevented the migration of the L1 proteins into the nucleus and that VLP did not assemble intracellularly. The L1 proteins were extracted and purified through a multistage process which, after self-assembly of L1 capsomeres, yields highly pure and immunogenic VLP. The physico-chemical and immunological properties of the HPV-16 and HPV-18 L1 VLPs have been thoroughly characterized [1], as well as their stability [2]. By negative staining EM, most VLPs appear single-shelled and subspherical in shape (Fig. 1). Their size, measured by disc centrifugation spectroscopy (DCS), was in good agreement with EM observations, ranging from 30 to 50 nm for HPV-16 L1 VLP, with a main peak at 40 nm. For HPV-18 VLP, the size ranged between 45 and 55 nm with a main peak at 50 nm. Both HPV-16 and HPV-18 L1 VLP populations had a small subset of multilayered VLPs with a size ranging from 60 to 75 nm. On average, the proportion of multilayered VLPs was 10.3 % and 7.8 % for HPV-16 and HPV-18 L1 VLP, respectively. Protein Tomography™ (SIDEC Technologies) was applied to cryo-EM analysis of the VLP. It revealed at the surface of the VLPs a combination of pentameric and hexameric organizations of the capsomeres with a typical icosahedral symmetry ( Fig. 2A). The 3D analysis also demonstrated that the structure of the large VLPs was either true concentric shells of capsomeres (2 or 3, rarely 4) or a compact spiral of capsomere ribbons. Protein Tomography™ also allowed visualizing the binding of monoclonal antibodies directed against conformational neutralizing VLP epitopes (Fig. 2B). Finally, direct imaging by negative staining in TEM showed that VLPs adsorbed on aluminum hydroxide were indistinguishable from their counterpart in bulk solution (Fig. 3). It demonstrated that adsorption on aluminum hydroxide in formulated vaccine did not alter the structure of VLPs.

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

Rotavirus (Reoviridae) viral particles consist of capsids with 3 concentric protein layers surrounding the virus genetic material. The outer layer is composed of the VP4 and VP7 proteins, which independently elicit neutralizing antibodies and induce protective immunity.Rotarix™, a live attenuated rotavirus vaccine, has been characterized by EM. Both negative staining ( Fig. 1) and cryo-EM ( Fig. 2) were applied. The observations showed that the vaccine contained both triple-layered particles (TLPs) containing all 3 protein layers and double-layered particles (DLPs) which lack the outer capsid proteins VP4 and VP7. Their apparent diameter difference of about 10 nm was well correlated in both techniques.As DLPs are non-infectious, it may be of importance to address the question of their abundance vs TLPs in bulk preparations. Although such information can be obtained by EM, the method is relatively impractical for this purpose, particularly for routine analysis, as high number of particles should be observed and sized to get accurate relative abundances.To determine the size and quantify the relative abundance of TLPs and DLPs on large populations of viral particles in bulk preparations, two size-based, orthogonal analytical techniques were applied to detect these particles: capillary electrophoresis (CE) and disc centrifuge (DC). To confirm the capacity of both methods to distinguish clearly the two types of capsids, TLPs and DLPs were partially purified using CsCL2 gradient. The profiles (Fig. 3) show the well-resolved identification.