No abstract

No abstract

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.

No abstract

The aim of this study was to evaluate the potential of poly(D,L-lactide-co-glycolide) nano-and microspheres, with a mean diameter of 220 nm and 8 microm, respectively, to enhance the nasal and systemic immune responses against influenza virus antigen. High encapsulation levels of antigen were achieved in all cases. Neither the molecular weight nor the antigenicity of the entrapped antigen were affected by the encapsulation procedure. Following nasal immunization, the nasal washes IgA and the serum IgG responses were evaluated. With the soluble antigen, relatively high immune responses were observed. With nanospheres, nasal washes IgA levels were significantly lower (p<0.01) and serum IgG levels were not significantly different (p>0.05) from those obtained with the soluble antigen. With microspheres, both nasal washes IgA and serum IgG levels were significantly lower (p<0.01 and <0.05, respectively) as compared to the levels found for the soluble antigen. In addition, fluorescent microspheres administered intranasally failed to reach the nasal-associated lymphoid tissue (NALT). This lack of particle uptake by NALT and the high immunogenicity of the antigen used in this study, could explain the absence of enhancement of the immune responses by the polymeric particles.