2004;321:205C216. of prophylactic HPV vaccines and measurement of their immunogenicity. The licensed preventive HPV vaccines are based on the seminal discovery that the L1 major capsid protein alone or co-expression with the minor capsid L2 protein will co-assemble into VLP (Kirnbauer et al., 1992; Zhou et al., 1993). Under both circumstances, these L1 or L1/L2 VLPs resemble empty HPV virions upon examination by an electron microscope, and when used for vaccination, elicit high titer type-specific neutralizing antibody responses (Harro et al., 2001). Early research on the biology and immunology of HPV virions was hampered by the inability to readily generate quantities of virions in standard tissue culture, the absence of a simple and quantitative readout for infectivity and their inability to produce disease in an animal model. HPV PsVs carrying reporter plasmids have proved as a valuable surrogate of authentic papillomavirus for many such studies (Buck and Thompson, 2007) because they can be readily manufactured, have simple and flexible readouts of infectivity both in tissue culture and animals, including mice (Roberts et al., 2007). Although highly successful and efficacious, the current licensed HPV vaccines provide mostly type-restricted and Igfbp6 there remain logistical challenges for their global implementation including cost, and need for a cold chain, and needle-based delivery. Consequently there are ongoing efforts to develop second-generation HPV vaccines based upon L1, L2 or both capsid proteins. L2 is a promising vaccine antigen because of its potential to protect against multiple oncogenic HPV types (Alphs et al., 2008; Gambhira et al., 2007a; Gambhira et al., 2007b; Pastrana et al., 2005; Roden et al., 1994). However, because L2 does not form a VLP, it is weakly immunogenic relative to L1. Nonetheless, preclinical vaccination studies with L2 are sufficient to provide full prophylaxis against a large inoculum of HPV genotypes. The standard neutralization assay developed by Buck and colleagues (Pastrana et al., 2004) often shows serum titers from L2 vaccinated mice to either be very low, or sometimes undetectable. Yet, passive transfer of the same anti-L2-serum antibodies can confer complete protection against experimental viral challenge in naive mice (Wang et al., 2014a). The discrepant findings for L2-specific antibodies between the neutralization assay (Pastrana et al., 2004) and animal protection studies (Roberts et al., 2007) suggest that the currently utilized PsV neutralization assay is not sensitive for anti-L2 neutralizing antibodies, although it detects L1-specific neutralizing antibodies with great sensitivity. Studies by Day utilizing HPV PsV identified spatio-temporal differences in the HPV L2-epitope exposure between the infection of 293TT cells used for neutralization studies (Day et al., 2008a; Day et al., 2007), and UNC569 infection of basal keratinocytes in the mouse challenge model (Day et al., 2008a). This may reflect differences in the primary receptor in 293TT monolayers versus the basement membrane versus neutralization assay, limiting the detection sensitivity of anti-L2 neutralizing antibodies (Day et al., 2012). To improve the detection of anti-L2 antibodies, we first developed maturation step. During the maturation step, we have found that raising the calcium concentration increases the activity of furin in the 293TTF cells (Anderson et al., 2002; Anderson et al., 1997), and cleavage of L2 in the PsV UNC569 (Wang et al., 2014a) (Figure 1B). In this section, we describe 1) transfection of 293TTF cells with HPV codon-optimized viral capsids and reporter plasmid; 2) harvesting/lysing of transfected 293TTF cells; and 3) maturation and purification of fcPsV particles (or studies. The method involves extraction of reporter plasmid from a given volume of HPV fcPsV virions and performing qPCR analysis to detect encapsidated reporter plasmid molecules versus a UNC569 known standard. Below, we outline the steps to perform quantitative polymerase chain reaction (qPCR) analysis to detect HPV fcPsV viral reporter plasmid genomic equivalents with a known standard. Additional Materials Machine able to perform qPCR Known quantity UNC569 (i.e 5C50 nanograms) of UNC569 reporter DNA to be utilized for the standard curve during qPCR analysis. (e.g. SEAP: pYSEAP construct, or LUCIFERASE: pcDNA-luciferase plasmid from: under under for 1 minute to bring down liquid that may have formed on the walls of the wells because of condensation. 40. Peel-off the plate sealer, and add 25 L of substrate. challenge studies. Addition materials Clear plastic 96 well plates Black.