A common goal for all vaccine programs is to identify clinically relevant correlates of protection including, specifically, laboratory parameter(s) that have been shown to be associated with protection from clinical disease. For many infectious diseases, neutralization antibody assays are used to assess the immunogenicity of prophylactic vaccines. For HPVs, the “gold standard” for measuring neutralizing antibody titers is the athymic mouse xenograft system (7
). In this assay, sera from individuals is mixed with infectious HPV and added to foreskin tissue, which is then implanted under the renal capsule of athymic mice. The implants are monitored for histological changes and for HPV DNA by in situ hybridization, which can take several months to develop. Because of the technical difficulties in testing a large number of sera in the athymic mouse xenograft system (7
), several complementary assays have been developed to measure neutralizing and non-neutralizing antibody titers. These include in vitro pseudoneutralization assays (7
), cRIAs (28
), and VLP-based enzyme-linked immunosorbent assays (43
). However, studies have shown that the pseudoneutralization assay can be 20- to 30-fold less sensitive than HPV-VLP enzyme-linked immunosorbent assays (24
) and up to a 100-fold less sensitive than a cRIA (28
). The pseudoneutralization assay is also a complex cell-based assay that requires dilution of the serum sample to avoid interference (44
). Since an immunoassay that measures HPV type-specific antibodies simultaneously is preferred to running multiple separate tests, we employed the Luminex LabMAP3 technology to develop a competitive immunoassay that simultaneously measures type-specific antibodies to neutralizing epitopes on HPV-6, -11, -16, and -18.
The rationale for developing a multiplexed competitive immunoassay, rather than a capture assay, was that capture assays to VLPs measure a combination of antibodies to both neutralizing and non-neutralizing epitopes and, potentially, other antibodies to yeast-derived proteins. Other advantages of the competitive assay format are that the serum sample does not need to be diluted and the assay measures antibodies specific to HPV type-specific neutralizing epitopes. Multiplexing the assay had little to no effect on the standard curves for HPV-11, -16, and -18 but did affect the HPV-6 standard curve by shifting the curve to the left (Fig. ). This effect on the HPV-6 curve was most likely due to the cross-reactivity of the HPV-11 sera to the HPV-6 VLP. This cross-reactivity was not unexpected, since HPV-6 and HPV-11 have 93% amino acid identity in the L1 protein (9
) and animals or individuals immunized with VLP-11 can neutralize HPV-6 (44
). Nevertheless, the HPV-Luminex assay showed good correlation with the competitive HPV type-specific RIAs (Pearson correlation coefficients of between 0.751 and 0.837). It is also noted that changing the number of microspheres analyzed did not affect the MFI values (data not shown), which confirms the observation by Vignali (40
). Since the HPV-Luminex assay correlated well with the currently used cRIAs, the Luminex assay should be a useful tool for analyzing antibody titers to different HPV genotypes.
The main advantage of the LabMAP3 technology is that it provides a sensitive and precise method to simultaneously measure antibodies to HPV type-specific, neutralizing epitopes against multiple HPV genotypes. Because multiple tests can be run on a single serum sample, LabMAP3 technology has the advantage of being ideal for use on pediatric samples for which serum volumes are often limiting. This has been shown previously by Pickering et al., who used the Luminex technology to simultaneously quantitate antibodies to 14 different pneumococcus polysaccharides in pneumococcal vaccine studies (30
). Bellisario et al. have also used Luminex technology to simultaneously measure antibodies to human immunodeficiency virus type 1 p24, gp160, and gp120 eluted from dried blood-spot specimens from newborns (2
). Luminex LabMAP3 technology has also been used to test serum samples for antibodies to a panel of seven respiratory viruses, including influenza A and B viruses; adenovirus; parainfluenza viruses 1, 2, and 3; and respiratory syncytial virus (21
). In this respiratory panel Luminex assay, internal controls were also added to test for rheumatoid factor and sufficient quantities of IgG to assure sample addition (21
). Because an effective HPV vaccine may include additional genotypes to cover more of the known cancer-causing viruses, LabMAP3 technology provides a platform for adding additional HPV genotypes to the assay. In addition to the flexibility of adding other VLP genotypes to the assay, Luminex platform technology provides the ability to add other assays, such as viral load measurements or cytokine assays, to the same test.
Although the assay described here included a wash step, a number of investigators have developed rapid, no-wash Luminex assays that can be performed more quickly (27
). A no-wash assay would certainly be advantageous, especially since the sensitivity and the precision of the HPV-Luminex assay were not compromised (Fig. ). However, the high concentrations of serum appeared to clog the Luminex100
and caused many of the VLP-microspheres to fall outside their specified calibration gates, lengthening the read times to greater than 1 h. An advantage of the 96-well plate Luminex assay format is that it avails itself to automation. We have used a Tecan Genesis liquid handler to automate the assay, which has saved time and has the added value of preventing accidental exposure of laboratory personal to potentially infectious serum. In summary, LabMAP3 technology provided a sensitive and robust system to develop a competitive immunoassay that accurately and precisely measures antibodies to neutralizing epitopes on HPV-6, -11, -16, and -18.