Development and application of a GuHCl-modified ELISA to measure the avidity of anti-HPV L1 VLP antibodies in vaccinated individuals.

Antibody responses against infectious agents are an important component in the prevention of disease. The avidity of antibodies for their antigens relates to their functional efficiency, and is a fundamental aspect in the investigation of humoral responses. Modified ELISAs are used to estimate avidity through the use of chaotropic agents and the measurement of the degree to which they disrupt the interaction between antibody and antigen. The theory behind the assay is the higher the avidity of an interaction the less susceptible it is to the effects of the chaotropic agent. The goal of this study was to generate a modified ELISA where a complex, multimeric coating-antigen, human papillomavirus (HPV) virus-like particles (VLP), was used to measure the avidity of anti-HPV antibodies generated following vaccination with HPV VLPs. A series of chaotropic agents were evaluated in the assay for their effectiveness in measuring avidity. Guanidine hydrochloride (GuHCl) was selected as a chaotropic reagent with the ability to disrupt antibody and antigen interactions, while not affecting the integrity of the plate-bound VLP. Two methods of determining the avidity index were assessed and shown to be comparable. This assay was then successfully applied to measure the avidity of anti-HPV VLP serum antibodies in samples from an HPV L1 VLP vaccine clinical trial. Overall, the assay was highly reproducible and captured a wide range of antibody avidities. Therefore, a GuHCl-modified ELISA is an acceptable method that can be used to determine HPV-specific antibody avidity indices within a clinical trial setting.

HPV16/18 L1 VLP vaccine induces cross-neutralizing antibodies that may mediate cross-protection.

Human papillomavirus (HPV) L1 VLP-based vaccines are protective against HPV vaccine-related types; however, the correlates of protection have not been defined. We observed that vaccination with Cervarix™ induced cross-neutralizing antibodies for HPV types for which evidence of vaccine efficacy has been demonstrated (HPV31/45) but not for other types (HPV52/58). In addition, HPV31/45 cross-neutralizing titers showed a significant increase with number of doses (HPV31, p<0.001; HPV45, p<0.001) and correlated with HPV16/18 neutralizing titers, respectively. These findings raise the possibility that cross-neutralizing antibodies are effectors of cross-protection observed for the HPV16/18 vaccine.

Increased plasma levels of adipokines and inflammatory markers in older women with persistent HPV infection.

We observed diminished lymphoproliferation to multiple stimuli in older women with persistent cervical human papillomavirus (HPV) infection. Adipokines are a class of inflammatory cytokines that are altered in some persistent infections. The objective was to compare the level of adipokines and inflammatory cytokines in heparinized plasma from women with persistent HPV cervical infection (Cases, N=50, oversampled for their weak lymphoproliferation responses) with women with no evidence of persistent HPV cervical infection (Controls, N=50, oversampled for their strong lymphoproliferation responses). Plasma samples were analyzed with multiplex assays for adipokines and inflammatory cytokines. Cases had significantly elevated plasma levels of resistin (p<0.0001) and sFas (p=0.0038) as compared to controls. Risk of persistent HPV infection increased significantly with increasing levels of resistin and 8Fas. This is the first study to demonstrate elevated levels of resistin and sFas in HPV persistently infected, older women with decreased immune function expanding the understanding of the systemic inflammation and immune alterations in individuals persistently infected with HPV. Further studies within a larger cohort are needed to define the generalities of these findings and any role adipokines have in persistent HPV infection.

Characterization of the HPV-specific memory B cell and systemic antibody responses in women receiving an unadjuvanted HPV16 L1 VLP vaccine.

Human papillomavirus (HPV)-specific antibodies are proposed to be the correlate of protection afforded by HPV L1 virus-like particle (VLP) vaccines. Previous studies have characterized the systemic antibody response to immunization in terms of both the quantity and the ability to neutralize HPV. Here, we have adapted a generalized memory B cell ELISPOT to the HPV16 system and expanded the analysis of the systemic antibody response to include an avidity measurement of HPV L1 VLP-specific antibodies. We show the results of the memory B cell ELISPOT significantly correlated with IgG and neutralizing antibody titers, but not with the avidity measurement. This is the first comprehensive study to correlate a variety of humoral aspects potentially associated with protective immunity following vaccination with a HPV16 L1 VLP vaccine.

Perfusion fixation preserves enhanced yellow fluorescent protein and other cellular markers in lymphoid tissues.

Fluorescent proteins are increasingly being used to analyze cellular gene expression and to facilitate tracking of cell lineages in vivo. One of these, enhanced yellow fluorescent protein (EYFP) has several properties such as intense fluorescence and little to no toxicity in cells, which makes it an excellent molecule to label proteins and cells of interest. In live cells, visualization of EYFP has been highly successful; however, detection of EYFP in lymphoid tissue sections, particularly in combination with other markers of interest has been difficult. This is because of the enhanced solubility of EYFP in the absence of fixation. When extended fixation protocols are employed, EYFP is preserved but detection of other cellular antigens becomes problematic due to over fixation. Here we demonstrate that EYFP-expressing T and B cells can be efficiently visualized in lymphoid tissue sections without compromising the ability to detect other cellular markers.

Differential microenvironment localization of effector and memory CD8 T cells.

CD8 T cells are critical for the clearance of intracellular pathogens. Upon infection, naive CD8 T cells differentiate into effector cells that target and eliminate infected cells. Following clearance of the pathogen, most effector cells die, although a small fraction survives to establish a memory population. Subsequent exposure to the same pathogen induces a rapid response of memory T cells and efficient elimination of the pathogen. Although much is known about the CD8 T cell response, the precise microenvironment location of effector and memory CD8 T cells in secondary lymphoid organs is not well characterized. In this study, we present an in situ analysis of the localization of effector and memory CD8 T cells during the murine immune response to lymphocytic choriomenginits virus. We identified the location of these cells using a transgenic mouse model system in which CD8 T cells are irreversibly tagged with yellow fluorescent protein (YFP) after activation. After infection, YFP+ CD8 T cells were initially observed within T cell zones. Later, these cells were found in the red pulp and a disruption of all CD8 T cell zones was observed. After resolution of the immune response, YFP+ memory CD8 T cells were observed primarily in T cells zones. Thus, in the spleens of mice, effector CD8 T cells localize to the red pulp and memory CD8 T cells localize to the T cell zones. Upon rechallenge, memory CD8 T cells rapidly proliferate and the secondary effector CD8 T cells are found in the red pulp.

The nonclassical MHC class I molecule Qa-1 forms unstable peptide complexes.

The MHC class Ib molecule Qa-1 is the primary ligand for mouse CD94/NKG2A inhibitory receptors expressed on NK cells, in addition to presenting Ags to a subpopulation of T cells. CD94/NKG2A receptors specifically recognize Qa-1 bound to the MHC class Ia leader sequence-derived peptide Qdm. Qdm is the dominant peptide loaded onto Qa-1 under physiological conditions and this peptide has an optimal sequence for binding to Qa-1. Peptide dissociation experiments demonstrated that Qdm dissociates from soluble or cell surface Qa-1(b) molecules with a t(1/2) of approximately 1.5 h at 37 degrees C. In comparison, complexes of an optimal peptide (SIINFEKL) bound to the MHC class Ia molecule H-2K(b) dissociated with a t(1/2) in the range from 11 to 31 h. In contrast to K(b), the stability of cell surface Qa-1(b) molecules was independent of bound peptides, and several observations suggested that empty cell surface Qa-1(b) molecules might be unusually stable. Consistent with the rapid dissociation rate of Qdm from Qa-1(b), cells become susceptible to lysis by CD94/NKG2A(+) NK cells under conditions in which new Qa-1(b)/Qdm complexes cannot be continuously generated at the cell surface. These results support the hypothesis that Qa-1 has been selected as a specialized MHC molecule that is unable to form highly stable peptide complexes. We propose that the CD94/NKG2A-Qa-1/Qdm recognition system has evolved as a rapid sensor of the integrity of the MHC class I biosynthesis and Ag presentation pathway.