Janine T. Bryan

A Novel Staphylococcus aureus Vaccine: Iron Surface Determinant B Induces Rapid Antibody Responses in Rhesus Macaques and Specific Increased Survival in a Murine S. aureus Sepsis Model

ABSTRACT Staphylococcus aureus is a major cause of nosocomial infections worldwide, and the rate of resistance to clinically relevant antibiotics, such as methicillin, is increasing; furthermore, there has been an increase in the number of methicillin-resistant S. aureus community-acquired infections. Effective treatment and prevention strategies are urgently needed. We investigated the potential of the S. aureus surface protein iron surface determinant B (IsdB) as a prophylactic vaccine against S. aureus infection. IsdB is an iron-sequestering protein that is conserved in diverse S. aureus clinical isolates, both methicillin resistant and methicillin sensitive, and it is expressed on the surface of all isolates tested. The vaccine was highly immunogenic in mice when it was formulated with amorphous aluminum hydroxyphosphate sulfate adjuvant, and the resulting antibody responses were associated with reproducible and significant protection in animal models of infection. The specificity of the protective immune responses in mice was demonstrated by using an S. aureus strain deficient for IsdB and HarA, a protein with a high level of identity to IsdB. We also demonstrated that IsdB is highly immunogenic in rhesus macaques, inducing a more-than-fivefold increase in antibody titers after a single immunization. Based on the data presented here, IsdB has excellent prospects for use as a vaccine against S. aureus disease in humans.

Human papillomavirus type 11 (HPV-11) neutralizing antibodies in the serum and genital mucosal secretions of African green monkeys immunized with HPV-11 virus-like particles expressed in yeast.

It has been shown previously that immunization of animals with recombinant virus-like particles (VLPs) consisting of the viral capsid proteins L1 or L1 plus L2 protected animals against experimental viral challenge. However, none of these experimental models addresses the issue of whether systemic immunization with VLPs elicits a neutralizing antibody response in the genital mucosa. Such a response may be necessary to protect the uterine cervix against infection with genital human papillomavirus (HPV) types. African green monkeys systemically immunized with HPV-11 VLPs expressed in Saccharomyces cerevisiae and formulated on aluminum adjuvant elicited high-titered HPV-11 VLP-specific serum antibody responses. Sera from these immunized monkeys neutralized HPV-11 in the athymic mouse xenograft system. Significant levels of HPV-11-neutralizing antibodies also were observed in cervicovaginal secretions. These findings suggest that protection against HPV infection of the uterine cervix may be possible through systemic immunization with HPV VLPs.

Evolution of type-specific immunoassays to evaluate the functional immune response to GARDASIL®, a vaccine for Human Papillomavirus types 16, 18, 6, and 11

Epidemiological studies and clinical trials of vaccines depend on the accurate measurement of antibodies within the polyclonal response to infection or vaccination. The assay currently used to measure the antibody response to vaccination with GARDASIL® [Quadrivalent Human Papillomavirus (Types 6, 11, 16, 18) Recombinant Vaccine]―a quadrivalent vaccine used against human papillomavirus (HPV) types 6, 11, 16, and 18―is a competitive Luminex® assay (cLIA) that uses multiplex technology to detect type-specific neutralizing antibodies against all four HPV types in a single serum sample. Here we describe how the cLIA was developed, as well as how the monoclonal antibodies (mAbs), used as competitors in the assay, were characterized. An enzyme-linked immunosorbent assay (ELISA) was used to screen eight previously-identified mAbs for their ability to bind to HPV virus-like particles (VLPs) in a type-specific and conformation-dependent manner. Four of these mAbs, H6.M48, K11.B2, H16.V5, and H18.J4, met our specifications and were shown to have the potential to neutralize HPV infection in hemagglutination inhibition and pseudovirus neutralization assays. The competitive immunoassay format was able to distinguish type-specific antibodies in the sera of nonhuman primates vaccinated with HPV VLPs, whereas a traditional direct-bind ELISA could not. In addition, the serum antibodies measured by the competitive assay are known to be neutralizing, whereas the ELISA does not distinguish neutralizing and non-neutralizing antibodies in a serum sample. By detecting antibodies to neutralizing epitopes, the competitive assay both demonstrates sero-conversion and provides a potential functional link between sero-conversion and protective immunity in response to vaccination with GARDASIL®.

Infectious Virions Produced from a Human Papillomavirus Type 18/16 Genomic DNA Chimera

ABSTRACT The organotypic raft culture system has allowed the study of the differentiation-dependent aspects of the human papillomavirus (HPV) life cycle. However, genetic strategies to more completely understand the HPV life cycle are limited. The generation of chimeric viruses has been a useful tool in other virus systems to analyze infection and replication. To investigate the specificity of the interaction of nonstructural genes of one HPV type with the structural genes of another HPV type, we have replaced the L2 and L1 open reading frames (ORFs) of HPV type 18 (HPV18) with the L2 and L1 ORFs of HPV type 16 (HPV16). The resulting HPV18/16 chimeric construct was introduced into primary keratinocytes, where it was stably maintained episomally at a range of 50 to 100 copies of HPV genomic DNA, similar to that typically found in HPV-infected cells in vivo. The integrity of the HPV18/16 genomic DNA chimera was demonstrated. Upon differentiation in raft cultures, late viral functions, including viral DNA amplification, capsid gene expression, and virion morphogenesis, occurred. Chimeric HPV18/16 virions were purified from the raft cultures and were capable of infecting keratinocytes in vitro. Additionally, infection was specifically neutralized with human HPV16 virus-like particle (VLP)-specific antiserum and not with human HPV18 VLP-specific antiserum. Our data demonstrate that the nonstructural genes of HPV18 functionally interact with the structural genes of HPV16, allowing the complete HPV life cycle to occur. We believe that this is the first report of the propagation of chimeric HPV by normal life cycle pathways.

Human papillomavirus type 11 neutralization in the athymic mouse xenograft system: Correlation with virus-like particle IgG concentration

Neutralization of virus is likely to be necessary for development of an effective prophylactic vaccine against genital human papillomavirus (HPV) infection. Two New Zealand white rabbits were immunized with purified HPV type 11 (HPV 11) virions in Freunds adjuvant. An enzyme linked immunoassay (ELISA) was used to determine the quantity of IgG which recognized the HPV 11 major capsid protein (L1 protein) virus‐like particles (VLPs) in the two anti‐HPV 11 sera (serum A and serum B). The concentration of HPV 11 L1 VLP‐specific IgG in the A and B sera were determined to be 37 and 90 μg per ml, respectively. The A and B sera were used in neutralization experiments in the athymic mouse xenograft system with known quantities of purified HPV 11 virions. The concentration of HPV 11 L1 VLP‐specific IgG required to neutralize HPV 11 was determined for each antiserum. This concentration of IgG was approximately 700 to 900 ng per ml. This study demonstrates a positive correlation between the level of HPV 11 L1 VLP‐specific IgG in animals immunized with HPV 11 virions and neutralization of HPV 11 in the athymic mouse model. Further studies are needed 1) to determine if sera or genital secretions from other species are neutralizing in the athymic mouse xenograft system, and 2) to determine if the VLP ELISA can be used as a reliable substitute for more cumbersome neutralization assays. J. Med. Virol. 53:185–188, 1997.

H5N1 virus causes significant perturbations in host proteome very early in influenza virus-infected primary human monocyte-derived macrophages.

H5N1 influenza viruses, which cause disease in humans, have unusually high pathogenicity. The temporal response of primary human monocyte-derived macrophages infected with highly pathogenic H5N1 and seasonal H1N1 influenza viruses was evaluated using mass spectrometry-based quantitative proteomic profiling. This was done in order to demonstrate significant perturbation of the host proteome upon viral infection, as early as 1 hour after infection. This early host response distinguished H5N1 infection from H1N1 infection, the latter inducing less of a response. The most pronounced effect was observed on the translational machinery, suggesting that H5N1 might gain advantage in replication by using the cell protein synthesis machinery early in the infection.

Human papillomavirus detection by hybrid capture in paired cervicovaginal lavage and cervical biopsy specimens

Infection of the uterine cervix with human papillomavirus (HPV) is associated with dysplastic lesions that may progress to malignancy. Certain HPV types are associated with higher risk of cervical cancer than other genital HPVs. The goal of this study was to determine if cells obtained by cervicovaginal lavage contain similar HPV types as paired cervical biopsy in women referred because of abnormal cervical cytology. Thirty‐four paired lavage and biopsy samples were analyzed for HPV DNA by hybrid capture, using “low risk” (HPV types 6. 11, and related types and “high risk” group (HPV types 16, 18, and related types) HPV. HPV was detected in 24 lavage samples and 18 biopsies. High risk types were predominant. In 14 of 18 HPV‐positive biopsies, the paired lavage was also positive for the same HPV group. Four biopsies were HPV‐positive at low levels, and the paired lavage was HPV‐negative. The mean viral copy numbers of the biopsies from patients with positive and negative lavage samples were 2.7 and 0.1, respectively (P = .02). Ten low level HPV infections were detected by lavage that were not detected by biopsy. HPV detection by hybrid capture in cells obtained by cervicovaginal lavage reflects the results of HPV testing in cervical biopsies.

High-grade dysplasia in genital warts from two patients infected with the human immunodeficiency virus.

Cancer‐associated human papillomavirus (HPV) types are detected in genital warts removed from immunosuppressed individuals more commonly than from those occurring in otherwise healthy individuals. The prognosis of genital warts containing cancer‐associated HPV types is not known. Because it is assumed that genital warts are benign lesions, they are usually treated by destructive therapies without prior knowledge of histopathology. The aim of the present study was to determine whether genital warts from individuals with or without human immunodeficiency virus (HIV) contain high‐risk HPV types or areas of dysplasia. The study design was a nonrandomized analysis of genital warts removed by excision biopsy from 15 HIV‐infected patients and 15 HIV‐negative patients. The tissue was analyzed for HPV DNA by hybrid capture, and microscopic sections of each biopsy were examined for areas of dysplasia. Genital warts from HIV‐infected patients contained cancer‐associated (“high risk”) HPV types in 9 of 15 cases, including 1 that contained only a high‐risk type. High‐grade dysplastic abnormalities were present in 2 of the 15 lesions from this group, both of which contained high‐risk HPV types. Four genital warts removed from HIV‐negative patients contained high‐risk HPV types, but none contained dysplastic abnormalities. It is concluded that genital warts from HIV‐infected patients often contain high‐risk HPV types. Such lesions may exhibit dysplastic changes. The frequency of dysplastic changes in genital warts from HIV‐infected patients is not known. Biopsy of genital warts may be indicated prior to additional therapy in HIV‐infected patients, and surgical removal should be considered as a preferred treatment option in these patients. J. Med. Virol. 54:69–73, 1998.

Propagation of Human Papillomavirus Type 59 in the Athymic Mouse Xenograft System

Studies of human papillomavirus (HPV) infection are hampered by the lack of an adequate culture system. The athymic mouse xenograft system permits propagation of HPV, but only a few HPV types have been grown in this manner. To produce an oncogenic type for studies of HPV pathogenesis, a condylomata acuminata lesion from an immunosuppressed patient was used to prepare an infectious extract. The patient’s lesion was shown by PCR analysis to contain abundant HPV 59 (an oncogenic type) and a lesser amount of HPV 11, a nononcogenic type. The extract was used to infect human foreskin tissue which was subsequently implanted into athymic mice. Characterization of implants recovered after 3–4 months of growth revealed the presence of HPV 59 exclusively. A second extract was prepared from one of these implants and used in an additional experiment to demonstrate the passage of HPV 59. Compared to the histopathologic changes induced by the prototypic nononcogenic HPV 11, infection with HPV 59 caused a higher degree of basal cell crowding, less acanthosis, minimal papillomatosis and less pronounced koilocytosis.

Temporal and histologic relationships of proliferating cell nuclear antigen and human papillomavirus type 11 in the mouse xenograft system

Proliferating cell nuclear antigen (PCNA) is an accessory protein of DNA polymerase delta. This protein is associated with cell cycle progression and can be detected in the replicating cells of normal tissues. Condylomata acuminata are benign epithelial tumors caused by infection with human papillomaviruses and are characterized by abnormal cell proliferation. The athymic mouse xenograft model of HPV 11 infection was used to test the hypothesis that PCNA is induced early in the course of HPV 11 infection and to study the temporal and histologic relationships between detection of PCNA and HPV DNA. Human foreskin tissue was infected with HPV 11 and implanted under the renal capsules of 10 athymic mice. Pairs of mice were sacrificed every week beginning four weeks after implantation. HPV DNA was detected in sections of foreskin implants by in situ hybridization. PCNA was as or more abundant in implants removed at earlier time points than at later time points, whereas HPV DNA became increasingly more abundant with time. PCNA was detected only in basal cells in areas of histologically normal epithelium that were also negative for HPV DNA. In contrast, PCNA was present throughout the epithelium in regions that were HPV DNA‐positive. HPV DNA was detected only in differentiated epithelial cells in implants removed at all five time points, but in HPV DNA‐positive regions, PCNA was detected with equal intensity in differentiated and undifferentiated cells. The foci of PCNA‐positive cells were well demarcated and were larger than, but included, the foci of HPV DNA‐positive cells. PCNA may be induced maximally in differentiated epithelium by HPV 11 prior to significant HPV DNA replication.