Project NexGen: Developing the Next Generation of COVID-19 Vaccines and Therapeutics to Respond to the Present and Prepare for the Future.

COVID-19 epidemiology and product landscapes have changed considerably since onset of the pandemic. Safe and effective vaccines and therapeutics are available, but the continual emergence of SARS-CoV-2 variants introduce limitations in our ability to prevent and treat disease. Project NextGen is a collaboration between the Biomedical Advanced Research and Development Authority (BARDA), part of the Administration for Strategic Preparedness and Response (ASPR), and the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health, that is leveraging public-private partnerships to address gaps in the nation's COVID-19 vaccine and therapeutic capabilities. Targeted investments will advance promising next-generation candidates through the most difficult phases of clinical development to encourage further private sector interest for later stage development and commercial availability. New commercial vaccines and therapeutics that are more durable and effective across variants will improve our fight against COVID-19 and transform our response to future threats.

Advancing development of medical countermeasures: Incorporating COVID-19 lessons learned into future pandemic preparedness planning.

The COVID-19 pandemic profoundly disrupted, and, out of necessity, accelerated innovation of research and development of medical countermeasures to combat COVID-19. Although countermeasures were developed with unprecedented speed as a result of decades of long-term Federal investments in platform technologies and existing partnerships, the pandemic also revealed gaps in our preparedness and response capabilities that threaten our readiness posture. Challenges include limited federal funding that hinders sustainable development and manufacturing of, and equitable access to, medical countermeasures. Here we discuss lessons learned from the development and production efforts of medical countermeasures, such as vaccines and immunotherapeutics, to combat COVID-19. This commentary highlights some of the key gaps and challenges that must be addressed to ensure preparation for future outbreaks caused by viruses of pandemic potential.

Seasonal influenza vaccination is associated with reduced risk of death among Medicare beneficiaries☆.

BACKGROUND: Influenza causes substantial mortality, especially among older persons. Influenza vaccines are rarely more than 50% effective and rarely reach more than half of the US Medicare population, which is primarily an aged population. We wished to estimate the association between vaccination and mortality reduction. METHOD: We used the US Center for Medicare and Medicaid Services (CMS) DataLink Project to determine vaccination status and timing during the 2017-2018 influenza season for more than 26 million Medicare enrollees. Patient-level demographic, health, co-morbidity, hospitalization, vaccination, and healthcare utilization claims data were supplied as covariates to general linear models in order to isolate and estimate the association between participation in the vaccination program and relative risk of death. FINDINGS: The 2017-2018 seasonal influenza vaccine reduced (Relative Risk Ratio [RRR] 0.936 [95% CI = 0.918-0.954]) the risk of all-cause death among beneficiaries following a hospitalization for sepsis and moreover the risk of death without a prior hospitalization during the 2.5-month outcome window (RRR 0.870 [95% CI = 0.853-0.887]). We estimate the number needed to vaccinate (NNV) to prevent a death in the ten-week outcome window is between 1,515 beneficiaries (95% CI = 1,351-1,754; derived from the average treatment effect of augmented inverse probability weighting) and 1,960 beneficiaries (95% CI = 1,695-2,381; derived from the average marginal effect of logistic regression). Among beneficiaries requiring hospitalization, the greatest death risk reduction accrued to those 85 + years of age who were hospitalized with sepsis, RRR 0.92 [95% CI = 0.89-0.95]. No apparent benefit was realized by beneficiaries who required custodial (nursing home) care. INTERPRETATION: Seasonal influenza immunization is associated with relative reduction of death risk among non-institutionalized Medicare beneficiaries. FUNDING: All authors are full-time or contractual employees of the United States Federal Government, Department of Health and Human Services, the funding agency.

Sepsis Among Medicare Beneficiaries: 4. Precoronavirus Disease 2019 Update January 2012-February 2020.

OBJECTIVES: To provide updated information on the burdens of sepsis during acute inpatient admissions for Medicare beneficiaries. DESIGN: Analysis of paid Medicare claims via the Centers for Medicare and Medicaid Services DataLink Project. SETTING: All U.S. acute-care hospitals, excluding federally operated hospitals (Veterans Administration and Defense Health Agency). PATIENTS: All Medicare beneficiaries, January 2012-February 2020, with an explicit sepsis diagnostic code assigned during an inpatient admission. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The count of Medicare Part A/B (fee-for-service) plus Medicare Advantage inpatient sepsis admissions rose from 981,027 (CY2012) to 1,700,433 (CY 2019). The proportion of total admissions with sepsis in the Medicare Advantage population rose from 21.43% to 35.39%, reflecting the increasing beneficiary proportion enrolled in Medicare Advantage. In CY2019, 6-month mortality rates in Medicare fee-for-service beneficiaries for sepsis continued to decline, but remained high: 59.9% for septic shock, 35.5% for severe sepsis, 30.8% for sepsis attributed to a specific organism, and 26.5% for unspecified sepsis. Total fee-for-service-only inpatient hospital costs rose from $17.79B (CY2012) to $22.98B (CY2019). We estimated that the aggregate cost of sepsis hospital care for the entire U.S. population was at least $57.47B in 2019. Inclusion of 14 months' (January 2019-February 2020) newer data exposed new trends: the cost per patient, number of admissions, and fraction of patients with sepsis labeled as present on admission inflected around November 2015, coincident with the change to International Classification of Diseases, 10th Edition, and introduction of the Severe Sepsis and Septic Shock Management Bundle (SEP-1) metric. CONCLUSIONS: Sepsis among Medicare beneficiaries precoronavirus disease 2019 imposed immense burdens upon patients, their families, and the taxpayers.

Sepsis Among Medicare Beneficiaries: 1. The Burdens of Sepsis, 2012-2018.

OBJECTIVES: To provide contemporary estimates of the burdens (costs and mortality) associated with acute inpatient Medicare beneficiary admissions for sepsis. DESIGN: Analysis of paid Medicare claims via the Centers for Medicare & Medicaid Services DataLink Project. SETTING: All U.S. acute care hospitals, excluding federally operated hospitals (Veterans Administration and Defense Health Agency). PATIENTS: All Medicare beneficiaries, 2012-2018, with an inpatient admission including one or more explicit sepsis codes. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Total inpatient hospital and skilled nursing facility admission counts, costs, and mortality over time. From calendar year (CY)2012-CY2018, the total number of Medicare Part A/B (fee-for-service) beneficiaries with an inpatient hospital admission associated with an explicit sepsis code rose from 811,644 to 1,136,889. The total cost of inpatient hospital admission including an explicit sepsis code for those beneficiaries in those calendar years rose from $17,792,657,303 to $22,439,794,212. The total cost of skilled nursing facility care in the 90 days subsequent to an inpatient hospital discharge that included an explicit sepsis code for Medicare Part A/B rose from $3,931,616,160 to $5,623,862,486 over that same interval. Precise costs are not available for Medicare Part C (Medicare Advantage) patients. Using available federal data sources, we estimated the aggregate cost of inpatient admissions and skilled nursing facility admissions for Medicare Advantage patients to have risen from $6.0 to $13.4 billion over the CY2012-CY2018 interval. Combining data for fee-for-service beneficiaries and estimates for Medicare Advantage beneficiaries, we estimate the total inpatient admission sepsis cost and any subsequent skilled nursing facility admission for all (fee-for-service and Medicare Advantage) Medicare patients to have risen from $27.7 to $41.5 billion. Contemporary 6-month mortality rates for Medicare fee-for-service beneficiaries with a sepsis inpatient admission remain high: for septic shock, approximately 60%; for severe sepsis, approximately 36%; for sepsis attributed to a specific organism, approximately 31%; and for unspecified sepsis, approximately 27%. CONCLUSION: Sepsis remains common, costly to treat, and presages significant mortality for Medicare beneficiaries.

Sepsis Among Medicare Beneficiaries: 2. The Trajectories of Sepsis, 2012-2018.

OBJECTIVES: To distinguish characteristics of Medicare beneficiaries who will have an acute inpatient admission for sepsis from those who have an inpatient admission without sepsis, and to describe their further trajectories during and subsequent to those inpatient admissions. DESIGN: Analysis of paid Medicare claims via the Centers for Medicare and Medicaid Services DataLink Project. SETTING: All U.S. acute care hospitals, excepting federal hospitals (Veterans Administration and Defense Health Agency). PATIENTS: Medicare beneficiaries, 2012-2018, with an inpatient hospital admission including one or more explicit sepsis codes. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Prevalent diagnoses in the year prior to the inpatient admission; healthcare contacts in the week prior to the inpatient admission; discharges, transfers, readmissions, and deaths (trajectories) for 6 months following discharge from the inpatient admission. Beneficiaries with no sepsis inpatient hospital admission for a year prior to an index hospital admission for sepsis were nearly indistinguishable by accumulated diagnostic codes from beneficiaries who had an index hospital admission without sepsis. Although the timing of healthcare services in the week prior to inpatient hospital admission was similar among beneficiaries who would be admitted for sepsis versus those whose inpatient admission did not include a sepsis code, the setting differed: beneficiaries destined for a sepsis admission were more likely to have received skilled nursing or unskilled nursing (e.g., nursing aide for activities of daily living) care. In contrast, comparing beneficiaries who had been free of any inpatient admission for an entire year and then required an inpatient admission, acute inpatient stays that included a sepsis code led to more than three times as many deaths within 1 week of discharge, with more admissions to skilled nursing facilities and fewer discharges to home. Comparing all beneficiaries who were admitted to a skilled nursing facility after an inpatient hospital admission, those who had sepsis coded during the index admission were more likely to die in the skilled nursing facility; more likely to be readmitted to an acute inpatient hospital and subsequently die in that setting; or if they survive to discharge from the skilled nursing facility, they are more likely to go next to a custodial nursing home. CONCLUSIONS: Although Medicare beneficiaries destined for an inpatient hospital admission with a sepsis code are nearly indistinguishable by other diagnostic codes from those whose admissions will not have a sepsis code, their healthcare trajectories following the admission are worse. This suggests that an inpatient stay that included a sepsis code not only identifies beneficiaries who were less resilient to infection but also signals increased risk for worsening health, for mortality, and for increased use of advanced healthcare services during and postdischarge along with an increased likelihood of an inpatient hospital readmission.

Sepsis Among Medicare Beneficiaries: 3. The Methods, Models, and Forecasts of Sepsis, 2012-2018.

OBJECTIVE: To evaluate the impact of sepsis, age, and comorbidities on death following an acute inpatient admission and to model and forecast inpatient and skilled nursing facility costs for Medicare beneficiaries during and subsequent to an acute inpatient sepsis admission. DESIGN: Analysis of paid Medicare claims via the Centers for Medicare & Medicaid Services DataLink Project (CMS) and leveraging the CMS-Hierarchical Condition Category risk adjustment model. SETTING: All U.S. acute care hospitals, excepting federal hospitals (Veterans Administration and Defense Health Agency). PATIENTS: All Part A/B (fee-for-service) Medicare beneficiaries with an acute inpatient admission in 2017 and who had no inpatient sepsis admission in the prior year. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Logistic regression models to determine covariate risk contribution to death following an acute inpatient admission; conventional regression to predict Medicare beneficiary sepsis costs. Using the Hierarchical Condition Category risk adjustment model to illuminate influence of illness on outcome of inpatient admissions, representative odds ratios (with 95% CIs) for death within 6 months of an admission (referenced to beneficiaries admitted but without the characteristic) are as follows: septic shock, 7.27 (7.19-7.35); metastatic cancer and acute leukemia (Hierarchical Condition Category 8), 6.76 (6.71-6.82); all sepsis, 2.63 (2.62-2.65); respiratory arrest (Hierarchical Condition Category 83), 2.55 (2.35-2.77); end-stage liver disease (Hierarchical Condition Category 27), 2.53 (2.49-2.56); and severe sepsis without shock, 2.48 (2.45-2.51). Models of the cost of sepsis care for Medicare beneficiaries forecast arise approximately 13% over 2 years owing the rising enrollments in Medicare offset by the cost of care per admission. CONCLUSIONS: A sepsis inpatient admission is associated with marked increase in risk of death that is comparable to the risks associated with inpatient admissions for other common and serious chronic illnesses. The aggregate costs of sepsis care for Medicare beneficiaries will continue to increase.

Progress towards a vaccine against Ebola to meet emergency medical countermeasure needs.

The Ebola virus epidemic in West Africa proved to be the largest in the history of filovirus outbreaks, causing the World Health Organization to declare a public health emergency of international concern in August of 2014. In collaboration with domestic and international partners, the Biomedical Advanced Research and Development Authority (BARDA) initiated several vaccine development projects in support of the overall response efforts. The urgency associated with the epidemic triggered the clinical evaluation of lead vaccine candidates starting in late 2014. Here we will discuss development of the lead vaccine candidates for Ebola virus, specifically Zaire ebolavirus.

The U.S. government's medical countermeasure portfolio management for nuclear and radiological emergencies: synergy from interagency cooperation.

Following the attacks of 11 September 2001, emergency preparedness within the U.S. Department of Health and Human Services, as well as at the Department of Defense and other federal agencies, received higher visibility, new mandates and increased funding. Emergency deployment teams increased the frequency of drills to enable better response to the health consequences of mass-casualty incidents. Interagency coordination has also continued to increase to more efficiently and effectively leverage federal resources toward emergency medical preparedness for both civilian and military populations.

Immortalization and transformation of human mammary epithelial cells by a tumor-derived Myc mutant.

The Myc transcription factor is commonly dysregulated in many human cancers, including breast carcinomas. However, the precise role of Myc in the initiation and maintenance of malignancy is unclear. In this study we compared the ability of wild-type Myc (wt Myc) or Myc phosphorylation deficient mutants (T58A, S62A or T58A/S62A) to immortalize and transform human mammary epithelial cells (HMECs). All Myc constructs promoted cellular immortalization. As previously reported in other cells, the Myc T58A mutant tempered apoptotic responses and increased Myc protein stability in HMEC cells. More importantly, we now show that HMECs overexpressing the Myc T58A mutant acquire a unique cellular phenotype characterized by cell aggregation, detachment from the substrate and growth in liquid suspension. Coincident with these changes, the cells become anchorage-independent for growth in agarose. Previous studies have shown that wt Myc can collaborate with hTERT in inducing HMEC anchorage-independent growth. We have verified this observation and further shown that Myc T58A was a stronger facilitator of such co-transformation. Thus, our findings indicate that differences in Myc protein phosphorylation modulate its biological activity in human breast epithelial cells and specifically that the T58A mutation can facilitate both cellular immortalization and transformation. Finally, we used the isogenic cell lines generated in this study to identify a subset of genes whose expression is greatly altered during the transition from the immortal to the anchorage-independent states.

Cell-restricted immortalization by human papillomavirus correlates with telomerase activation and engagement of the hTERT promoter by Myc.

The high-risk human papillomaviruses (HPVs) are the causative agents of nearly all cervical cancers and are etiologically linked to additional human cancers, including those of anal, oral, and laryngeal origin. The main transforming genes of the high-risk HPVs are E6 and E7. E6, in addition to its role in p53 degradation, induces hTERT mRNA transcription in genital keratinocytes via interactions with Myc protein, thereby increasing cellular telomerase activity. While the HPV type 16 E6 and E7 genes efficiently immortalize human keratinocytes, they appear to only prolong the life span of human fibroblasts. To examine the molecular basis for this cell-type dependency, we examined the correlation between the ability of E6 to transactivate endogenous and exogenous hTERT promoters and to immortalize genital keratinocytes and fibroblasts. Confirming earlier studies, the E6 and E7 genes were incapable of immortalizing human fibroblasts but did delay senescence. Despite the lack of immortalization, E6 was functional in the fibroblasts, mediating p53 degradation and strongly transactivating an exogenous hTERT promoter. However, E6 failed to transactivate the endogenous hTERT promoter. Coordinately with this failure, we observed that Myc protein was not associated with the endogenous hTERT promoter, most likely due to the extremely low level of Myc expression in these cells and/or to differences in chromatin structure, in contrast with hTERT promoters that we found to be activated by E6 (i.e., the endogenous hTERT promoter in primary keratinoctyes and the exogenous hTERT core promoter in fibroblasts), where Myc is associated with the promoter in either a quiescent or an E6-induced state. These findings are consistent with those of our previous studies on mutagenesis and the knockdown of small interfering RNA, which demonstrated a requirement for Myc in the induction of the hTERT promoter by E6 and suggested that occupancy of the promoter by Myc determines the responsiveness of E6 and the downstream induction of telomerase and cell immortalization.

Myc and human papillomavirus type 16 E7 genes cooperate to immortalize human keratinocytes.

The E6 protein of the oncogenic human papillomaviruses (HPVs), in combination with the E7 protein, is essential for the efficient immortalization of human foreskin keratinocytes (HFKs). Since we recently demonstrated that E6 activates the human telomerase reverse transcriptase (hTERT) promoter via a Myc-dependent mechanism, we speculated that overexpressed Myc might be able to substitute for E6 in cell immortalization. Myc (similar to E6) was unable to immortalize HFKs when transduced alone, despite inducing high levels of telomerase activity. However, when transduced with E7, Myc immortalized HFKs following a brief but detectable crisis period. In contrast to E6 + E7-immortalized cells, the Myc + E7-immortalized cells expressed high levels of p53 protein as well as two p53-regulated proteins, p21 and hdm-2. The increase in p21 and hdm-2 proteins correlated directly with their mRNA levels, suggesting transcriptional activation of the respective genes by the overexpressed p53 protein. Interestingly, a significant proportion of the p53 protein in the Myc + E7-immortalized cells was localized to the cytoplasm, potentially due to interactions with the overexpressed hdm-2 protein. Regardless, cell immortalization by the Myc + E7 genes occurred independently of p53 degradation. Since we have already observed high-efficiency cell immortalization with the hTERT + E7 or E6 mutant (p53 degradation-defective) + E7 genes (i.e., no crisis period) that proceeds in the presence of high levels of p53, we hypothesize that the crisis period in the Myc + E7 cells is due not to the levels of the p53 protein but rather to unique properties of the Myc protein. The common factor in cell immortalization by the three gene sets (E6 + E7, Myc + E7, and hTERT + E7 genes) is the induction of telomerase activity.

Dihydroartemisinin is cytotoxic to papillomavirus-expressing epithelial cells in vitro and in vivo.

Nearly all cervical cancers are etiologically attributable to human papillomavirus (HPV) infection and pharmaceutical treatments targeting HPV-infected cells would be of great medical benefit. Because many neoplastic cells (including cervical cancer cells) overexpress the transferrin receptor to increase their iron uptake, we hypothesized that iron-dependent, antimalarial drugs such as artemisinin might prove useful in treating HPV-infected or transformed cells. We tested three different artemisinin compounds and found that dihydroartemisinin (DHA) and artesunate displayed strong cytotoxic effects on HPV-immortalized and transformed cervical cells in vitro with little effect on normal cervical epithelial cells. DHA-induced cell death involved activation of the mitochondrial caspase pathway with resultant apoptosis. Apoptosis was p53 independent and was not the consequence of drug-induced reductions in viral oncogene expression. Due to its selective cytotoxicity, hydrophobicity, and known ability to penetrate epithelial surfaces, we postulated that DHA might be useful for the topical treatment of mucosal papillomavirus lesions. To test this hypothesis, we applied DHA to the oral mucosa of dogs that had been challenged with the canine oral papillomavirus. Although applied only intermittently, DHA strongly inhibited viral-induced tumor formation. Interestingly, the DHA-treated, tumor-negative dogs developed antibodies against the viral L1 capsid protein, suggesting that DHA had inhibited tumor growth but not early rounds of papillomavirus replication. These findings indicate that DHA and other artemisinin derivatives may be useful for the topical treatment of epithelial papillomavirus lesions, including those that have progressed to the neoplastic state.

Requirement of epidermal growth factor receptor for hyperplasia induced by E5, a high-risk human papillomavirus oncogene.

Multicellular organisms rely on complex networks of signaling cascades for development, homeostasis, and responses to the environment. These networks involve diffusible signaling molecules, their receptors, and a variety of downstream effectors. Alterations in the expression or function of any one of these factors can contribute to disease, including cancer. Many viruses have been implicated in cancer, and some of these modulate cellular signal transduction cascades to carry out their life cycles. High-risk human papillomaviruses (HPVs), the causative agents of most cervical and anogenital cancers, encode three oncogenes. One of these, E5, has been postulated to transform cells in tissue culture by modulating growth factor receptors. In this study, we generate and characterize transgenic mice in which the E5 gene of the most common high-risk HPV, HPV16, is targeted to the basal layer of the stratified squamous epithelium. In these mice, E5 alters the growth and differentiation of stratified epithelia and induces epithelial tumors at a high frequency. Through the analysis of these mice, we show a requirement of the epidermal growth factor receptor for the hyperplastic properties of E5.

Endoplasmic reticulum-localized human papillomavirus type 16 E5 protein alters endosomal pH but not trans-Golgi pH.

The human papillomavirus type 16 (HPV-16) E5 protein is a small, hydrophobic polypeptide that is expressed in virus-infected keratinocytes and alters receptor signaling pathways, apoptotic responses, and endosomal pH. Despite its ability to inhibit endosomal acidification, the HPV-16 E5 protein is found predominantly in the endoplasmic reticulum (ER), suggesting that its effect may be indirect and perhaps global. To determine whether E5 alters the pHs of additional intracellular compartments, we transduced human keratinocytes with a codon-optimized E5 vector and then quantified endosomal and trans-Golgi pHs using sensitive, compartment-specific, ratiometric pHluorin constructs. E5 protein increased endosomal pH from 5.9 to 6.9 but did not affect the normal trans-Golgi pH of 6.3. Confirming the lack of alteration in trans-Golgi pH, we observed no alterations in the acidification-dependent processing of the proH3 protein. C-terminal deletions of E5, which retained normal expression and localization in the ER, were defective for endosomal alkalization. Thus, E5 does not uniformly alkalinize intracellular compartments, and its C-terminal 10 amino acids appear to mediate interactions with critical ER targets that modulate proton pump function and/or localization.

The E6AP ubiquitin ligase is required for transactivation of the hTERT promoter by the human papillomavirus E6 oncoprotein.

Most human cancer cells display increased telomerase activity that appears to be critical for continued cell proliferation and tumor formation. The E6 protein of malignancy-associated human papillomaviruses increases cellular telomerase in primary human keratinocytes at least partly via transcriptional activation of the telomerase catalytic subunit, hTERT. In the present study, we investigated whether E6AP, a ubiquitin ligase well known for binding and mediating some of the activities of the E6 oncoprotein, participated in the transactivation of the hTERT promoter. Our results demonstrate that E6 mutants that fail to bind E6AP are also defective for increasing telomerase activity and transactivating the hTERT promoter. More importantly, E6AP knock-out mouse cells and small interfering RNA techniques demonstrated that E6AP was required for hTERT promoter transactivation in both mouse and human cells. Neither E6 nor E6AP bound to the hTERT promoter or activated the promoter in the absence of the partner protein. With all transactivation-competent E6 proteins, induction of the hTERT promoter was dependent upon E box elements in the core promoter. It appears, therefore, that E6-mediated activation of the hTERT promoter requires a complex of E6-E6AP to engage the hTERT promoter and that activation is dependent upon Myc binding sites in the promoter. The recruitment of a cellular ubiquitin ligase to the hTERT promoter during E6-mediated transcriptional activation suggests a role for the local ubiquitination (and potential degradation) of promoter-associated regulatory proteins, including the Myc protein.

Are transforming properties of the bovine papillomavirus E5 protein shared by E5 from high-risk human papillomavirus type 16?

The E5 proteins of bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 16 (HPV-16) are small (44-83 amino acids), hydrophobic polypeptides that localize to membranes of the Golgi apparatus and endoplasmic reticulum, respectively. While the oncogenic properties of BPV-1 E5 have been characterized in detail, less is known about HPV-16 E5 due to its low expression in mammalian cells. Using codon-optimized HPV-16 E5 DNA, we have generated stable fibroblast cell lines that express equivalent levels of epitope-tagged BPV-1 and HPV-16 E5 proteins. In contrast to BPV-1 E5, HPV-16 E5 does not activate growth factor receptors, phosphoinositide 3-kinase or c-Src, and fails to induce focus formation, although it does promote anchorage-independent growth in soft agar. These variant activities are apparently unrelated to differences in intracellular localization of the E5 proteins since retargeting HPV-16 E5 to the Golgi apparatus does not induce focus formation.

IGFBP-3, a marker of cellular senescence, is overexpressed in human papillomavirus-immortalized cervical cells and enhances IGF-1-induced mitogenesis.

Human ectocervical cells, following retroviral transduction with the human papillomavirus type 16 E6/E7 oncogenes, are altered in their array of transcribed cellular genes, including increased mRNA for the insulin-like growth factor binding protein 3 (IGFBP-3). IGFBP-3 expression is associated with cellular senescence, and its addition to many cell types inhibits growth or induces apoptosis. By immunoblotting and enzyme-linked immunosorbent assay methods, we demonstrate that late-passage, immortalized E6/E7-transduced cells secrete high levels of IGFBP-3 (25 ng/ml), which represent a 500-fold increase compared to levels in early-passage, nonimmortalized transduced cells (<0.05 ng/ml). Concomitantly, these late-passage cervical cells exhibit an increase in sensitivity to IGF-1, including enhanced phosphorylation of the IGF receptor (IGF-R) and insulin receptor substrate as well as increased DNA synthesis (5-fold) and cell proliferation (3.7-fold). However, there was no change in the level of IGF-R in these cells (surface or total), and the cells did not synthesize IGF-1, indicating that these arms of the IGF pathway were independently regulated and not responsible for the augmented signaling. Consistent with a causal relationship between IGFBP-3 expression and enhanced IGF-1 responses, we found that early-passage cells could be converted to the late-passage, IGF-1-responsive phenotype by preincubation with IGFBP-3. Thus, in contrast to findings with some cell types, IGFBP-3 expression in cervical cells is associated with augmented IGF-1 signaling and cell proliferation and correlates with the timing of cellular immortalization.