Unfinished Business

The following is excerpted from an address given by HEW Secretary Joseph A. Califano, Jr., at the 111 th Convocation of Howard University, September 22, 1978

Immunodominant Antigens of Actinobacillus Actinomycetemcomitans in Antibody Positive Subjects

This study was initiated to look for characteristics of the immunodominant antigen(s) of Actinobacillus actinomycetemcomitans (Aa) that might help explain the high antibody titers reactive with this organism in periodontitis patients. Radioimmunoassays (RIA) and limiting dilution analysis on western blots were used to identify and characterize the immunodominant antigens of Aa in high responder patients (i.e. patients with high antibody titers). Additional experiments focused on specific responding subjects with a range of antibody titers to serotype b (AaY4). This group included both white and black subjects. Similar studies then examined the immunodominant antigens of Aa serotypes a and c. The Aa serotype specificity of the high responders was determined and further work was confined to individuals responding specifically to serotypes a or c. The results indicated that the immunodominant antigen of Aa Y 4 in all high responders was heat and papain stable. Limiting dilution analysis on western blots showed that a large diffuse band resembling carbohydrate was the immunodominant antigen. Partially purified AaY4 carbohydrate also contained the large diffuse band. Double immunodiffusion (Ouchterlony) tests indicated that the dominant antigen recognized in the carbohydrate extract by the patients\u27 sera was the same antigen recognized by serotype b specific rabbit antiserum which has been previously shown to react with the serotype specific carbohydrate of AaY4. When the subjects with specific antibody responses covering a range of positive antibody titers were examined, 95% of the subjects were responding to the smeared serotype specific carbohydrate antigen on western blots. For serotype-a, no immunodominant antigen was common to the majority of high responding subjects. For serotype-c two antigens were immunodominant in the majority of the high responding patients: 92% had an antigen with a diffuse smeared appearance on western blots typical of a carbohydrate antigen and 67% had an approximately 15Kd antigen. The smeared antigen gave a reaction of identity with patient serum and serotype c specific rabbit antiserum in double immunodiffusion assays. These findings indicated that the immunodominant antigen for serotypes-b and-c in high responding subjects was the serotype specific carbohydrate. The same antigen was immunodominant for subjects with a range of antibody titers to AaY4. The dominant antigen was highly variable for serotype-a. These antigens were highly immunogenic. This immunogenicity may explain the enormous antibody titers observed for Aa in response to what appears to be a small quantity of antigen

A vaccine for Ebola virus – approaches and results of accelerated process development and characterization studies

In 2014, West Africa experienced the worst outbreak of Ebola virus in history with 10 times more cases than in all previous outbreaks combined. In response to this public health emergency, MSD and a global network of partners collaborated to speed the research, development, and deployment of a candidate vaccine that ultimately provided the first evidence of efficacy in human subjects for any Ebola vaccine. While work continues to ultimately license the candidate vaccine, a risk-based approach to process development and characterization was used to accelerate and prioritize the study of parameters. Risk was evaluated by experts familiar with unit operations and parameters in similar licensed live viral vaccines and resulted in an overall plan of study encompassing five major areas – cell expansion, viral infection, purification, formulation, and general robustness. In parallel to batch size scale-up to support commercial production, a scale-down model comparable to commercial scale production was developed and enabled high-throughput experimentation. This approach reduced experiment cycle time from eight weeks to three weeks, reduced process volumes enabling design of experiments, and resulted in high-throughput execution of lab-scale studies. Typically, potency is extremely sensitive to multiplicity of infection (MOI); this vaccine is capable of producing acceptable potencies during viral infection with a 1000x range of MOIs. The most critical parameter during purification is digestion, which results in a ~10-fold increase in product potency. The final tangential flow filtration unit operation is extremely robust with no critical process parameters while still being capable of effectively clearing residual enzyme. Additionally, the implementation of a fully disposable single-use drug substance manufacturing process also helped accelerate process development and characterization activities. Component user requirements and schematic drawings were used to design prototypes which were evaluated using innovative shake-down studies. This approach resulted in a rugged system of end-to-end, single-use disposable components with 42 modular, “plug-and-play” designs available to support \u3e500 single-use assemblies needed in production. Components were delivered to the commercial manufacturing site within 15 months with no required design changes following water-run testing. Taken together these approaches helped accelerate process development and characterization studies that will expedite the licensure of an Ebola virus vaccine

Computational methods reveal novel functionalities of PIWI-interacting RNAs in human papillomavirus-induced head and neck squamous cell carcinoma.

Human papillomavirus (HPV) infection is the fastest growing cause of head and neck squamous cell carcinoma (HNSCC) today, but its role in malignant transformation remains unclear. This study aimed to conduct a comprehensive investigation of PIWI-interacting RNA (piRNA) alterations and functionalities in HPV-induced HNSCC. Using 77 RNA-sequencing datasets from TCGA, we examined differential expression of piRNAs between HPV16(+) HNSCC and HPV(-) Normal samples, identifying a panel of 30 HPV-dysregulated piRNAs. We then computationally investigated the potential mechanistic significances of these transcripts in HPV-induced HNSCC, identifying our panel of piRNAs to associate with the protein PIWIL4 as well as the RTL family of retrotransposon-like genes, possibly through direct binding interactions. We also recognized several HPV-dysregulated transcripts for their correlations with well-documented mutations and copy number variations in HNSCC as well as HNSCC clinical variables, demonstrating the potential ability of our piRNAs to play important roles in large-scale modulation of HNSCC in addition to their direct, smaller-scale interactions in this malignancy. The differential expression of key piRNAs, including NONHSAT077364, NONHSAT102574, and NONHSAT128479, was verified in vitro by evaluating endogenous expression in HPV(+) cancer vs. HPV(-) normal cell lines. Overall, our novel study provides a rigorous investigation of piRNA dysregulation in HPV-related HNSCC, and lends critical insight into the idea that these small regulatory transcripts may play crucial and previously unidentified roles in tumor pathogenesis and progression

Chronic CSE Treatment Induces the Growth of Normal Oral Keratinocytes via PDK2 Upregulation, Increased Glycolysis and HIF1α Stabilization

Exposure to cigarette smoke is a major risk factor for head and neck squamous cell carcinoma (HNSCC). We have previously established a chronic cigarette smoke extract (CSE)-treated human oral normal keratinocyte model, demonstrating an elevated frequency of mitochondrial mutations in CSE treated cells. Using this model we further characterized the mechanism by which chronic CSE treatment induces increased cellular proliferation.We demonstrate that chronic CSE treatment upregulates PDK2 expression, decreases PDH activity and thereby increases the glycolytic metabolites pyruvate and lactate. We also found that the chronic CSE treatment enhanced HIF1α accumulation through increased pyruvate and lactate production in a manner selectively reversible by ascorbate. Use of a HIF1α small molecule inhibitor blocked the growth induced by chronic CSE treatment in OKF6 cells. Furthermore, chronic CSE treatment was found to increase ROS (reactive oxygen species) production, and application of the ROS scavengers N-acetylcysteine abrogated the expression of PDK2 and HIF1α. Notably, treatment with dichloroacetate, a PDK2 inhibitor, also decreased the HIF1α expression as well as cell proliferation in chronic CSE treated OKF6 cells.Our findings suggest that chronic CSE treatment contribute to cell growth via increased ROS production through mitochondrial mutations, upregulation of PDK2, attenuating PDH activity thereby increasing glycolytic metabolites, resulting in HIF1α stabilization. This study suggests a role for chronic tobacco exposure in the development of aerobic glycolysis and normoxic HIFα activation as a part of HNSCC initiation. These data may provide insights into development of chemopreventive strategies for smoking related cancers