Abstract
An Escherichia coli-expressed recombinant bivalent human papillomavirus (types 16 and 18) vaccine candidate has been shown to be safe and immunogenic in preclinical trials. The safety of this vaccine was analyzed in an open-label phase I clinical trial in Jiangsu province, China. Thirty-eight healthy women from 18 to 55 y of age were enrolled and vaccinated at 0, 1, and 6 mo. Adverse events that occurred within 30 d after each injection and serious adverse events that occurred throughout the study were recorded. In addition, blood parameters were tested before and after each injection. All but one woman received all 3 doses. Thirty-two (84.2%) of the participants reported adverse events, all adverse events of which were mild, of short duration and resolved spontaneously. No serious adverse events occurred during the study. Changes in blood parameters after each injection were random, mild, and not clinically significant. These preliminary results show that a new Escherichia coli-expressed recombinant HPV 16/18 bivalent vaccine is well tolerated in healthy women and support further immunogenicity and efficacy studies for this HPV vaccine candidate.
Introduction
An estimated 529 800 new cases of cervical cancer occur worldwide, with 275 800 deaths due to cervical cancer in 2008. More than 85% of these cases and related deaths occurred in developing countries.Citation1 In 54 countries, cervical cancer is the leading cause of years-of-life-lost among women.Citation2 Persistent infection with oncogenic human papillomavirus (HPV) is the most common cause of cervical cancer.Citation3-Citation5 HPV16 and HPV18 are the most prevalent HPV types associated with cervical cancer, accounting for approximately 70% of cervical cancer cases worldwide.Citation6-Citation8
Two prophylactic HPV L1 virus-like particle (VLP) vaccines are currently marketed in more than 100 countries. The first is a quadrivalent (types 6, 11, 16, and 18) vaccine expressed in yeast and absorbed into an alum adjuvant (Gardasil®, Merck and Co.).Citation9,Citation10 The second is a bivalent (types 16 and 18) vaccine expressed in insect cells and absorbed into a combination of aluminum hydroxide and monophosphoryl lipid ASO4 adjuvant (Cervarix®, GlaxoSmithKline Biologicals).Citation11,Citation12 The safety and efficacy of these two vaccines have been demonstrated in several clinical trials.Citation13 However, the current cost of these licensed HPV vaccines produced by yeast or baculovirus systems is a significant barrier to their sustained global implementation. This obstacle has led to efforts to create a low-cost HPV vaccine using an Escherichia coli (E. coli) system. HPV16 and 18 L1 VLPs have been successfully expressed in E. coli and have been shown to be highly immunogenic in mice, rabbits, and goats. These antigens have been shown to elicit high titers of specific neutralizing antibody that can effectively neutralize infection of pseudovirus.Citation14,Citation15 Based on these VLPs, a bivalent (types 16 and 18) HPV vaccine candidate was developed and shown to be safe and highly immunogenic in preclinical studies (unpublished data). This vaccine was approved for human trials by the China Food and Drug Administration (CFDA). Here, we report the findings from a Phase I trial of the vaccine candidate conducted in eastern China.
Results
Study population
In December 2010, 40 women from 18 to 55 y of age were recruited. This age range covers most of the sexually active period. Thirty-eight healthy subjects were enrolled and received the study vaccine; the other two women were excluded from the study due to current pregnancy or a serious allergy history (). All 38 participants received the first two doses at months 0 and 1. One participant moved away from the study area and therefore withdrew from the study before the third dose. The other 37 participants received the third dose at month 6. Blood samples were taken before and two days after each injection. The participants had a mean age of 39.4 y (SD 9.7)().
Figure 1. Trial profile. One participant withdrew from the study before her third dose because she had moved away from the study area.
Figure 2. Age distribution of the participants.
Clinical toxicity
No serious adverse events (SAE) occurred during the trial. shows the solicited and unsolicited clinical adverse events (AEs) after vaccination. Thirty-two participants (84.2%) reported solicited and/or unsolicited AEs throughout the study. The rate of local AEs after each vaccination was similar (P = 0.1143). Systemic AEs were less frequent after the last vaccination than they were after the first vaccination (P = 0.0405). The rate of unsolicited AEs was lower after the last vaccination than it was after the first and the second vaccinations (P = 0.0006 and 0.0239, respectively).
Table 1. AEs reported after each vaccination
shows the observed clinical symptoms and their severities after vaccination. All AEs were mild or moderate—80.8% were grade 1 and 19.2% were grade 2. No unsolicited AEs of grade 3 or higher were reported. The most common local, systemic, and unsolicited AEs were pain in injection site (17/38, 44.7%), raised body temperature (11/38, 28.9%), headache (7/38, 18.4%), fatigue (6/38, 15.8%), nausea (5/38, 13.2%), and upper respiratory tract infection (10/38, 26.3%). There was no significant difference between women less than 40 y old and women over 40 y old with regard to the rates of solicited AEs (P = 0.4384) and unsolicited AEs (P = 0.1786).
Figure 3. Solicited local and systemic adverse events, unsolicited AEs and SAEs after vaccinations. The x-axis shows the number of participants experiencing AEs, with the highest severity of AEs experienced by each participant after each vaccination shown. Solicited AEs were monitored for seven days. Unsolicited AEs were monitored for 30 d. SAEs were monitored throughout the 7 months’ study. AE: adverse event; SAE: serious adverse event.
Laboratory parameters
summarizes the changes in laboratory parameters before and after vaccination. Thirty-three hematological parameters were analyzed for each blood sample, resulting in a total of 3729 pairs of results. Most pairs (3512, 94.2%) were within normal ranges. Eighty-six pairs (2.3%) shifted from normal to abnormal ranges after vaccination, and 70 pairs (1.9%) shifted from abnormal to normal. These changes were not significant (P = 0.2297). Similar results were observed when the analyses were performed on classified indexes including routine blood tests, blood coagulation function, liver function, and kidney function. Therefore, the changes in laboratory parameters were determined to be physiological rather than caused by the vaccine.
Table 2. Laboratory parameters*
The changes in laboratory parameters were classified into three categories: “remaining” indicates that no change in grade was observed; “worsen” indicates either a change from normal to abnormal or an increase in grade; and “improving” indicates a change from abnormal to normal or a decrease in grade. As shown in , most fluctuations were “remaining,” including normal to normal (3512 pairs), grade 1 to 1 (53 pairs), and 2 to 2 (3 pairs). Some post-vaccination samples were worse than the pre-vaccination sample, including 68 pairs that changed from normal to grade 1, 2 pairs that changed from normal to grade 2, 1 that changed from grade 1 to grade 2 and 1 that changed from grade 2 to grade 3.
Figure 4. Blood index fluctuations after the first, second, and third vaccinations. The x-axis shows the number of participants who experienced changes in blood parameters. For each participant, these changes were determined by comparing the results of paired blood samples obtained before and 2 d after each vaccination. The fluctuations were classified into three categories: “remaining” indicated no grade change observed; “worsen” indicated a change from normal to abnormal or an increase in grade; and “improving” indicated a change from abnormal to normal or a decrease in grade. a, routine blood test; b, blood coagulation function; c, liver function; d, kidney function; e, other indexes.
Most of the abnormal indexes were mild or moderate, with the exception of one 21-y-old woman who suffered from a grade 3 abnormality after the first vaccination. Her ALT level was 97 U/L (3.1 times over the upper limit of normal range [ULN], grade 2) at enrollment and increased to 167 U/L (5.4 ULN, grade 3) after the first vaccination. Without any medical intervention, her ALT level spontaneously recovered to within normal levels after one month (10 d later: 66 U/L, 20 d later: 44 U/L, 30 d later: 20 U/L).
Discussion
Two marketed HPV vaccines expressed by yeast and insect cells have been shown to be safe and efficacious in humans.Citation9-Citation12 In this phase I clinical trial of an E. coli-expressed HPV vaccine, the vaccine candidate was well tolerated in healthy women. All of the observed AEs were mild or moderate, and no SAEs occurred in the 38 participants who received 2 or 3 doses of the vaccine. These data pave the way for future immunogenicity and efficacy studies of this vaccine with the same or lower dosage.
A total of 113 doses of the HPV vaccine were given to 38 participants at months 0, 1, and 6. AEs were observed in 32 participants (84.2%). The most common solicited AEs were pain at the site of injection, followed by raised body temperature, headache, and fatigue. These results are similar to those of an E. coli-expressed, alum adjuvant VLP vaccine against hepatitis E.Citation16 The rate of fever was 28.9%, which is much higher than the observed fever rate (13%) of another alum adjuvant HPV vaccine, Gardasil®, as described in the Product Specification. This difference could be due to the different criteria for “raised temperature” used by the CFDA and FDA. The CFDA’s criterion for “raised temperature” is higher than 37.0 °C,Citation17 while the criteria for Gardasil® and Cervarix® in clinical trials are higher than 100 °F (37.8 °C) and 99.5 °F (37.5 °C), respectively.Citation18-Citation21 All but one of the 11 reported cases of fever in this study were lower than 37.8 °C.
In addition to routine safety observations, blood samples were collected before and 2 d after each dose. Thirty-three serum indexes indicating general healthy status were tested for each sample. The changes in blood parameters were random, mild, and not clinically significant.
The two currently commercialized HPV vaccines are both expressed in eukaryotic expression systems. One is expressed in insect cells, and the other is expressed in yeast cells. The HPV vaccine tested in this study is the only candidate E. coli-expressed recombinant vaccine currently in human trials. Due to the difficulty in expressing useable HBsAg particles, it was uncertain whether E. coli could be used to express VLP. Recombinant HBsAg particles consist of approximately 30% lipid, which might disrupt the expression in E. coli due to the unfavorable environmental conditions or lipid composition within the bacteria.Citation22 In contrast to HBV virions, HPV virions have nonenveloped icosahedral capsids.Citation23 The recombinant HPV L1 protein was shown to self-assemble into VLPs upon expression in E. coli. After a similar post-purification disassembly/reassembly procedure, the homogeneity and morphology of the E. coli-expressed HPV L1 VLPs improved greatly, as assessed by cryo EM. This phenomenon was very similar to the results observed in yeast.Citation24 Additionally, E. coli-derived VP1 of several polyomaviruses, which are structurally similar to papillomaviruses, have been shown to self-assemble into morphologically correct VLPs from capsomeric subunits in vitro.Citation25 The E. coli expression system is stable and easy to control and has been used to produce several recombinant protein drugs for many years.Citation26 The first commercialized recombinant VLP vaccine expressed by E. coli is a hepatitis E vaccine, Hecolin®, which was marketed in China after demonstrating safety and efficacy in a large randomized controlled clinical trial of 112 604 adults.Citation16,Citation27,Citation28 A new, low-cost, safe, and efficacious HPV vaccine would be a good supplement to the currently available HPV vaccines, especially for the developing world, in which resources are limited.
This study has some strengths and limitations. One strength is the wide range of blood parameters tested before and 2 d after each dose. These tests could unveil a subclinical impact of the vaccine on the body. The main limitation is the lack of direct comparison of the study vaccine to either of the other two HPV vaccines, Gardasil®, and Cervarix®, because these vaccines have not been licensed in China. After the safety, the immunogenicity and efficacy of the E. coli-expressed HPV vaccine candidate been evaluated in subsequent randomized controlled clinical trials, a head-to-head comparison of the safety and immunogenicity of the three HPV vaccines will be important. Another limitation of this study is the lack of a placebo or adjuvant control group. A control group will be a part of subsequent clinical trials.
In conclusion, the preliminary safety results from this study show that a new E. coli-expressed recombinant HPV 16/18 bivalent vaccine is well tolerated in healthy women. These results support further immunogenicity and efficacy studies of this HPV vaccine candidate.
Methods
Vaccine
The tested bivalent HPV vaccine candidate, Cecolin®, contained a mixture of two recombinant L1 VLPs of HPV 16 and 18 (Xiamen Innovax Biotech Co. Ltd) with diameters of 50 nm and 60 nm, respectively ().Citation14,Citation15 The production of this vaccine has been described previously.Citation14,Citation15 In brief, recombinant plasmids containing the HPV 16 or 18 L1 genes were transferred into E. coli. The bacteria were centrifuged and lysed, and the proteins were purified by sequential chromatography. After purification, HPV L1 VLPs were disassembled and reassembled. The final purity was greater than 95%. The E. coli-expressed and purified HPV type 16 and type 18 L1 VLPs were separately absorbed into hydroxy aluminum adjuvant and then mixed together. The vaccine formulation contained 60 μg HPV 16 L1 VLP and 30 μg HPV 18 L1 VLP with 208 μg aluminum adjuvant suspended in 0.5 mL phosphate buffered saline. The vaccine lot for the clinical trial was produced under the good manufacturing practice conditions according to the requirements of the CFDA.
Figure 5. Transmission electron micrograph of HPV16 and HPV18 L1 VLP ( × 25,000, Bar = 200 nm). (A). HPV16 L1 VLP; (B). HPV18 L1 VLP.
Study design
This was an open-label phase I clinical trial designed to assess the safety of the bivalent HPV 16/18 vaccine. Independent ethics committee approval was obtained from the Ethics Committee of the Jiangsu Provincial Centre for Disease Control and Prevention (JSCDC), and the study was performed in accordance with the principles of the Declaration of Helsinki, the standards of Good Clinical Practice, and Chinese regulatory requirements as stipulated by CFDA. This trial was registered with ClinicalTrials.gov, number NCT01263327.
Healthy adult women aged 18 to 55 y living in Dongtai City, Jiangsu Province, China, were enrolled. Informed consent was obtained from each subject before enrollment. Subjects were required to have a negative urine pregnancy test before inclusion and prior to each vaccination. Subjects were excluded if they had any preexisting severe, acute or chronic medical condition, current pregnancy or lactation, severe anaphylaxis history to medicines or vaccines, or any other condition that might interfere with the study objectives.
Eligible participants were vaccinated intramuscularly in the deltoid at months 0, 1, and 6. Participants were observed for 30 min after each dose for immediate adverse reactions and were assessed by study investigators at 6 h, 24 h, 48 h, 72 h, 7 d, 14 d, and 28 d after each dose. Any observed or reported adverse effects (AEs) within 30 d of each injection were assessed by trained field investigators and recorded on safety diary cards. All subjects were asked to report any SAEs to investigators during the trial until month 7. Before and 2 d after each injection, paired blood samples were collected from participants to assess the following indexes: (1) 14 types of routine blood tests: complete blood count including red blood cells (RBC), platelets (PLT), hemoglobin (HGB), white blood cells (WBC), neutrophils (NEU), lymphocytes (LYM), monocytes (MON), eosinophils (EO), basophils (BAS), and the ratio of each type of WBC including NEU, LYM, MON, EO, and BAS; (2) two types of blood coagulation function tests: activated partial thromboplastin time (APTT) and fibrinogen (FIB); (3) 12 types of blood chemistry tests to assess changes in liver function: prothrombin time (PT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP), glutamyltranspeptidase (GGT), total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin(IBIL), total protein (TP), albumin (ALB), globulin (GLB), ratio of ALB to GLB (A/G); (4) 2 tests for kidney function: urea nitrogen (UREA) and creatinine (CREA); and (5) 3 other tests: glucose (GLU), total cholesterol (TC), and triglycerides (TG). Each AE was graded according to the guidelines issued by the CFDA.
Statistical methods
This phase 1 trial was designed to obtain safety information and not to test for immunogenicity or efficacy. Summary and descriptive statistics and graphs were used to describe the results. All participants who received at least 1 dose of study vaccine were included in the safety analysis. The frequencies of AEs reported following each vaccination were first compared as a whole, followed by pair-wise comparisons when the discrepancy was significant. Changes in the blood test parameters for each participant were determined by comparing the results of paired blood samples obtained before and 2 d after each vaccination. The fluctuations were classified into three categories: “remaining” indicated no grade change; “worsen” indicated a change from normal to abnormal or an increase in grade; and “improving” indicated a change from abnormal to normal or a decrease in grade. The frequencies of indexes that worsened were compared using the McNemar test for the routine blood tests, coagulation function, liver function, kidney function, and other tests.
| Abbreviations: | ||
| A/G | = | ratio of ALB to GLB |
| AE | = | adverse effect |
| ALB | = | albumin |
| ALT | = | alanine aminotransferase |
| AP | = | alkaline phosphatase |
| APTT | = | activated partial thromboplastin time |
| AST | = | aspartate aminotransferase |
| BAS | = | basophils |
| CFDA | = | China Food and Drug Administration |
| CREA | = | creatinine |
| DBIL | = | direct bilirubin |
| E. coli | = | Escherichia coli |
| EO | = | eosinophils |
| FIB | = | fibrinogen |
| GGT | = | glutamyltranspeptidase |
| GLB | = | globulin |
| HGB | = | hemoglobin |
| HPV | = | human papillomavirus |
| IBIL | = | indirect bilirubin |
| JSCDC | = | the Jiangsu Provincial Centre for Disease Control and Prevention |
| LYM | = | lymphocytes |
| MON | = | monocytes |
| NEU | = | neutrophils |
| PLT | = | platelets |
| PT | = | prothrombin time |
| RBC | = | red blood cells |
| SAE | = | serious adverse event |
| TBIL | = | total bilirubin |
| TP | = | total protein |
| UREA | = | urea nitrogen |
| VLP | = | virus-like particle |
| WBC | = | white blood cells |
Disclosure of Potential Conflicts of Interest
M.G. and X.-H.L. are currently employees of Xiamen Innovax. H-J Huang was an employee of Xiamen Innovax. The other authors declare that they have no conflicts of interest.
Acknowledgments
This work was supported by the National Major Scientific and Technological Special Project (2012ZX09101316), the National High-tech R&D Program (863 Program) (2012AA02A408), the International Science and Technology Cooperation Program of China (2011DFG33050), the Xiamen Scientific Project (3502Z20127027).
References
- Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011; 61:69 - 90; http://dx.doi.org/10.3322/caac.20107; PMID: 21296855
- Soerjomataram I, Lortet-Tieulent J, Parkin DM, Ferlay J, Mathers C, Forman D, Bray F. Global burden of cancer in 2008: a systematic analysis of disability-adjusted life-years in 12 world regions. Lancet 2012; 380:1840 - 50; http://dx.doi.org/10.1016/S0140-6736(12)60919-2; PMID: 23079588
- Rodríguez AC, Schiffman M, Herrero R, Wacholder S, Hildesheim A, Castle PE, Solomon D, Burk R, Proyecto Epidemiológico Guanacaste Group. Rapid clearance of human papillomavirus and implications for clinical focus on persistent infections. J Natl Cancer Inst 2008; 100:513 - 7; http://dx.doi.org/10.1093/jnci/djn044; PMID: 18364507
- Woodman CB, Collins S, Winter H, Bailey A, Ellis J, Prior P, Yates M, Rollason TP, Young LS. Natural history of cervical human papillomavirus infection in young women: a longitudinal cohort study. Lancet 2001; 357:1831 - 6; http://dx.doi.org/10.1016/S0140-6736(00)04956-4; PMID: 11410191
- Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, Snijders PJ, Peto J, Meijer CJ, Muñoz N. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999; 189:12 - 9; http://dx.doi.org/10.1002/(SICI)1096-9896(199909)189:1<12::AID-PATH431>3.0.CO;2-F; PMID: 10451482
- Chen W, Zhang X, Molijn A, Jenkins D, Shi JF, Quint W, Schmidt JE, Wang P, Liu YL, Li LK, et al. Human papillomavirus type-distribution in cervical cancer in China: the importance of HPV 16 and 18. Cancer Causes Control 2009; 20:1705 - 13; http://dx.doi.org/10.1007/s10552-009-9422-z; PMID: 19705288
- Muñoz N, Bosch FX, de Sanjosé S, Herrero R, Castellsagué X, Shah KV, Snijders PJ, Meijer CJ, International Agency for Research on Cancer Multicenter Cervical Cancer Study Group. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003; 348:518 - 27; http://dx.doi.org/10.1056/NEJMoa021641; PMID: 12571259
- Lo KW, Wong YF, Chan MK, Li JC, Poon JS, Wang VW, Zhu SN, Zhang TM, He ZG, Wu QL, et al. Prevalence of human papillomavirus in cervical cancer: a multicenter study in China. Int J Cancer 2002; 100:327 - 31; http://dx.doi.org/10.1002/ijc.10506; PMID: 12115548
- FUTURE II Study Group. Quadrivalent vaccine against human papillomavirus to prevent high-grade cervical lesions. N Engl J Med 2007; 356:1915 - 27; http://dx.doi.org/10.1056/NEJMoa061741; PMID: 17494925
- Garland SM, Hernandez-Avila M, Wheeler CM, Perez G, Harper DM, Leodolter S, Tang GW, Ferris DG, Steben M, Bryan J, et al, Females United to Unilaterally Reduce Endo/Ectocervical Disease (FUTURE) I Investigators. Quadrivalent vaccine against human papillomavirus to prevent anogenital diseases. N Engl J Med 2007; 356:1928 - 43; http://dx.doi.org/10.1056/NEJMoa061760; PMID: 17494926
- Paavonen J, Jenkins D, Bosch FX, Naud P, Salmerón J, Wheeler CM, Chow SN, Apter DL, Kitchener HC, Castellsague X, et al, HPV PATRICIA study group. Efficacy of a prophylactic adjuvanted bivalent L1 virus-like-particle vaccine against infection with human papillomavirus types 16 and 18 in young women: an interim analysis of a phase III double-blind, randomised controlled trial. Lancet 2007; 369:2161 - 70; http://dx.doi.org/10.1016/S0140-6736(07)60946-5; PMID: 17602732
- Paavonen J, Naud P, Salmerón J, Wheeler CM, Chow SN, Apter D, Kitchener H, Castellsague X, Teixeira JC, Skinner SR, et al, HPV PATRICIA Study Group. Efficacy of human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine against cervical infection and precancer caused by oncogenic HPV types (PATRICIA): final analysis of a double-blind, randomised study in young women. Lancet 2009; 374:301 - 14; http://dx.doi.org/10.1016/S0140-6736(09)61248-4; PMID: 19586656
- Schiller JT, Castellsagué X, Garland SM. A review of clinical trials of human papillomavirus prophylactic vaccines. Vaccine 2012; 30:Suppl 5 F123 - 38; http://dx.doi.org/10.1016/j.vaccine.2012.04.108; PMID: 23199956
- Wei M-X, Li SW, Huang B, Shen WT, Su YZ, Zhang CH, Gu Y, Du HL, Zhang J, Xia NS. [Production of human papillomavirus type 16 virus-like particles and its immunogenicity]. [in Chinese] Bing Du Xue Bao 2009; 25:245 - 50; PMID: 19769155
- Xie M-H, Li S-W, Shen W-T, Li Z-Y, Zhuang Y-D, Mo X-B, Gu Y, Wu T, Zhang J, Xia N. [Expression, purification and immunogenicity analysis of HPV type 18 virus-like particles from Escherichia coli]. [in Chinese] Sheng Wu Gong Cheng Xue Bao 2009; 25:1082 - 7; PMID: 19835152
- Zhu FC, Zhang J, Zhang XF, Zhou C, Wang ZZ, Huang SJ, Wang H, Yang CL, Jiang HM, Cai JP, et al. Efficacy and safety of a recombinant hepatitis E vaccine in healthy adults: a large-scale, randomised, double-blind placebo-controlled, phase 3 trial. Lancet 2010; 376:895 - 902; http://dx.doi.org/10.1016/S0140-6736(10)61030-6; PMID: 20728932
- CFDA. Adverse reaction grading standards guideline for prophylactic vaccine clinical trial. 2005 Available from: http://www.sda.gov.cn/WS01/CL1616/83435.html.
- Corp. MSD. GARDASIL Prescribing Information.2013 Available from: http://www.merck.com/product/usa/pi_circulars/g/gardasil/gardasil_pi.pdf
- GlaxoSmithKline. CERVARIX Prescribing Information. 2012 Available from: http://us.gsk.com/products/assets/us_cervarix.pdf
- Miller NB. Clinical Review of Biologics License Application for Human Papillomavirus 6, 11, 16, 18 L1 Virus Like Particle Vaccine (S. cerevisiae) (STN 125126 GARDASIL), manufactured by Merck, Inc. 2006 Available from: http://www.fda.gov/downloads/BiologicsBloodVaccines/Vaccines/ApprovedProducts/UCM111287.pdf
- Nancy B. Miller, Roberts J. Clinical Review of Biologics License Application for Human Papillomavirus 16, 18 L1 Virus Like Particle Vaccine, AS04 Adjuvant-Adsorbed (Cervarix). 2009[http://www.fda.gov/downloads/biologicsbloodvaccines/vaccines/approvedproducts/ucm237976.pdf].
- Zhao Q, Li S, Yu H, Xia N, Modis Y. Virus-like particle-based human vaccines: quality assessment based on structural and functional properties. Trends Biotechnol 2013; In press http://dx.doi.org/10.1016/j.tibtech.2013.09.002; PMID: 24125746
- Baker TS, Newcomb WW, Olson NH, Cowsert LM, Olson C, Brown JC. Structures of bovine and human papillomaviruses. Analysis by cryoelectron microscopy and three-dimensional image reconstruction. Biophys J 1991; 60:1445 - 56; http://dx.doi.org/10.1016/S0006-3495(91)82181-6; PMID: 1663794
- Zhao Q, Modis Y, High K, Towne V, Meng Y, Wang Y, Alexandroff J, Brown M, Carragher B, Potter CS, et al. Disassembly and reassembly of human papillomavirus virus-like particles produces more virion-like antibody reactivity. Virol J 2012; 9:52; http://dx.doi.org/10.1186/1743-422X-9-52; PMID: 22356831
- Salunke DM, Caspar DL, Garcea RL. Self-assembly of purified polyomavirus capsid protein VP1. Cell 1986; 46:895 - 904; http://dx.doi.org/10.1016/0092-8674(86)90071-1; PMID: 3019556
- Ferrer-Miralles N, Domingo-Espín J, Corchero JL, Vázquez E, Villaverde A. Microbial factories for recombinant pharmaceuticals. Microb Cell Fact 2009; 8:17; http://dx.doi.org/10.1186/1475-2859-8-17; PMID: 19317892
- Zhang J, Shih JW, Wu T, Li SW, Xia NS. Development of the hepatitis E vaccine: from bench to field. Semin Liver Dis 2013; 33:79 - 88; http://dx.doi.org/10.1055/s-0033-1338116; PMID: 23564392
- Park SB. Hepatitis E vaccine debuts. Nature 2012; 491:21 - 2; http://dx.doi.org/10.1038/491021a; PMID: 23128204