[Human Vaccines 4:2, 143-147; March/April 2008]; ©2008 Landes Bioscience
Research Paper
Immunogenicity of a thermally inactivated rotavirus vaccine in mice Baoming Jiang,1,* Yuhuan Wang,1 Jean-Francois Saluzzo,2 Kristina Bargeron,1 Marie-Joelle Frachette2 and Roger I. Glass1,3 1Gastroenteritis
and Respiratory Virus Laboratory Branch; Division of Viral Diseases; Centers for Disease Control and Prevention; Atlanta, Georgia USA; 2Sanofi Pasteur; Lyon, France; 3Fogarty International Center; National Institutes of Health; Bethesda, Maryland USA
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just beginning or in progress for Rotarix® and RotaTeq® from Merck & Co., we will not know how effective these vaccines are for several years. Should they induce poor immune response or only weak protection in these settings, or should intussusception occur as an adverse event with these vaccines, the world will suffer a substantial setback in protecting children from diarrhea and deaths. We have pursued an alternative strategy to develop an inactivated rotavirus vaccine (IRV) that when administered parenterally, should induce an immune response in children of less developed countries comparable to those in developed countries. This approach would likely be free of the complication of intussusception and could lead to a product that contains rotavirus as part of a combination child‑ hood vaccine. Experimental IRV when administered intramuscularly have induced potent systemic and local immune responses and solid protection against rotavirus infection in mice and rabbits.3,4 However, in the gnotobiotic piglet model of rotavirus disease the results have been ambiguous, ranging from little or no protection to complete protective immunity.5,6 (Nakagomi, personal communica‑ tion) Furthermore, passively acquired circulating antibody in serum protected calves, piglets and monkeys from rotavirus challenge, suggesting that a parenteral vaccine which induces strong serum antibody response could do the same in children.7‑10 Strategies for virus inactivation have been the use of traditional chemicals (e.g., formaldehyde, b‑propiolactone, and aziridines) and biophysical treatment (e.g., UV irradiation, pressure and heat).11‑13 Formaldehyde has been the most commonly used to prepare inac‑ tivated vaccines against viruses, such as polio, influenza, Japanese encephalitis, and Hantaan virus.14‑17 This chemical agent has been demonstrated to be fairly effective in inactivating virus infectivity, but such inactivation procedures typically have an extended incuba‑ tion period, are not a linear or first‑order reaction, and often are associated with residual infectivity in some vaccines.13,18 In addition, the use of formaldehyde in the manufacture and preparation of certain vaccines has resulted in unwanted toxicity, disease enhance‑ ment, and even deaths in children who received vaccines against respiratory syncytial virus (RSV) and measles virus.19,20 Furthermore, formaldehyde is classified as a carcinogen and thus can not be used as an inactivating agent for new vaccines in some countries. Last, chemical approach is a challenge for the inactivation of rotavirus. The two most commonly used chemicals formaldehyde and b‑pro‑ piolactone have been shown to cause damage to the integrity of rotavirus particles, alter the structure of viral proteins, and reduce
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Current approaches to the prevention of severe rotavirus diar‑ rhea and deaths in children have all been through the use of live oral vaccines. To develop a safe and effective inactivated rotavirus vaccine (IRV), a new simple, rapid and robust method for the inactivation is critical and essential because chemical inactivation commonly used for a number of killed vaccines has been a chal‑ lenge and problematic for rotavirus. We have examined an array of thermal conditions and demonstrated that purified YK‑1 rotavirus in diluent buffer can be completely inactivated by heat treat‑ ment, as evidenced by the lack of virus growth in two successive passages in cell culture. Unlike chemical treatment that often causes physical and biochemical damages to viruses, thermally inactivated rotavirus particles maintained their structural, biochemical and antigenic integrity. A two‑dose intramuscular administration of thermally inactivated YK‑1 rotavirus without adjuvant resulted in high titers of total and neutralizing antibody in serum of mice. Adjuvant Al(OH)3 further led to enhanced antibody titers and also dramatically lowered the amount of antigens in the vaccine formu‑ lation. Our results demonstrate the potential of heat inactivation as a novel approach to the manufacture of a safe and efficacious parenteral rotavirus vaccine, which should serve as an important addition to and back up for live oral rotavirus vaccine in children.
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Key words: rotavirus, thermal inactivation, parenteral vaccine, immunogenicity
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Introduction
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To date, all rotavirus vaccines that have entered clinical trials in children have been live oral strains and several of these have proven effective and become licensed. However, none of these vaccines have been tested in the target populations in poor countries of Africa and Asia where rotavirus remains a prime killer of children.1 Past experience with live oral vaccines (polio, cholera, typhoid, and early candidate rotavirus vaccines) and the recent immunogenicity data with the GlaxoSmithkline vaccine Rotarix® all indicate that the immune responses and protection in these populations are less robust than in children of more developed settings.2 Since clinical trials are *Correspondence to: Baoming Jiang; Viral Gastroenteritis Team; Mailstop G04; National Center for Immunization and Respiratory Diseases; 1600 Clifton Road; Atlanta, Georgia 30333 USA; Tel.: 404.639.2861; Fax: 404.639.3645; Email:
[email protected] Submitted: 08/16/07; Accepted: 11/04/07 Previously published online as a Human Vaccines E-publication: www.landesbioscience.com/journals/vaccines/article/5263 www.landesbioscience.com
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YK‑1 rotavirus was purified from infected Vero cell cultures and only triple‑layered particles were used for the inactivation study (Fig. 1). After thermal treatment, YK‑1 particles were found to maintain biophysical integrity, as evidenced by the preservation of triple‑layered structures that were morphologically similar to live native virions (Fig. 1). Heat‑treated virus particles contained almost all structural viral proteins—VP1, VP2, VP4, VP6 and VP7, and were antigenic, as demonstrated by the detection of major proteins in Western blot analysis using rabbit hyperimmune serum to RRV rotavirus (Fig. 2). Of note, a few bands with high molecular mass (> 125 kDa) were detected in heat‑treated YK‑1 sample. The origin of these large‑size bands is not clear, but might result from heat‑in‑ duced protein aggregates. In addition, we observed an approximately 60 kDa protein in the original but not in heat treated vaccine mate‑ rials. Whether this protein is a part of viral particle is not known and will be investigated. Inactivated particles also possessed dsRNA genome like native virions (data not shown). Inactivation of rotavirus in heat‑treated samples was confirmed by the lack of virus growth following two sequential passages in Vero cells (data not shown). In controls, robust virus growth was observed in cells infected with the original non heat‑treated YK‑1 rotavirus. We inoculated mice I.M. with our thermally inactivated rotavirus without adjuvant and demonstrated that the killed vaccine was highly immunogenic (Fig. 3). Mice in groups of seven mounted low to moderate rotavirus‑specific total antibody response in serum if they received only one‑dose immunization with 20 mg or 2 mg of killed YK‑1 rotavirus (Fig. 3A). After two‑dose immunizations with 20 mg of antigens, high total antibody titers were detected. Comparable though lower (two to eight‑fold) antibody titers were seen in mice that were inoculated twice with 2 mg of antigens. These high levels of antibody were sustained two weeks later in the final serum specimens when the mice were euthanized. No antibody titers (
