HSV-1-Based Replication-defective particles and amplicons as transgenic vectors expressing antigens from human and animal pathogens

PALACIOS, CA; CLAUS JUAN; MATTION NORA

Ciudad Autónoma de Buenos Aires

Workshop; 3rd ICGEB Workshop on Human RNA Viruses; 2012

Fundación Instituto Leloir

Diarrheal diseases remain one of the leading causes of morbidity and mortality among young children worldwide, accounting for an estimated 1.34 million deaths annually among children younger than 5 years of age. Rotavirus (RV) gastroenteritis is the leading cause, resulting in an estimated 2 million hospitalizations and 527,000 deaths each year. Because of the widespread nature of rotaviruses, the development of vaccines against this pathogen is considered a key control measure. Killed rotavirus vaccines and subunit vaccines have been developed, but these types of vaccines do not provide endogenously synthesized proteins and generally do not elicit cytotoxic T lymphocyte (CTL) responses that may be important in controlling rotavirus infection. Development of live attenuated vaccines based in human or animal RV strains, has been influenced by views regarding the importance of serotype-specific neutralizing antibody. Recently by large-scale sequencing results, it was revealed that the genomes of human RVs typically consist of phylogenetically linked constellations of eleven dsRNA segments, indicating that RV genes co-evolved to produce a pattern that supports the viral replication, showing the complexity of circulating RVs. The recent introduction of two live attenuated RV vaccines (RotaTeqTM and RotarixTM) into the childhood vaccination programs of various countries has been highly effective in reducing the incidence of RV diarrheal disease. Whether the widespread use of these vaccines will introduce selective pressures on human RVs, triggering genetic and antigenic changes that undermine the effectiveness of vaccination programs, is uncertain and will require continue surveillance of human RVs. On the other hand, the epidemiology of the disease which differs in developed and developing countries can affect decisions about vaccine composition and delivery. In order to overcome these hurdles, it is important to support the development of new, non-replicating vaccines, which will not suffer the potential disadvantages of the present vaccines. In this context, the use of HSV-1-based vectors offers an attractive alternative strategy for rotavirus antigens expression or display in animal models. Advantages of defective HSV-1 vectors include: (i) HSV-1 can infect most cell types, quiescent or in proliferation, including antigen-presenting cells, from most mammalian species, including humans, mice, rabbits and pigs, (ii) HSV-1 vectors have a very large transgene capacity (150-kbp foreign DNA), thus allowing the simultaneous delivery of multiple transgene sequences, (iii) HSV-1 vectors can be designed to cause no toxicity, (iv) Recent studies from several groups showed that HSV-1 vectors can induce very strong and long-term protective immune responses, even in the presence of pre-existing antibodies to HSV-1. The overall scientific goal of the present project is to contribute to a better understanding of the immune biology of rotavirus infections using a novel generation of gene transfer vectors derived from HSV-1, as a first step towards the development of innovative genetic vaccines. The capability of these vectors to support the expression of different murine RV proteins in mammalian cells has been demonstrated, as well as the generation of specific RV immune responses in inoculated animals. A collection of HSV-1-based vectors expressing human and mouse rotavirus antigens were constructed and will be evaluated in mice. They include defective HSV-1 based vectors expressing Rhesus RV VP6, and polycistronic amplicon vectors expressing different combinations of VP6, VP2, VP7, VP4 and NSP4 from the human Wa RV strain. Control vectors expressing reporter genes (ß-gal or GFP) were also constructed. We were able to demonstrate in vitro the functionality of these vectors, detecting the expression of VP6, VP2, VP4 and VP7 by immunofluorescence; VP6, VP2 and NSP4 by Western blot, ß-gal activity by X-gal staining, and in vivo GFP expression. The final part of this project will be to evaluate the contribution of the different RV antigens to the protective immunity, after inoculation of groups of mice with combinations of these vectors. Humoral and cellular immune responses will be assessed.