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Herpesvirus-based vaccines against Rotavirus infections

Final Report Summary - HEVAR (Herpesvirus-based vaccines against Rotavirus infections)

HEVAR is a collaborative project involving four academic laboratories from four European countries (France, Switzerland, Germany, Italy) and four academic laboratories belonging to three South American countries (2 x Argentine, Brazil, Uruguay). The overall scientific goal of HEVAR was to contribute to a better understanding of the immune biology of rotavirus infections using a novel generation of gene transfer vectors derived from herpes virus simplex type 1 (HSV-1), as a first step towards the development of innovative genetic vaccines to fight against these pathogens, which are the most common and important cause of severe dehydrating diarrhoea in young children of developing countries.

In addition to contributing to a better understanding of the immune biology of rotavirus infection and of evaluating the feasibility of using HSV-1 vectors as anti-rotavirus vaccines, the main deliverables of HEVAR are a set of toolboxes containing a large collection of HSV-1-based and DNA-based vectors expressing mouse, monkey and human rotavirus antigens, that we have immunologically evaluated and rendered accessible to any academic team wishing to use them for vaccine development or fundamental research on rotaviruses.

A last set of deliverables consisted in a series of scientific meetings and events required to implement the transfer of knowledge and complex technology that is necessary to generate, produce, and evaluate, HSV-1-based gene transfer vectors in South America (including the implementation of a local platform for viral vector production and distribution), therefore improving the human capital and the technological competence of these countries, as well as the reciprocal transfer to European teams of knowledge on the biology of rotavirus and other endemic viruses with high social cost in South America, as a way to strengthen the awareness to, and the understanding of, these neglected diseases.

Work planned, performed, results achieved

The overall objectives of HEVAR were therefore:
(i) to generate a battery of HSV-1-based vectors expressing antigens from different rotavirus strains, either individually or in combination;
(ii) to evaluate the ability of these vectors to elicit humoral and cellular immune responses, and particularly neutralising and protective responses, in model animals (mice); and
(iii) to implement the transfer of knowledge and technologies required to generate and produce the viral vectors in South America.
To achieve these major tasks, the HEVAR project was divided into three scientific work packages (WPs):
- Vector construction (WP1);
- Evaluation of immune responses (WP2); and
- Transfer of knowledge and technology (WP3).

For WP1, the first major task was to individually clone into different types of plasmids the genes encoding the four structural proteins (VP2, VP4, VP6 and VP7) and one non-structural protein (NSP4) from four different rotavirus strains: mice EC, monkey RRV, and human Wa and DS-1 strains, and to validate the identity of these genes through sequencing. This task was fully achieved, and the 20 rotavirus genes were cloned and validated. A second major task of WP1 was to generate HSV-1 vector backbones able to simultaneously express or display one or more rotavirus antigens from different loci and under the control of various types of regulatory sequences. This task was also fully achieved. Furthermore, we have generated more vector backbones than planned. A third major task was to use the backbones and the cloned rotavirus genes to generate HSV-1-based vectors expressing rotavirus proteins (both amplicon and recombinant vectors). As planned, the rotavirus genes were used to generate a large battery of HSV-1 vectors expressing single or combined rotavirus antigens. The expression profiles of these vectors, and the localisation and properties of the expressed rotavirus proteins, have been characterised in infected cells, both by western blotting and by immune-fluorescence studies. Importantly, in the last part of HEVAR we demonstrated that vectors simultaneously expressing three different rotavirus proteins (RRV VP2/VP6/VP7) were able to induce the in situ assembly of empty rotavirus-like particles, which is a major achievement of our project. Lastly, these vectors were sent to our immunology partners for immune-evaluation in mice. We consider therefore that all major tasks of WP1 were successfully accomplished and the WP1 deliverables were satisfactorily achieved.

For WP2, the main tasks were to evaluate the toxicity and the immune responses elicited in model animals (mice) by the different vectors produced in WP1. Towards this end, during the first year of the period, WP2 partners had set up the experimental conditions and developed the tools and approaches required to perform the immuneevaluation.

In the second and third years of the project, this evaluation took place. The most important aspects of the immune evaluation were to study whether the vectors were able to elicit both humoral and cellular specific responses, to compare the relative efficacy of different routes of immunisation, to characterise the nature of the humoral responses (type of immunoglobulins, neutralizing antibodies), and to explore if the vectors were able to induce protection to rotavirus challenge. Several different vectors, expressing mouse and monkey rotavirus antigens have been already immunologically evaluated in this way. Our results indicate that most of these vectors, but particularly amplicon vectors, elicited significant levels of humoral and / or cellular responses. Furthermore, some of the antibodies, raised by vectors expressing VP4 or VP7 antigens, displayed neutralising activity and we also observed a significant level of protection against high-dose challenge with live rotavirus when using vectors expressing three different rotavirus proteins. These results are quite encouraging in the perspective of generating novel genetic vaccines. As above quoted, the number of HSV-1 vectors generated in WP1 is large, and immuneevaluation of other vectors is still ongoing. In addition, as we have described in the second intermediary report, we had accumulated some delay in WP1 as some rotavirus genes were strikingly difficult to be cloned and, furthermore, when introduced into HSV-1 vectors, some unexpected toxic effect resulted in low-level production of the vectors, particularly in the case of recombinant vectors. Although most of these difficulties were later resolved, the delay accumulated in vector production resulted in a concomitant delay in vector evaluation, which explains why some vectors are still being evaluated now. This task will therefore be continued after the end of the project.

Regarding WP3, the main goals of the project were:
(i) to establish a technological vector platform in South-America, able to locally generate and produce viral vectors for South American partners;
(ii) to implement the transfer of knowledge and methodologies between partners, based on trainings and exchanges between HEVAR laboratories and on the implement a PhD programme that allowed several South-American PhD students to accomplish part of their work in European laboratories; and
(iii) to organise courses, conferences, and seminars, at the time of our general meetings.
All these tasks have been satisfactorily accomplished. It is important to stress that the quality of the links established between South-American and European teams was very good and that in most cases the scientific collaborations and the training of young researchers will continue after the end of the present project.

In conclusion, virtually all tasks and deliverables planned at the beginning of the project have been successfully achieved, in spite of some difficulties described in the first and second intermediary reports. As a result, we feel that the work and results achieved by HEVAR constitute a significant advance in the comprehension of many aspects of the immune biology of rotavirus infection. The large set of available DNA-based and HSV-1 based vaccines that we have developed and are available to any academic laboratory willing to use them for their research will certainly boost further experimentation and novel approaches in the domain of rotavirus immune biology. The neutralising and protective antibodies elicited, as well as the cellular responses we observed, demonstrate the feasibility and the interest of exploring innovative ways to immunise against rotaviruses, and represents a first significant step in the development of HSV-1-based vector vaccines to fight against these agents. Lastly, the implementation of a local platform for HSV-1 vector production in South America, as well as the training of many young South American researchers and technicians, who have learnt how to develop and to apply these vectors, will certainly contribute to further developments and collaborations in the field of viral vectors for gene transfer and as vector vaccines.

To finish, we would like to stress that the collaborations and links established between European and South American partners were both delightful in social terms and fruitful in scientific terms, and will, without any doubt and funded by other means, be continued in the future.

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