Development of new type anti-HCV therapeutic individualized vaccine prototype
Project is being carried out within the ERDF 126.96.36.199. activity „Support for Science and Research”
Project agreement Nr. 2010/0233/2DP/188.8.131.52.0/10/APIA/VIAA/164
Project duration: 36 months (01.10.2010.-30.09.2013.)
Project costs: 266419 LVL
Project leader: Dr. biol. Irina Sominska
Collaboration partner - State Agency “Latvian Infectology Centre”
The general aim – Enlargement of chronic HCV patient's treatment effectiveness.The specific aims of the project - a new type of individual therapeutic vaccine prototypes development and universal laboratory technology development for such type of vaccine production.
Industrial research - research work in order to obtain the knowledge and skills for a new product - development of a new type of individual therapeutic vaccine prototypes and development of universal laboratory technology for the production of such type of vaccine;
2. guarantee of publicity and availability of the research results;
3. consolidation of industrial property rights for the research results.
Implementation site of the project - Latvian Biomedical Research and Study Centre, a cooperation partner - Latvian Infectology Centre
Our project provides development of an individual anti-HCV therapeutic vaccine prototype. The idea is based on combination of the HCV variable structural part and conservative non-structural regions. This type of vaccine could be especially effective for chronic infections.
Prototype of the vaccine protein component is represented by highly variable region of virus envelope protein E2 (hypervariable region HVR1). HVR1 sequences will be selected individually by using HCV genomic material of the individual patient's serum.
Prototype of the vaccine DNA component includes CTL and T-helper epitopes from the highly conservative non-structural protein NS3/4a. As an alternative option, a plasmid containing the full NS3/4a protein encoding sequences will be used. NS3/4a sequence will be designed on the basis of consensus sequences representing combination of Latvian major subtype 1b HCV isolates.
High genetic variability of HCV HVR1 region demands, from our point of view, an urgent need to elaborate personalized, or individual, vaccines to be effective against a particular patient's hepatitis C virus. After determination of HVR1 structures of this particular virus, it will be possible to constitute vaccine models, tailored to the expression of this sequence and to the production of virus neutralizing antibodies. It was shown in studies with chimpanzees that the HVR1 region contains the virus neutralizing epitopes. Recently, the relationship was demonstrated between decline of acute viremia and antibody levels, which are able to neutralize HCV pseidovirions. In rare cases, spontaneous absorption of chronic infection is associated with high anti-E2 antibody titres, which are able to block virus interaction with the major components of the receptor. Protective role of antibodies is approved by the fact that significantly more severe chronic disease course is observed in hypogammaglobulinemia patients. Complete prevention of HCV infection in chimpanzees was possible by inoculation of rabbit anti-HCV antiserum.
NS3/4a protein, one of the most conservative proteins among different HCV genotypes, provides serine protease and helicase activity. Protein NS3 region contains CD4 + T helper epitopes, and some CTL epitopes. Speed and intensity of cellular immune response against NS3/4a proteins determine the body's release from HCV infection. Therefore, NS3/4a can serve as an essential component of the HCV vaccine candidate. Recently, high immunogenicity of NS3/4a-containing DNA vaccine has been demonstrated in mice and also in HCV-infected humans.
As the most appropriate VLP vector, we plan to use recombinant HBc, which is expressed in E. coli system and is able to form VLPs with high efficiency. In this project, HVR1- containing chimeras will be constructed on the basis of the HBc VLPs. Such HBc-HVR1 structures will ensure high immunogenicity of the heterologous HVR1 fragments, due to VLP molecular organization and their high intrinsic immunogenicity per se. Recombinant HBc as a carrier of heterologous sequences efficiently activates T-helper 1 like alpha-IFN response. Using unique experience of the BMC Protein engineering in the design of chimeric VLPs, it is possible to create an universal set of vectors, which will allow designing vaccine models and prototypes by inclusion of definite HCV protein fragments into chimeric VLPs without interference with ability of the HBc to self-assemble. Genetic immunization with DNA in which is included the HBc gene is most effective to activate CTL response. We plane to insert NS3/4a epitopes at the 3' end of the HBc gene.
It is very essential that we have the capabilities to carry out a full cycle of research, including vaccine candidate design, modeling, construction of variants and their structurally functional investigation. Technology assumes optimization of chimeric VLP expression, development of the purification cycle by using of advanced protein purification techniques. Laboratory equipment ensures full GLP (Good Laboratory Practice) conditions.
Current results (August of 2011)
Research activity 1. Construction and characterization of protein and DNA vaccine components.
Variants of the recombinant procariotic expression constructs on the modified human hepatitis B virus core antigen (HBcAg) base were developed to obtain protein component of the vaccine. Based on the literature data HVR1 region sequences of hepatitis C virus (HCV) with high potential of cross-reactivity were selected. In parallel, HVR1 regions of HCV genome form selected sera samples of chronic HCV patients were amplified, cloned and sequenced. Obtained sequences were cloned into the HBcAg vectors. Obtained recombinant constructs were expressed in E.coli cells using different conditions of cultivation. Constructs with the highest expression level and optimal conditions of expression were selected.
Different schemes of recombinant protein purification were developed, possibility to form virus-like particles were studied. Recombinant proteins HBc-HVR1 sequences were obtained in amount and quality required for immunization experiments.
Data about the circulating in Latvia HCV NS3/4a region sequences were collected. For this purpose NS3/4a regions of HCV genome form selected chronic HCV patient's sera samples were amplified, cloned and sequenced. Nucleotide and protein consensus sequences of NS3/4a region that are typical for HCV genotype 1b viruses circulating in Latvia were created. NS3/4a nucleotide consensus sequence was optimized for expression in the mice and human cell cultures.
Research activity 2. Immunization of the small laboratory animals
The immunization experiments in mice using chimeric HBcAg/ HVR1 virus-like particles were just started.