Design Of Epitope-based Immunogen And Study On Functional Activities Based On Conserved Stem Fragment Of Hemagglutinin

Influenza virus can be divided into three types(A, B and C). Among these three types, Influenza A virus(IAV), as the main type of influenza pandemic, is one of the major pathogens which threate hunman health. IAV has the highly contagious in the crowd of all ages and has characteristic of the upper respiratory tract infection, especially in the elderly, children and immunocompromised patients. According to the records, only in the IAV pandemic of three years(1918, 1957 and 1968), about 50 million people were killed. According to the world health organization(WHO) report, there are 5% ~ 15% of the people are infected with influenza virus every year all over the world, including 3 million~5 million severe cases and of 250,000~500,000 people dead due to its complication.Medicine and vaccine are used for inhibiting influenza virus. Neuraminidase inhibitors and M2 ion channel inhibitors are mainly used to treat IAV. However, virus resistance comes in to being with the wide use or abuse of these antiviral drugs. These have affected the therapeutic effect of the IAV drugs. So the application of the vaccine against the infection of IAV is more significant.The present influenza vaccine mainly include the inactivated influenza virus vaccine(IIV), subunit vaccine, cracking influenza virus vaccine and live attenuated influenza virus vaccine(LAIV). Each vaccine contains antigen components from H1, H3 subtypes of A type and B type of influenza virus. Due to the broader coverage and the perfect production technology, this kind of trivalent vaccine is becoming the mainstream of influenza virus vaccine. However, because of the high mutation of virus antigen, the prediction of virus strains becomes the main stage of vaccine design. Currently, the virus strains of seasonal influenza vaccine were predicted by the world health organization(WHO) based on circulating antigen of the previous season. However, it is difficult to predict the coverage of the antigen shift of influenza viruses, which could cause a pandemic. Because of the above deficiency of trivalent influenza vaccine, it is a very urgent issue for researchers to develop a broad spectrum and durable universal influenza vaccine.In recent years, with the increasing of pandemic of H5N1, H1N1 and H7N9, research and development of broad-spectrum influenza vaccine is particulary important to enhance the ability of precention and control of influenza virus in China. Previous study showed that the candidate vaccine based on the conservative protein of influenza virus(matrix protein and surface protein) could induce high level of neutralizing antibodies in mice. It provides a new fundamental of influenza broad-spectrum vaccine. Because hemagglutinin(HA) protein is the main antigen sites which induce a protective immune response with the significant conservative stem sequence, we develop the candidate vaccine based on HA. However, defect still exist in vaccine candidates based on the high conservative HA. First, many of broad spectrum neutralizing antibodies(n Abs) depend on a structural/non-contiguous epitope of the HA stem for binding, which cannot be utilized for immunogen design. Second, the mechanism of n Abs is not clear, which cannot be utilized for immunogen design, either. At the same time, the immunogenicity of high conservative protein subunit vaccine and epitope-based vaccine is queit low, so the problem how to improve the immunogenicity of candidate vaccine can not be ignored. Therefore, it is very important to develop high immunogenicity and broad spectrum protection to make the broad-spectrum flu vaccine be successful.Based on the neutralizing mechanism of inhibiting conformational change by anchoring the HA1 and HA2, the study aims at designing an epitope-based immunogen which is made up with short and linear epitope. The immunogen should also have strong immunogenicity and neutralizing activity. Then, the immunogenicity, broad spectrum neutralization and protective activity can be also increased by optimization. This thesis is divided into two parts:Part one: Design of the immunogen and study of the neutralizing mechanism based on conserved stem fragment from H3 influenza hemagglutininCurrently available influenza vaccines typically fail to elicit/boost broadly neutralizing antibodies due to the mutability of virus sequences and conformational changes during protective immunity, thereby limiting their efficacy. This problem needs to be addressed by further understanding the mechanisms of neutralization and finding the desired neutralizing site during membrane fusion. This study specifically focused on viruses of the H3N2 subtype, which have persisted as a principal source of influenza-related morbidity and mortality in humans since the 1968 influenza pandemic. Through sequence alignment and epitope prediction, a series of highly conserved stem fragments(97.59%±0.62%, spanning 47 years) were found and coupled to the Keyhole Limpet Hemocyanin(KLH) protein. By application of a combinatorial display library and crystal structure modeling, a stem fragment immunogen, located at the turning point of the HA neck undergoing conformational change during membrane fusion with both B- and T-cell epitopes, was identified. After synthesis of the optimal stem fragment using a multiple antigen peptide(MAP) system, strong humoral immune responses(log104.14 ~ 4.74) and cross-clade neutralizing activities(ID50:564.57~1408,P<0.005) against strains from the H3 subtype of group 2 influenza viruses after animal immunizations were observed. By detection of nuclear protein immunofluorescence with acid bypass treatment, antisera raised against MAP4 immunogens of the stem fragment showed the potential to inhibit the conformational change of HA in stem-targeted virus neutralization. The identification of this conserved stem fragment provides great potential for exploitation of this site of vulnerability in therapeutic and vaccine design.Part two: Development of chimeric Norovirus P particle to induce influenza broad neutralizing antibodyIn this study, through sequence alignment and epitope prediction, potential B-cell epitopes were found in the highly conserved HA2:90-105 stem fragment among three virus subtypes H1 and B(97±0.123% and 99.98±0.003%). A Norovirus(No V) P particle platform was used to express 24 copies of the HA2:90-105 sequences of H1, H3 and B subtypes in loop 1, loop 2 and loop 3, respectively. Through mouse immunization and microneutralization assays, immunogenicity and protective efficacy of the chimeric No V P particle(trivalent HA2-PP) were tested against infection with IAV of three subtypes(H1N1, H3N2 and B) in MDCK cells. The protective efficacy of the trivalent HA2-PP was also evaluated preliminarily in vivo by virus challenge in the mouse model. The trivalent HA2-PP immunogen induced significant Ig G antibody responses(log104.39~5.15), which could be increased by virus booster vaccination. Besides, the trivalent HA2-PP immunogen also induced in vitro neutralization of H3 and B virus(ID50:45~656,P<0.05) and in vivo protection against H3 virus. Our results support that the broad protective vaccine approach using the HA2-based vaccine in the No V P particle platform can provide cross-protection against challenge viruses of different HA subtypes. The efficacy of the immunogen should be enhanced further to be practical, and a better understanding of the protective immune mechanism will be critical for the development of a HA2-based multivalent vaccine.In conclusion, we identified a conserved stem fragment with both B- and T-cell epitopes. The stem fragment, which is located in the turning point of the HA neck undergoing conformational change during membrane fusion, could induce a strong humoral immune response and broad neutralizing activities against strains of the H3 subtype in group 2 IAV. Most importantly, the potent neutralizing activity of the elicited antisera was carried out by inhibiting conformational change of the HA stem during membrane fusion. Based on this study, we evaluated the No V P particle vaccine platform expressing the HA2:90-105 sequence of influenza H1, H3 and B subtypes in the mouse model. The trivalent HA2-PP was found to be immunogenic when administered via the subcutaneous route with Freund’s adjuvant and could provide protective antibody responses as assessed in vitro and in vivo against H3 and B subtypes of IAV. Evaluating other possible innate and/or adaptive protection mechanisms induced by HA2 stem in the mouse model would be worthwhile in future work. With further improvement in the trivalent HA2-PP immunogen construct and optimization of the dose and vaccination strategy, this vaccine candidate has the potential to be applied in the clinic as one component of a universal vaccine.