| The influenza virus, the causative agent of flu and other acute respiratory infections, can spread in human populations. Based on the viral internal nucleoprotein (NP) structure, influenza viruses are divided into three different antigenic types, designated as A, B, and C. They are further divided into different subtypes based on two virus surface proteins called hemagglutinin (HA) and neuraminidase (NA). There are 16 HA subtypes and 9 NA subtypes. Three subtypes of influenza A, namely H1N1, H3N2, and H2N2, as well as influenza B mainly infect humans. Influenza may also be categorized as seasonal influenza and pandemic influenza. Pandemic influenza strain can spread worldwide and has a very high mortality rate. Avian influenza viruses mainly include subtypes H5, H7, and H9, of which H5N1, H7N7, and other avian influenza virus subtypes have crossed the species barrier, causing human infections and several deaths. This phenomenon could trigger the next influenza pandemic.Vaccination is an effective method for controlling the influenza virus. At present, the traditional influenza vaccine is widely used, but it has several limitations, including a long production period, a complicated production processes, high dependence on embryonated eggs, costly, and limited strain-specific protection. Multiple subtypes of influenza virus coexist but the vaccines provide weak cross-protection. Furthermore, sequences of the major protective antigen, HA, are quite different from each other. The development of a new influenza vaccine that can protect against different influenza virus subtypes and can be simply mass-produced independent of embryonated eggs is increasingly important. Currently, vaccine research mainly focuses on genetic engineering of new influenza vaccines, including recombinant subunit vaccines, virus-like particle vaccines, DNA vaccines, and recombinant viral vector vaccines.DNA vaccine and adenovirus vaccine offer good prospects for developing new influenza vaccine. Nucleic acid vaccines, also known as DNA vaccines, are relatively easy to prepare, safe, and stable. It also has long-term storage stability and provides non-carrier immunity and repeated immunity. DNA vaccines stimulate cellular and humoral immune responses. These advantages made DNA vaccines the focus of research. Compared with subunit vaccines and recombinant viral vector vaccines, DNA vaccines elicit a weaker immunogenic response. Vaccine efficacy can be enhanced by adding different types of adjuvants, electroporation-assisted vaccination, gene gun, and other methods. Adenoviruses have an excellent safety profile, a wide range of host cells, high viral titers, and high cell transduction efficiency, which can accommodate large segments of foreign genes. Furthermore, it is stable and can stimulate the body to produce strong cellular and humoral immune responses.A multi-epitope influenza vaccine against different influenza virus subtypes was prepared by screening the epitopes of major protective antigen genes. Different specific epitopes are connected by a linker to form the multi-epitope expression cassette, which is inserted alone or combined with other genes into the corresponding expression vector.In this study, the eukaryotic expression vector pVAX1 and the replication-defective human adenovirus type 5 vector constructs were used to design a multi-epitope DNA vaccine and adenovirus vaccine specifically against seasonal influenza H3 subtype and pandemic influenza H1 subtype HA (HA1). The genes of these subtypes were combine with the HA antigen Th and B cell epitopes cassette (EHA) of avian influenza subtypes such as H5, H7. and H9, which have crossed species barrier to infect humans, and single expression control groups were constructed. The components of antigen transcription and antigenicity were determined by reverse transcription polymerase chain reaction, indirect immunofluorescence, and western blot method. The results show that the constructed potential DNA vaccines and recombinant adenovirus vaccines effectively expressed components of antigen, thus, these are good vaccines.Potential DNA vaccines, which include pV-H3-EHA-H1HA1 new, pV-H1HA new, pV-H1HA1 new, pV-H3HA, and pV-EHA, were used to immunize mice through electroporation-assisted immunization. The levels of cellular and humoral immunity of each immunized group were determined. The results show that upon specific antigen stimulation, the group treated with the multiple epitope vaccine through electroporation-assisted immunization, had the same levels of humoral immunity as the single-expression immunized group, and had significantly higher levels than that of the group without electroporation-assisted immunization (P<0.05). In addition, the IgG antibody levels of the multi-epitope immunized group were approximately twice than that of the single-expression group upon antigen stimulation with the H5/H7/H9 epitope peptides (P<0.05). Moreover, groups treated with the multiple epitope vaccine through electroporation-assisted immunization had significantly higher IL-2 and IL-4 levels than that of the single-expression group and the non-electroporation groups (P<0.05). The multi-epitope immunization with electroporation also induced better immune protection (90%), which was significantly higher than that of the single-expression and the non-electroporation groups (P<0.05) based on the lethal dose challenge (10 LD50) with the H5 subtype influenza virus on immunized mice.Mice were immunized using potential recombinant influenza adenovirus vaccine: pacAd5-H3HA, pacAd5-H1HA1, pacAd5-H1HA1 new, pacAd5-H3-H1HA1, and pacAd5-H3-EHA-H1HA1 new. The results show that all immunized mice produced specific humoral and cellular immune response, in which the multi-epitope recombinant group had the highest level of immune response. Single and double immunization with the expressed H3HA recombinant adenovirus was used as the control. Based on the results, the single-immunized group induced considerable levels of immunity compared with the twice-immunized group. Immunized mice were challenged with a lethal dose of the H3 subtype of influenza virus 14 days after the booster immunization. The results show that the mice in the multi-epitope recombinant adenovirus group, the co-expressed H3-H1HA1 group, and the single expression group acquired complete protection, which were significantly higher than that produced in the H1 subtype single expression group and the control group (P<0.05). The body weight changes were relatively mild and quickly recovered among the mice in the multi-epitope recombinant adenovirus-immunized group.Combined immunity was achieved using DNA vaccine and recombinant vaccine based on the Prime-Boost Immunization Strategy. Prime immunization with the DNA vaccine and boost immunization with recombinant adenovirus vaccine were performed. The results show that the level of antibodies induced in the multi-epitope combined immunization group and the single combined immune-related group was not significantly different (P>0.05), but both were significantly higher than that of the single immunized with DNA vaccine and adenovirus vaccine (P<0.05). Furthermore, the multi-epitope combined immunized group exhibited a strong immune response, which was significantly higher than that of the single combined immunization group and the single-expression immunized group (P<0.05). Based on the H3/H5/H1 subtype influenza virus challenge experiment, the multi-epitope combined immunization group had the highest immune protection rates. The group was not only fully protected against the major antigenic components associated with the epitopes of the H3 and H5 subtypes, but also elicited strong cross-protective immunity against heterologous subtypes of seasonal H1N1 influenza viruses (90%) that were significantly higher than that in the combined immunization group and the single-expression immunized groups (P<0.05).In summary, the potential multi-epitope DNA vaccine and the recombinant adenovirus vaccine conferred strong humoral and cellular immunity in mice. The epitopes of the H5/H7/H9 subtypes provided enhanced immune protection against avian influenza virus subtypes that can cause cross-species infection. Combined immunization with multi-epitope vaccines effectively protects mice against heterologous subtype virus challenge. Multi-epitope DNA vaccine and adenovirus vaccine show good prospects in the study of universal influenza vaccine.
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