Induction of broadly neutralizing antibodies against measles virus mutants using a polyepitope vaccine strategy

Chimeric molecules expressing multiple copies of the loop-forming hemagglutinin noose epitope (designated as "L"; aa386-400), a protective B cell epitope of the measles virus were generated by recombinant technology. The recombinant polyepitope [L4T4]2 combining two sets of four repeats of the L epitope and with two sets of four repeats of the human promiscuous T cell epitope of tetanus toxoid ("T", tt830-844) was produced in transgenic carrot plants. After intraperitoneal immunization of mice with plant membrane extract, sera neutralized all wild-type viruses. In a modified plaque reduction neutralization assay based on CD150-transfected Vero cells anti-[L4T4]2 sera neutralized all field isolates, irrespective of mutations in the L epitope. Even viruses with a mutation in the contact residues of a neutralizing L-specific monoclonal antibody or two mutations in other positions of the epitope were equally sensitive to neutralization. These results suggest that the multiple copies of the L epitope fold into different conformations that induce a repertoire of B cells diverse enough to overcome the genetic diversity of field viruses.     

Plant-derived measles virus hemagglutinin protein induces neutralizing antibodies in mice

Measles remains a significant problem in both the developed and developing world, and new measles vaccination strategies need to be developed. This paper examines the strategy of utilizing transgenic plants expressing a measles antigen for the development of an oral sub-unit measles vaccine. A 1.8 kb fragment encompassing the coding region of the measles virus hemagglutinin (H) protein was cloned into a plant expression cassette. Three different expression constructs were tested: pBinH (H gene alone), pBinH/KDEL (addition of a C-terminal endoplasmic reticulum-retention sequence SEKDEL) and pBinSP/H/KDEL (further addition of an authentic N-terminal plant signal peptide). The highest levels of recombinant H protein production were observed in plants transformed with pBinH/KDEL. Mice inoculated intraperitoneally with transgenic plant derived recombinant H protein produced serum anti-H protein antibodies that neutralized the measles virus (MV) in vitro. Mice gavaged with transgenic tobacco leaf extracts also developed serum H protein-specific antibodies with neutralizing activity against MV in vitro. These results indicate that the plant-derived measles H protein is immunogenic when administered orally and that, with further development, oral vaccination utilizing transgenic plants may become a viable approach to measles vaccine development.     

A hemagglutinin-derived peptide-vaccine ignored by virus-neutralizing passive antibodies, protects against murine measles encephalitis.

The neutralizing and protective monoclonal antibody BH47 defines the sequential epitope H236-255 of the measles virus hemagglutinin protein (MV-H). The objective of this study was to design peptides combining this B cell epitope (BCE) with different T cell epitopes (TCE) to obtain protective immunity. Most TTB peptides based on the 15mer BCE H236-250 induced MV-crossreactive antibodies, but only certain TCE induced virus neutralizing antibodies. The shortest BCE required for MV-reactivity and -neutralization was the 8mer H243-250 containing residue R243 implicated in CD46 down-regulation. Sera obtained after immunization with the TTB peptide containing the MV-derived TCE F421-435 protected mice against a lethal challenge with a neuro-adapted MV strain. Our results further demonstrate that this TTB peptide is fully immunogenic, even in the presence of protective levels of pre-existing MV-specific antibodies, suggesting that subunit vaccines based on such peptides could potentially be used to immunize infants in the presence of persisting maternal antibodies. It is therefore interesting that neutralizing antibodies were also obtained with a TTB peptide comprising a human promiscuous TCE (tt830). However, our results also emphasize the need to test sera induced with epitope-based vaccines against different virus strains, in particular if the epitope is not fully conserved.     

A recombinant live attenuated measles vaccine vector primes effective HLA-A0201-restricted cytotoxic T lymphocytes and broadly neutralizing antibodies against HIV-1 conserved epitopes

Live attenuated measles vaccine (MV) could provide a safe and efficient pediatric vaccination vector to immunize children simultaneously against measles and human immunodeficiency virus type 1 (HIV-1). To evaluate the capacity of a vector derived from the certified Schwarz measles vaccine (MVSchw) to prime effective cytotoxic T cells (CTL) and broad neutralizing antibodies against HIV-1 conserved epitopes, we generated recombinant MVSchw viruses expressing HIV-1 antigens. We demonstrate that a recombinant MVSchw virus expressing an HIV-1-derived CTL polyepitope primes effective HLA-A0201-restricted CTLs against multiple conserved HIV-1 epitopes in mice susceptible to measles and humanized for the major histocompatibility complex (MHC) class-I molecule HLA-A0201. Additionally, we show that a recombinant MVSchw virus expressing an HIV-1(89.6) gp140 glycoprotein whose hyper variable V1, V2 and V3 loops were deleted (DeltaV1V2V3gp140), induces broadly neutralizing antibodies against HIV-1 primary isolates. These results show that the MVSchw pediatric vaccination vector induces efficient cellular and humoral HIV-specific immune responses.     

Immunogenicity and in vitro protective efficacy of a polyepitope Plasmodium falciparum candidate vaccine constructed by epitope shuffling

A polyepitope chimeric antigen incorporating multiple protective and conservative epitopes from multiple antigens of Plasmodium falciparum has been considered to be more effective in inducing multiple layers of immunity against malaria than a single stage- or single antigen-based vaccine. By modifying the molecular breeding approach to epitope shuffling, we have constructed a polyepitope chimeric gene that encodes 11 B-cell and T-cell proliferative epitope peptides derived from eight key antigens mostly in the blood stage of Plasmodium falciparum. A 35-kDa antigen encoded by this gene, named Malaria RCAg-1, was purified from an E. coli expression system. Immunization of rabbits and mice with the purified protein in the presence of Freund's adjuvant strongly generated long-lasting antibody responses that recognized the corresponding individual epitope peptide in this vaccine as well as blood stage parasites. CD4(+) T-cell responses were also elicited as shown by the enhancement of T-cell proliferation, IFN-gamma and IL-4 level. In vitro assay of protection revealed that vaccine-elicited antibodies could efficiently inhibit the growth of blood-stage parasites. Additionally, the chimeric antigen was recognized by human serum specimens from malaria patients and individuals living in the endemic area. Our studies indicate the potential of M.RCAg-1 recombinant protein as malaria candidate vaccines as well as the rationale of the epitope shuffling technology applied in designing malaria vaccines.     

Crossreactivity of mimotopes and peptide homologues of a sequential epitope with a monoclonal antibody does not predict crossreactive immunogenicity.

The sequence H236-256 of the measles virus (MV) hemagglutinin (H) contains the sequential epitope of the neutralizing and protective monoclonal antibody (mAb) BH129 with the minimal epitope E(245)L-QL(249). Using this mAb, we have recently developed 7mer mimotopes binding up to 135x better than the corresponding 7mer epitope H244-250. In this study, we combined T cell epitopes (TCE) with either highly crossreactive 7mer mimotopes, 13mer mimotopes or less crossreactive MV-derived B cell epitopes (BCE). Antigenicity of these TBB, TTB and TTBB peptides was determined with BH129 in a competition ELISA against MV. We found that chimeric peptides including mimotopes were up to 80x better binders to the mAb than peptides containing the original BCEs. All peptides irrespective of their antigenicity were used for immunization to compare their virus- crossreactive immunogenicity. Unexpectedly, none of the highly antigenic mimotope-based peptides induced MV-crossreactive antibodies. In contrast, a number of peptides with the viral BCE sequence that did not bind to the mAb, induced MV-crossreactive and even neutralizing antibodies.This report describes a striking example of disparity between antigenicity and crossreactive immunogenicity and casts considerable doubt on the predictive value of antigenicity in immunogenicity studies, considerably complicating the selection of potential vaccine candidates.     

Expression in plants and immunogenicity of plant virus-based experimental rabies vaccine

A new approach to the production and delivery of vaccine antigens is the use of engineered amino virus-based vectors. A chimeric peptide containing antigenic determinants from rabies virus glycoprotein (G protein) (amino acids 253-275) and nucleoprotein (N protein) (amino acids 404-418) was PCR-amplified and cloned as a translational fusion product with the alfalfa mosaic virus (AlMV) coat protein (CP). This recombinant CP was expressed in two plant virus-based expression systems. The first one utilized transgenic Nicotiana tabacum cv. Samsun NN plants providing replicative functions in trans for full-length infectious RNA3 of AlMV (NF1-g24). The second one utilized Nicotiana benthamiana and spinach (Spinacia oleracea) plants using autonomously replicating tobacco mosaic virus (TMV) lacking native CP (Av/A4-g24). Recombinant virus containing the chimeric rabies virus epitope was isolated from infected transgenic N. tabacum cv. Samsun NN plants and used for parenteral immunization of mice. Mice immunized with recombinant virus were protected against challenge infection. Based on the previously demonstrated efficacy of this plant virus-based experimental rabies vaccine when orally administered to mice in virus-infected unprocessed raw spinach leaves, we assessed its efficacy in human volunteers. Three of five volunteers who had previously been immunized against rabies virus with a conventional vaccine specifically responded against the peptide antigen after ingesting spinach leaves infected with the recombinant virus. When rabies virus non-immune individuals were fed the same material, 5/9 demonstrated significant antibody responses to either rabies virus or AlMV. Following a single dose of conventional rabies virus vaccine, three of these individuals showed detectable levels of rabies virus-neutralizing antibodies, whereas none of five controls revealed these antibodies. These findings provide clear indication of the potential of the plant virus-based expression systems as supplementary oral booster for rabies vaccinations.     

The rationale of a peptide-conjugate vaccine against measles

The live-attenuated measles vaccine is poorly immunogenic in infants because of immune suppressive maternal antibodies and immaturity of the infant's immune system. Selected peptides corresponding to sequential, subdominant B cell epitopes of measles virus (MV) glycoproteins have been shown to induce neutralizing and protective antibodies even in the presence of whole virus antibodies. Similar to polysaccharide-conjugate vaccines, which are highly effective in infants a peptide-conjugate vaccine against measles is proposed. Such a vaccine induces carrier-specific T cells, avoiding measles-specific Th2 cells associated with the risk of atypical measles. This article discusses the rationale of such a strategy and its future potential.     

Humoral immune responses to a protective peptide-conjugate against measles after different prime-boost regimens

The current live-attenuated measles vaccine leaves many children unprotected until they reach the recommended age of vaccination. We have previously shown that the short peptide corresponding to the hemagglutinin noose epitope (HNE) of the measles virus (MV) hemagglutinin protein induced virus-neutralizing antibodies even in the presence of protective levels of anti-whole virus-specific antibodies. Here we investigate the immunogenicity of HNE peptide-conjugates of diphtheria or tetanus toxoid in mice after active and passive priming with antibodies against the peptide, toxoids and conjugates. Both conjugates induced high titers of peptide antibodies which crossreacted with the virus and protected against a lethal intracranial challenge with a rodent-adapted measles virus, even after active priming with homologous or heterologous toxoid or conjugate. Peptide-specific epitopic suppression was stronger after passive priming with carrier or conjugate antibodies, but diphtheria toxoid as a carrier was less susceptible to suppression than tetanus toxoid and suppression was overcome by an additional boost. Furthermore, prior immunization with peptide-conjugate did not interfere with the development of a complete response to a subsequent injection of MV, suggesting that the benefits of a follow-up vaccination with the current live-attenuated vaccine would not be lost. These results underline the potential of these peptide-based conjugates as vaccine candidates for use in early infancy to close the window of susceptibility before the live-attenuated vaccine can be administered.     

Immunocompetent truncated E2 glycoprotein of bovine viral diarrhea virus (BVDV) expressed in Nicotiana tabacum plants: a candidate antigen for new generation of veterinary vaccines

The bovine viral diarrhea virus (BVDV) is the etiological agent responsible for a wide spectrum of clinical diseases in cattle. The glycoprotein E2 is the major envelope protein of this virus and the strongest inductor of the immune response. There are several available commercial vaccines against bovine viral diarrhea (BVD), which show irregular performances. Here, we report the use of tobacco plants as an alternative productive platform for the expression of the truncated version of E2 glycoprotein (tE2) from the BVDV. The tE2 sequence, lacking the transmembrane domain, was cloned into the pK7WG2 Agrobacterium binary vector. The construct also carried the 2S2 Arabidopsis thaliana signal for directing the protein into the plant secretory pathway, the Kozak sequence, an hexa-histidine tag to facilitate protein purification and the KDEL endoplasmic reticulum retention signal. The resulting plasmid (pK-2S2-tE2-His-KDEL) was introduced into Agrobacterium tumefaciens strain EHA101 by electroporation. The transformed A. tumefaciens was then used to express tE2 in leaves of Nicotiana tabacum plants. Western blot and ELISA using specific monoclonal antibodies confirmed the presence of the recombinant tE2 protein in plant extracts. An estimated amount of 20 μg of tE2 per gram of fresh leaves was regularly obtained with this plant system. Injection of guinea pigs with plant extracts containing 20 μg of rtE2 induced the production of BVDV specific antibodies at equal or higher levels than those induced by whole virus vaccines. This is the first report of the production of an immunocompetent tE2 in N. tabacum plants, having the advantage to be free of any eventual animal contaminant.     

A novel methodology to develop a foot and mouth disease virus (FMDV) peptide-based vaccine in transgenic plants.

The expression of antigens in transgenic plants has been increasingly used as an alternative to the classical methodologies for antigen expression in the development of experimental vaccines. However, an important limitation in most cases is the low concentration of the recombinant antigens in the plant tissues, which reduces the possibilities of practical applications. Because the site of insertion of the transferred DNA into the cellular chromosomal DNA is at random, different levels of foreign protein expression in independent transformants is expected. Strategies to allow the evaluation of a high number of the transgenic individuals, usually an expensive and very time consuming process, would permit the selection of those plants presenting the highest levels of recombinant protein expression. Here, we present the development of an experimental immunogen based in the expression of a highly immunogenic epitope from foot and mouth disease virus (FMDV) fused to the glucuronidase (gus A) reporter gene, which allows selection of the transgenic plants by the ss-glucuronidase (ssGUS) enzymatic activity. We produced transgenic plants of alfalfa expressing the immunogenic site between amino acid residues 135-160 of structural protein VP1 (VP135-160), fused to the ssGUS protein. Plants expressing the highest levels of the immunogenic epitope VP135-160, analyzed by Western blot, were efficiently selected based on their levels of ssGUS enzymatic activity. The FMDV epitope expressed in plants was highly immunogenic in mice which developed, after immunization, a strong anti-FMDV antibody response against a synthetic peptide representing the region VP135-160, to native virus VP1, and to purified FMDV particles. Additionally, these mice were completely protected against experimental challenge with the virulent virus. To our knowledge, this constitutes the first report of a peptide-based vaccine produced in transgenic plants that induces a protective immune response when used in experimental hosts. Also, these results demonstrated the possibility of using a novel and simple methodology for obtaining transgenic plants expressing high levels of foreign immunogenic epitopes, which could be directly applied in the development of plant-based vaccines.     

Immunogenicity of peptides of measles virus origin and influence of adjuvants

Epitope-based peptide antigens have been under development for protection against measles virus. The immunogenicity of five peptides composed of the same B cell epitope (BCE) (H236-250 of the measles virus hemagglutinin), and different T cell epitopes of measles virus fusion protein (F421-435, F256-270, F288-302) and nucleoprotein (NP335-345) was studied in mice (subcutaneous immunisation). The adjuvant effects of peptidoglycan monomer (PGM), Montanide ISA 720 and 206 were also investigated. Results showed basic differences in peptide immunogenicity that were consistent with already described structural differences. PGM elevated peptide-specific IgG when applied together with four of five tested peptides. A strong synergistic effect was observed after co-immunisation of mice with a mixture containing all five chimeric peptides in small and equal amounts. Results revealed for the first time that immunisation with several peptides having the common BCE generated significantly higher levels of both anti-peptide and anti-BCE IgG in comparison to those obtained after immunisation with a single peptide in much higher quantity. Further improvement of immune response was obtained after incorporation of such a peptide mixture into oil-based adjuvants.     

Immunogenicity of polyepitope libraries assembled by epitope shuffling: an approach to the development of chimeric gene vaccination against malaria

Developing a polyepitope vaccine which contains diverse antigenic types is a promising strategy to cope with the problem of malaria variation and diversity. However, arranging the peptides to produce the most effective immunogenicity remains a hurdle. In an attempt to develop an effective complex antigenic gene vaccine, we constructed a polyepitope library by randomly assembling epitopes using the epitope shuffling technique. The polyepitope library, which contains epitopes from different antigens of Plasmodium falciparum, was divided into five sub-libraries based on the size of chimeric genes. Here we report that higher antibody titers were observed in mice with immunized with sub-libraries containing genes >1200 bp, using enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IFAT) assay to determine both individual epitope peptides and the natural parasites at the erythrocyte stage. Different levels of IgG subtypes and cytokines were elicited by different sub-library and administration times. In a rodent malaria model, some groups of immunized mice were partially cross-protected against a lethal challenge from Plasmodium yoelii. These results suggest that the immunogenicity of a polyepitope chimeric antigen is essentially conformation- and length-dependent, and demonstrates that the promising advantage of epitope shuffling technology is that it allows us to randomly assemble many polyepitope molecules in tandem format. This finding also indicates that polyepitope library vaccination is a suitable approach for screening optimized chimeric gene vaccines against malaria and other diseases.     

Expression of the synthetic neutralizing epitope gene of porcine epidemic diarrhea virus in tobacco plants without nicotine

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea and dehydration in pigs, leading to death with a high mortality. In this study, the synthetic neutralizing epitope gene of PEDV, which was optimized based on the coding sequence of tobacco plant genes being modified, was expressed in the no-nicotine tobacco plants. The synthetic gene was cloned into the plant expression vector under the control of CaMV 35S promoter and transformed by an Agrobacterium-mediated transformation. The amount of synthetic epitope protein of PEDV detected in the transgenic tobacco plants was approximately 2.1% of the total soluble plant protein, which was approximately five-fold higher than that expressed with the native gene.     

Induction of antigen-specific systemic and mucosal immune responses by feeding animals transgenic plants expressing the antigen

A report from that the presence of lactogenic immunity in pigs protected suckling piglets from porcine epidemic diarrhea virus (PEDV) infection suggested that inducing mucosal immune responses in lactating pigs is an effective way of protecting swine from PEDV infection. In this study, we developed transgenic tobacco plants that express the antigen protein corresponding to the neutralizing epitope of PEDV spike protein, and tested whether feeding the plants to pigs induced an effective immune response against PEDV infection. First, we confirmed the immunogenicity of the plant-derived antigen by using a plaque reduction neutralization assay with serum obtained after injecting mice with protein extracted from the transgenic plants. Feeding the transgenic plants to mice induced both systemic and mucosal immune responses against the antigen. The induced antibodies inhibited virus infection in the plaque reduction neutralization assay. These results suggest that feeding animals transgenic plants carrying antigen genes is an effective strategy to induce protective immune responses against PEDV infection.     

Immunogenicity of polyepitope libraries assembled by epitope shuffling: an approach to the development of chimeric gene vaccination against malaria

Developing a polyepitope vaccine which contains diverse antigenic types is a promising strategy to cope with the problem of malaria variation and diversity. However, arranging the peptides to produce the most effective immunogenicity remains a hurdle. In an attempt to develop an effective complex antigenic gene vaccine, we constructed a polyepitope library by randomly assembling epitopes using the epitope shuffling technique. The polyepitope library, which contains epitopes from different antigens of Plasmodium falciparum, was divided into five sub-libraries based on the size of chimeric genes. Here we report that higher antibody titers were observed in mice with immunized with sub-libraries containing genes >1200 bp, using enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IFAT) assay to determine both individual epitope peptides and the natural parasites at the erythrocyte stage. Different levels of IgG subtypes and cytokines were elicited by different sub-library and administration times. In a rodent malaria model, some groups of immunized mice were partially cross-protected against a lethal challenge from Plasmodium yoelii. These results suggest that the immunogenicity of a polyepitope chimeric antigen is essentially conformation- and length-dependent, and demonstrates that the promising advantage of epitope shuffling technology is that it allows us to randomly assemble many polyepitope molecules in tandem format. This finding also indicates that polyepitope library vaccination is a suitable approach for screening optimized chimeric gene vaccines against malaria and other diseases.     

Deconstructing the antigenic profile of a protective epitope from measles virus fusion protein using overlapping peptides.

Three different approaches of using overlapping peptides have been compared to analyse the fine specificity of the antibody response to a protective epitope from measles virus (MV) fusion protein, spanning residues 397-420. Anti-peptide antibodies raised in BALB/c, CBA and C57BL/6 mice were shown to react with the homologous peptide and the MV by ELISA. Results from indirect ELISA using 15mer peptides (overlapping by one residue) as solid phase antigens have shown that anti-peptide antibodies from CBA and C57BL/6 mice recognised the same B-cell epitope(s) located within the 398-414 region, whereas BALB/c mice predominantly recognised epitopes located within the 400-417 region. When the 15mer peptides were used as fluid phase antigens in an inhibition ELISA, peptide 405-419 was shown to be the most effective inhibitor in all three strains of mice. Analysis of serum samples by SPOTs ELISA has shown that the region 407-417 was predominantly recognised by BALB/c mice, whereas antibodies from C57BL/6 mice recognised the 408-420 region. No reactivity was observed with serum samples from CBA mice.Although the majority of the identified B-cell epitopes were shown to overlap by the three methods, the identified boundaries of these epitopes differed, suggesting that the size and the mode of peptide presentation affects their antigenicity.     

Production of dengue 2 envelope domain III in plant using TMV-based vector system

The envelope protein of dengue virus is the major protein involved in host cell receptor binding for viral entry and induction of immunity. A gene fragment encoding domain III of the dengue 2 envelope protein (D2EIII, amino acids 298-400) was successfully expressed in Nicotinana benthamiana plant using a tobacco mosaic virus (TMV)-based transient expression system. The N-terminal 5' untranslated region-omega sequence located upstream of D2EIII increased protein production in infected plant tissues. The recombinant protein was reactive with anti-D2EIII polyclonal and anti-His tag antibodies. The intramuscular immunization of mice with D2EIII induced the production of the anti-dengue virus antibody. The induced antibody demonstrated neutralizing activity against dengue type 2 virus. The result indicates that the TMV expression system produces the dengue virus antigen in plant, which possesses appropriate antigenicity and immunogenicity.     

麻疹病毒抗原性变异及免疫保护效果研究

目的 了解浙江省麻疹野毒株的抗原性变异状况以及麻疹疫苗的免疫保护效果。方法 采用麻疹国际标准株Edmonston株、疫苗株沪19l与近几年浙江省分离的麻疹野毒株(浙98-5和浙00-4)分别制备免疫血清，与各毒株进行交叉中和试验，并分别对不同来源的人群血清测定中和抗体滴度。结果 麻疹野毒株浙98-5与疫苗株沪川和Edm株的抗原比分别为3．0和2．6，浙00-4与上述两毒株的抗原比分别为7．30和5．65，其抗原性存在着明显的差异。麻疹疫苗初免儿童和麻疹急性期病人血清对麻疹野毒株的中和抗体几何平均滴度(GMT)分别为15．03和5．04，明显低于疫苗株沪19l，GMT分别为68．12和11．76。但健康人群血清对两毒株的中和抗体滴度无显性差异。结论 浙江省麻疹野毒株已出现较明显的抗原性变异，现行麻疹疫苗的免疫保护效果也受到一定影响。     

麻疹病毒血凝素蛋白抗原表位的预测与分析

目的 探讨麻疹病毒(MeV)流行株血凝素蛋白(H)抗原表位上氨基酸(aa)变异对病毒抗原性的可能影响。方法 利用生物信息学软件预测MeV H蛋白上B细胞线性表位,设计并合成来源于疫苗株和流行株表位以及同一区域非表位上的多肽对。间接ELISA法检测合成多肽的免疫原性,并制备多肽免疫血清。采用交叉ELISA法分析两条多肽间的抗原性差异,计算抗原比。结果 合成的多肽均能与MeV免疫血清结合,其中设计在表位区的多肽对CW23/CW22(273～282 aa)结合能力最强,而非表位区多肽对CW150/CW151(418～427 aa)结合能力最弱。多肽对中来源不同两条多肽间抗原性差异较大,其中CW23(疫苗株来源)与CW22(流行株来源)间抗原比为16,CW123(疫苗株来源)与CW124(流行株来源)(236～246 aa)间的抗原比为2.877±0.583。非表位多肽对中,CW125与CW126(356～364 aa)间抗原比为1.631±0.481,而CW150与CW151间抗原比为10.367±1.617。结论 麻疹流行株上仍存在保守的抗原表位,但预测的抗原表位及非表位区上的部分aa变异导致疫苗株与流行株间抗原性存在差异。