Recombinant protein comprising multi-neutralizing epitopes induced high titer of antibodies against influenza A virus

In previous studies, we suggested that epitope-vaccine might be a new strategy against virus infection. Based on this hypothesis, we designed and expressed a recombinant immunogen (multi-epitope-peptide) comprising repeats of three neutralizing-epitopes (neutralizing epitopes: aa92-105, 127-133 and 183-195) of hemagglutininin (HA) of influenza virus (H3N2) in E. coli. After vaccination, the recombinant multi-epitope protein could induce a high level of antibodies with predefined multi-epitope-specificity in mice and rabbits. The epitope-specific antibodies in sera were tested using three different epitope-peptides (synthetic peptides) in ELISA assay, and the serum dilutions from 1 : 6400 to 1 : 25600 were confirmed. In western blot analysis, both the antiserum and the antibodies purified by synthetic epitope-peptide coupled sepharose columns could recognize natural HA from influenza virus particles (strain A/Wuhan/359/95 H3N2). In hemagglutination inhibition (HI) tests, these three antisera at the dilutions from 1 : 20 to 1 : 80 showed inhibitory activity. Interestingly, antisera and purified antibodies induced by the epitope-vaccine could partially inhibit plaque-formation of influenza virus (strain A/Wuhan/359/95) on MDCK cell monolayers. These results suggest that the recombinant multi-epitope vaccine can simultaneously induce multi-antiviral activities against influenza virus, which may provide a new way to develop effective vaccines against influenza virus.     

Immunogenicity of neutralizing epitopes on multiple-epitope vaccines against HIV-1

Based on our hypothesis that epitope vaccine may be a new strategy to induce high levels of neutralization antibodies against HIV-1, we prepared multiple-epitope vaccines using three neutralizing epitopes (GPGRAFY, RILAVERYLKD and ELDKWA) of HIV-1 gp160, and characterized their immunogenicity. Peptide 1 [C-G-(ELDKWA-GPGRAFY)(2)-K] and peptide 2 (CG-GPGRAFY-ELDKWA-G-RILAVERYLKD) were synthesized and conjugated with carrier protein bovine serum albumin (BSA). After vaccination antibody responses to these immunogens were induced and evaluated by ELISA. The C-G-(ELDKWA-GPGRAFY)(2)-K-BSA (BSA: carrier protein) multiple-epitope vaccine induced a strong antibody response to the C-G-(ELDKWA-GPGRAFY)(2)-K peptide (antibody titer: 1:25,600) and C-(ELDKWAG)(4) peptide (antibody titer: 1:12,800), but a weak antibody response to the C-(GPCGRAFY)(4) peptide. The CG-GPGRAFY-ELDKWA-G-RILAVERYLKD-K-BSA (BSA: carrier protein) multiple-epitope vaccine also induced strong antibody response to the CG-GPGRAFY-ELDKWA-G-RILAVERYLKD-K peptide (antibody titer: 1:25, 600) and C-(ELLDKWAG)(4) peptide (antibody titer: 1:6,400), a very strong response to C-(RIVALVERYLKD-G)(2)-K peptide (dilution: 1:102, 400), and a very weak response to the C-(GPGRAFY)(4) peptide (dilution: 1:400) in mice. Both antisera induced by both multiple-epitope vaccines interacted with the recombinant soluble gp41 (rgp41), but did not bind two control peptides. In comparison with both epitope vaccines, the rgp160 subunit vaccine could induce weak epitope-specific antibody response to these three epitopes on the three epitope peptides and V3, N-domain and C-domain peptides (dilution: 1:400-1:1,600). These results indicate that both multiple-epitope vaccines could induce high levels of antibodies to both neutralizing epitopes RILAVERYLKD and ELDKWA, while the GPGRAFY epitope on both vaccines appeared to have weak immunogenicity. Both multiple-epitope vaccines showed significant potency on inducing high levels of epitope-specific neutralization antibodies in comparison with rgp160 subunit vaccine.     

Differences in antibody responses of mice to intranasal or intraperitoneal immunization with influenza A virus and vaccination with subunit influenza vaccine

Two antigenically related but different influenza A virus strains of H3N2 subtype, A/Dunedin/ 4/73 (H3N2) (Dunedin) and A/Mississippi/1/85 (H3N2) (Mississippi), were used for intranasal (i.n.) and intraperitoneal (i.p.) immunization of mice and respective antibody responses were compared. In ELISA, using purified influenza A virus as antigen, the highest titer of antiviral antibodies was observed after a repeated i.n. infection, in which the Dunedin strain was followed by the Mississippi strain and vice versa. Similarly, in virus neutralization (VN) test, the highest titer of VN antibodies was found after a repeated i.n. infection. The subunit vaccine INFLUVAC, when administered intramuscularly (i.m.), induced only a poor antibody response as assayed by ELISA. Moreover, the INFLUVAC vaccination elicited a 100-fold lower titer of VN antibodies than the i.n. infection and an approx a 10-fold lower titer than the i.p. immunization. A repeated INFLUVAC vaccination did not lead to a significant increase of VN antibody titer. Also the antibody response to HA2gp--a conserved part of influenza hemagglutinin (HA) that might contribute to the induction of specific antiviral antibodies--was followed. Similarly to the VN antibody response, the highest HA2 antibody titer was induced after a repeated i.n. infection, whereas the lowest HA2 antibody titer was observed after a single or repeated INFLUVAC vaccination. Overall, the HA2 antibody titers remarkably well corresponded to the VN potential of the examined sera.     

Elicitation of broadly neutralizing influenza antibodies in animals with previous influenza exposure

The immune system responds to influenza infection by producing neutralizing antibodies to the viral surface protein, hemagglutinin (HA), which regularly changes its antigenic structure. Antibodies that target the highly conserved stem region of HA neutralize diverse influenza viruses and can be elicited through vaccination in animals and humans. Efforts to develop universal influenza vaccines have focused on strategies to elicit such antibodies; however, the concern has been raised that previous influenza immunity may abrogate the induction of such broadly protective antibodies. We show here that prime-boost immunization can induce broadly neutralizing antibody responses in influenza-immune mice and ferrets that were previously infected or vaccinated. HA stem-directed antibodies were elicited in mice primed with a DNA vaccine and boosted with inactivated vaccine from H1N1 A/New Caledonia/20/1999 (1999 NC) HA regardless of preexposure. Similarly, gene-based vaccination with replication-defective adenovirus 28 (rAd28) and 5 (rAd5) vectors encoding 1999 NC HA elicited stem-directed neutralizing antibodies and conferred protection against unmatched 1934 and 2007 H1N1 virus challenge in influenza-immune ferrets. Indeed, previous exposure to certain strains could enhance immunogenicity: The strongest HA stem-directed immune response was observed in ferrets previously infected with a divergent 1934 H1N1 virus. These findings suggest that broadly neutralizing antibodies against the conserved stem region of HA can be elicited through vaccination despite previous influenza exposure, which supports the feasibility of developing stem-directed universal influenza vaccines for humans.     

H1亚型流感病毒HA蛋白单克隆抗体的制备及部分特性鉴定

目的:制备针对H1亚型流感病毒HA蛋白的单克隆抗体(mAb),并分析其反应特性。方法:分别以2009年甲型H1N1、季节性A1流感病毒裂解疫苗为免疫原,常规法免疫、融合、克隆化,获得各抗原特异性mAb。应用ELISA、HI试验和Western blot等技术研究mAb的反应性和特异性。结果:获得稳定分泌抗H1亚型流感病毒HA蛋白的杂交瘤细胞97株。其中株特异性mAb39株,29株具有HI活性;亚型特异性mAb7株,5株具有HI活性;2009年流行株与季节性A1、A3流行株共同抗原的mAb16株,9株具有HI活性;针对流感病毒共同抗原mAb35株,22株具有HI活性。结论:两种疫苗均具有较好的免疫原性和免疫保护活性,这些mAb的获得为流感病毒株特异、亚型特异性诊断试剂盒及流感病毒通用诊断试剂盒的制备提供了实验资料,为进一步研究H1N1流感病毒HA的抗原表位奠定了基础。     

An MDCK Cell Culture-Derived Formalin-Inactivated Influenza Virus Whole-Virion Vaccine from an Influenza Virus Library Confers Cross-Protective Immunity by Intranasal Administration in Mice

It is currently impossible to predict the next pandemic influenza virus strain. We have thus established a library of influenza viruses of all hemagglutinin and neuraminidase subtypes and their genes. In this article, we examine the applicability of a rapid production model for the preparation of vaccines against emerging pandemic influenza viruses. This procedure utilizes the influenza virus library, cell culture-based vaccine production, and intranasal administration to induce a cross-protective immune response. First, an influenza virus reassortant from the library, A/duck/Hokkaido/Vac-3/2007 (H5N1), was passaged 22 times (P22) in Madin-Darby canine kidney (MDCK) cells. The P22 virus had a titer of >2 ×10⁸ PFU/ml, which was 40 times that of the original strain, with 4 point mutations, which altered amino acids in the deduced protein sequences encoded by the PB2 and PA genes. We then produced a formalin-inactivated whole-virion vaccine from the MDCK cell-cultured A/duck/Hokkaido/Vac-3/2007 (H5N1) P22 virus. Intranasal immunization of mice with this vaccine protected them against challenges with lethal influenza viruses of homologous and heterologous subtypes. We further demonstrated that intranasal immunization with the vaccine induced cross-reactive neutralizing antibody responses against the homotypic H5N1 influenza virus and its antigenic variants and cross-reactive cell-mediated immune responses to the homologous virus, its variants within a subtype, and even an influenza virus of a different subtype. These results indicate that a rapid model for emergency vaccine production may be effective for producing the next generation of pandemic influenza virus vaccines.     

Generation and characterization of an H5N1 avian influenza virus hemagglutinin glycoprotein pseudotyped lentivirus

An H5N1 avian influenza virus (AIV) hemagglutinin (HA) protein pseudotyped lentivirus, HIV/H5-HA, was generated, characterized in vitro and evaluated for its ability to induce protective immunity against virulent wild type AIV in mice. The HIV/H5-HA virus was able to infect 293T, BHK, Vero, PK-15, MDCK cells but not IBRS-2 cells and therefore demonstrated cell tropism similar to the wild type AIV. HIV/H5-HA agglutinated chicken erythrocytes and cell entry was blocked by ammonium chloride, indicating that the process is pH-dependent. In mice, HIV/H5-HA immunization resulted in low levels of virus in the lungs, elicited high levels of AIV HA-specific antibody as indicated by the hemagglutination inhibition (HI) test, and the antibody induction was both earlier and with a higher titer than that induced by the inactivated AIV vaccine. These results confirmed the roles played by HA in AIV infection and immunogenicity and suggested that the pseudotyped lentivirus is a good model for studying the functions of AIV HA.     

Influenza A (H1N1) vaccine efficacy in animal models is influenced by two amino acid substitutions in the hemagglutinin molecule

The immunogenicity and protective efficacy of formalin-inactivated vaccines prepared from influenza A (H1N1) viruses grown in MDCK cells and in eggs was compared in animal models. The A/Chr/157/83 virus grown in MDCK cells (157M) differed by two amino acid substitutions in the HA molecule from the corresponding virus grown in eggs (157E) and the two viruses could be distinguished antigenically by monoclonal and polyclonal antibodies. Following two intramuscular injections of vaccine in ferrets, guinea pigs, and hamsters, both vaccines were equally immunogenic when antibody was analyzed by hemagglutination inhibition using homologous virus. However, single radial hemolysis analysis following antibody cross-adsorption showed that antibody stimulated by 157E vaccine was exclusively strain specific whereas that produced by the 157M vaccine was more broadly reactive. When immunized hamsters were challenged with virus cultivated on mammalian (MDCK) cells, the homologous vaccine induced a higher degree of protection than the corresponding egg-grown vaccine.     

Distinct contributions of vaccine-induced immunoglobulin G1 (IgG1) and IgG2a antibodies to protective immunity against influenza

Vaccination represents the most effective form of protection against influenza infection. While neutralizing antibodies are typically measured as a correlate of vaccine-induced protective immunity against influenza, nonneutralizing antibodies may contribute to protection or amelioration of disease. The goal of this study was to dissect the individual contributions of the immunoglobulin G1 (IgG1) and IgG2a antibody isotypes to vaccine-induced immunity against influenza virus. To accomplish this, we utilized an influenza vaccine regimen that selectively enhanced IgG1 or IgG2a antibodies by using either DNA or viral replicon particle (VRP) vectors expressing influenza virus hemagglutinin (HA) (HA-DNA or HA-VRP, respectively). After HA-DNA vaccination, neutralizing antibodies were detected by both in vitro (microneutralization) and in vivo (lung viral titer) methods and were associated with increased IgG1 expression by enzyme-linked immunosorbent assay (ELISA). Vaccination with HA-VRP did not strongly stimulate either neutralizing or IgG1 antibodies but did induce IgG2a antibodies. Expression of IgG2a antibodies in this context correlated with clearance of virus and increased protection against lethal influenza challenge. Increased induction of both antibody isotypes as measured by ELISA was a better correlate for vaccine efficacy than neutralization alone. This study details separate but important roles for both IgG1 and IgG2a expression in vaccination against influenza and argues for the development of vaccine regimens that stimulate and measure expression of both antibody isotypes.     

Epitope-vaccines: a new strategy to induce high levels of neutralizing antibodies against HIV-1

Based on the experimental evidence that gp120 subunit vaccine did not protect individuals from HIV-1 infection, we suggested that epitope-vaccines of HIV-1 gp41 may be a new strategy to induce high levels of neutralizing antibodies against HIV-1, and characterised immunogenicity of epitope-vaccines. Two epitopes, RILAVERYLKD-epitope (aa586-596) on the N-domain and ELDKWA-epitope (aa669-674) on the C-domain of gp41, were demonstrated by us and others to induce protective activity. After vaccination course, the RILAVERYLKD-dimer epitope-vaccine [C(RILAVERYLKDG)2-BSA] induced strong epitope-specific antibody response by about 1:25,600 dilution, and the ELDKWA-tetramer epitope-vaccine [C-(ELDKWAG)4-BSA] could yet induce strong antibody response to ELDKWA-epitope by 1:12,800-25,600 dilution of antisera in mice, while rgp41 subunit vaccine induced very weak antibody response to both epitopes (1:400). In rabbit experiments, the titres of ELDKWA-epitope-specific antibody induced by ELDKWA-epitope-vaccine [C-(ELDKWAG)4-BSA] reached to 1:6,400, while rgp41 subunit vaccine induced very weak antibody response to this epitope and to P1 and P2 peptides (1:400). Moreover, the ELDKWA-epitope-specific antibodies in mice and rabbit antisera induced by epitope-vaccine could very strongly interact with P2 peptide sequence-corresponding to the C-domain of gp41 (dilution by 1:25,600), and the RILAVERYLKD-epitope-specific antibodies in mice antisera induced by epitope-vaccine could also very strongly interact with P1 peptide sequence-corresponding to the N-domain of gp41 (dilution by 1:102,400). All these results provided experimental evidence that epitope-vaccine may be a new general strategy to induce high levels of neutralizing antibodies against HIV-1 or other viruses.     

Antibodies to HA and NA augment uptake of influenza A viruses into cells via Fc receptor entry

The hemagglutinin (HA) and neuraminidase (NA) of influenza A viruses induce antibodies which augment the uptake of influenza A virus by antigen presenting cells via Fc receptor entry. Antibody-dependent enhancement of uptake of virus by cells was mediated by Fc receptors because F(ab')2 preparations of lgG mixed with virus did not enhance virus uptake. The enhanced infection was measured using a fluorescent focus assay and was confirmed by dot-blot hybridization analysis. A 25-fold increase in the number of cells containing influenza antigens was detected when virus was mixed with subneutralizing concentrations of immune serum to the homologous virus before adding to neuraminidase-treated cells. Infection was also augmented using reassortant viruses which shared only the HA or the NA of the virus used to induce antibodies. Specific antisera to purified HA or NA also enhanced virus uptake. These results indicate that both the HA and the NA induce antibodies that enhance uptake of virus by Fc receptor bearing cells. In addition we determined that the drift of neutralizing antigens occurred more quickly than the drift of infection-enhancing antigens during the evolution of virus strains of the H3 subtype. The increase in the number of antigen presenting cells as a result of uptake of virus complexed with cross-reactive enhancing antibodies may affect the T cell responses to influenza infection.     

Comparison of protection against H5N1 influenza virus in mouse offspring provided by maternal vaccination with HA DNA and inactivated vaccine

H5N1 influenza virus is one of the viruses that can potentially cause an influenza pandemic. Protection of newborns against influenza virus infection could be effectively provided by maternal immunization. In this study, female mice were immunized with H5N1 HA DNA vaccine or inactivated whole-virion vaccine, and the protection provided by maternal antibodies in their offspring against a lethal homologous influenza virus challenge was compared. The results showed that maternal antibodies, whether induced by a DNA vaccine or an inactivated vaccine, could completely protect offspring aged 1-4 weeks from a lethal influenza virus challenge. Breast-feeding was the major route of transfer for maternal antibodies. Milk-derived antibodies were able to effectively protect the offspring aged 1-4 weeks from lethal influenza virus infection, whereas maternal antibodies transferred through the placenta only partially protected the offspring 1-2 weeks of age. The milk- and placenta-transferred IgG2a antibody levels in offspring from their mothers, whether vaccinated with DNA vaccine or inactivated vaccine, were higher than the IgG1 levels. Our results indicated that maternal vaccination with HA DNA, as well as with whole-virion inactivated vaccine, could offer effective protection to offspring against H5N1 influenza virus infection.     

Antibody responses against epitopes on the F protein of bovine respiratory syncytial virus differ in infected or vaccinated cattle

Summary.  The fusion protein F of bovine respiratory syncytial virus (BRSV) is an important target for humoral and cellular immune responses, and antibodies against the F protein have been associated with protection. However, the F protein can induce antibodies with different biological activity, possibly related to distinct antigenic regions on the protein. Therefore, epitopes were mapped on the F protein using monoclonal antibodies. Two epitopes (A and B) were identified that induced neutralizing antibodies, and one epitope (C) that did not elicit neutralizing antibodies. Subsequently, antibody responses were analysed against these epitopes in cattle sera after natural infection, experimental infection or vaccination. After natural infection or reinfection, the antibody titres against epitope A were significantly higher than those against epitope B or C. After experimental infection and after vaccination with an inactivated vaccine, antibody titres against epitope B and C were significantly higher than after natural infection. Conversely, virus neutralizing antibody titres were significantly lower in these animals with higher antibody titres against epitopes B and C than in naturally infected cattle. Because after natural infection the epitope-specific-antibody titres against epitope A, B or C differed markedly between the cattle, the magnitude of the antibody titres against epitope A, B or C in relation to the major histocompatibility complex (MHC) genes of cattle (BoLA) was studied. The magnitude of the antibody responses against epitope A of the F protein, but not against the G protein, appeared to be associated with the bovine lymphocyte antigen (BoLA) haplotype. Received February 4, 1997 Accepted July 4, 1997     

Induction of high levels of antibodies recognizing the neutralizing epitope ELDKWA and the D- or K-position-mutated epitopes by candidate epitope vaccines against HIV-1

Monoclonal antibody 2F5 recognizing the ELDKWA epitope on HIV-1 gp41 has a significant neutralization potency against 90% of the investigated viruses of African, Asian, American, and European strains, but the antibody responses to the epitope 2F5 in HIV-1-infected individuals were very low. We attempted to induce high levels of epitope-specific antibodies to ELDKWA and its three mutated epitopes by candidate epitope vaccines. The four candidate epitope vaccines all induced strong antibody responses at dilutions from about 1:6,400 to 1:25,600. We tested the cross-reactions between these antisera and four epitope peptides. The ELDKWA-specific antisera showed strong cross-reactivity with three neutralizing-resistant mutated epitopes which contain changes in the D or K positions of the epitope sequence. Virus variants containing these changes could escape neutralization by monoclonal antibody 2F5. In immunoblotting analysis, the ELDKWA, ELDEWA, and ELEKWA epitope specific antibodies all recognized rsgp41 which confirms that the antibodies against both mutated epitopes, ELDEWA and ELEKWA, could cross-react with the native epitope on rsgp41. Although it is not clear whether the polyclonal antibodies induced by the ELDKWA epitope vaccine could neutralize the mutated viruses containing these mutated epitopes, it is conceivable that epitope vaccines based on mutated epitopes could induce strong antibody responses with predefined epitope specificity to neutralize mutated viruse containing the mutated epitope. An epitope vaccine, using different epitopes including mutated epitopes, could provide a new concept for developing a new vaccine against HIV-1.     

Chimaeric VP2 proteins from infectious bursal disease virus containing the N-terminal M2e of H9 subtype avian influenza virus induce neutralizing antibody responses to both viruses

Subunit vaccines capable of inducing antibody against both infectious bursal disease virus (IBDV) and H9 subtype avian influenza virus (AIV) were developed. The VP2 protein of IBDV was used as a cargo protein to display a 12-amino-acid immunodominant epitope derived from the N-terminal M2 extracellular domain (nM2e) of the H9 subtype AIV. Two chimaeric proteins were constructed by insertion of one copy of the nM2e into the P_BC region (VP2_BCnM2e(H9)) or by fusing four copies of nM2e to the carboxyl terminal (VP2-4nM2e(H9)) of VP2. Genes that encoded the VP2 chimaeras were subsequently cloned into a baculovirus vector and expressed in Spodoptera frugiperda cells. The recombinant proteins were used to vaccinate chickens at day 0 and again after 4 weeks. Blood was collected at 2-week intervals after primary and secondary vaccination to detect the antibody titre against VP2 or the nM2e via indirect enzyme-linked immunosorbent assay. Virus neutralization tests were also performed to measure anti-IBDV or anti-H9 AIV neutralizing antibodies in chick embryo fibroblasts. Oropharyngeal and cloacal swabs were collected 3, 5 and 7 days post H9 subtype AIV infection for virus isolation. Vaccination with VP2-4nM2e(H9) induced higher levels of antibody responses against IBDV or H9 subtype AIV, and provided better protection against an IBDV virulent challenge compared with vaccination with VP2BCnM2e(H9) vaccine, the wild-type VP2 subunit vaccine or the IBDV subunit commercial vaccines. Both chimaeric VP2 vaccines showed poor efficacy in inhibiting H9 virus replication post challenge. In summary, chimaeric proteins that contain the nM2e epitope were able to induce both IBDV and H9 subtype AIV-neutralizing antibody responses.     

Matrix-M™ adjuvant enhances immunogenicity of both protein- and modified vaccinia virus Ankara-based influenza vaccines in mice

Influenza viruses continuously circulate in the human population and escape recognition by virus neutralizing antibodies induced by prior infection or vaccination through accumulation of mutations in the surface proteins hemagglutinin (HA) and neuraminidase (NA). Various strategies to develop a vaccine that provides broad protection against different influenza A viruses are under investigation, including use of recombinant (r) viral vectors and adjuvants. The replication-deficient modified vaccinia virus Ankara (MVA) is a promising vaccine vector that efficiently induces B and T cell responses specific for the antigen of interest. It is assumed that live vaccine vectors do not require an adjuvant to be immunogenic as the vector already mediates recruitment and activation of immune cells. To address this topic, BALB/c mice were vaccinated with either protein- or rMVA-based HA influenza vaccines, formulated with or without the saponin-based Matrix-M? adjuvant. Co-formulation with Matrix-M significantly increased HA vaccine immunogenicity, resulting in antigen-specific humoral and cellular immune responses comparable to those induced by unadjuvanted rMVA-HA. Of special interest, rMVA-HA immunogenicity was also enhanced by addition of Matrix-M, demonstrated by enhanced HA inhibition antibody titres and cellular immune responses. Matrix-M added to either protein- or rMVA-based HA vaccines mediated recruitment and activation of antigen-presenting cells and lymphocytes to the draining lymph node 24 and 48 h post-vaccination. Taken together, these results suggest that adjuvants can be used not only with protein-based vaccines but also in combination with rMVA to increase vaccine immunogenicity, which may be a step forward to generate new and more effective influenza vaccines.     

抗AIV(H9)独特型单克隆抗体的制备及其免疫原性的研究

用H9N2亚型AIV作为抗原免疫接种SPF鸡制备高免血清,用饱和硫酸铵法从该血清中提取免疫球蛋白G(IgG)。以该IgG制备弗氏佐剂疫苗免疫SPFBALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞NS-1在聚乙二醇(PEG)作用下融合,采用间接ELISA检测法,筛选获得3株(2H1D1、2H1G4、3H2D7)分泌特异性单克隆抗体的杂交瘤细胞。3株融合细胞培养上清液中的单克隆抗体效价均为2-4,小鼠腹水中的单抗效价是细胞培养上清液中的300～500倍。经检测表明,3株细胞系产生的单克隆抗体仅与相应的鸡抗H9N2亚型AIV血清发生特异性反应。用2H1D1分泌的抗独特型单克隆抗体制备疫苗与抗AIV灭活疫苗同时免疫SPF鸡收获的高免血清与AIV在MDCK细胞上进行中和实验,中和效价分别为10-1.8/10μl、10-1.6/10μl。由此表明,该抗独特型抗体疫苗具有良好的免疫原性。     

Intranasal Immunization with a Formalin-Inactivated Human Influenza A Virus Whole-Virion Vaccine Alone and Intranasal Immunization with a Split-Virion Vaccine with Mucosal Adjuvants Show Similar Levels of Cross-Protection

The antigenicity of seasonal human influenza virus changes continuously; thus, a cross-protective influenza vaccine design needs to be established. Intranasal immunization with an influenza split-virion (SV) vaccine and a mucosal adjuvant induces cross-protection; however, no mucosal adjuvant has been assessed clinically. Formalin-inactivated intact human and avian viruses alone (without adjuvant) induce cross-protection against the highly pathogenic H5N1 avian influenza virus. However, it is unknown whether seasonal human influenza formalin-inactivated whole-virion (WV) vaccine alone induces cross-protection against strains within a subtype or in a different subtype of human influenza virus. Furthermore, there are few reports comparing the cross-protective efficacy of the WV vaccine and SV vaccine-mucosal adjuvant mixtures. Here, we found that the intranasal human influenza WV vaccine alone induced both the innate immune response and acquired immune response, resulting in cross-protection against drift variants within a subtype of human influenza virus. The cross-protective efficacy conferred by the WV vaccine in intranasally immunized mice was almost the same as that conferred by a mixture of SV vaccine and adjuvants. The level of cross-protective efficacy was correlated with the cross-reactive neutralizing antibody titer in the nasal wash and bronchoalveolar fluids. However, neither the SV vaccine with adjuvant nor the WV vaccine induced cross-reactive virus-specific cytotoxic T-lymphocyte activity. These results suggest that the intranasal human WV vaccine injection alone is effective against variants within a virus subtype, mainly through a humoral immune response, and that the cross-protection elicited by the WV vaccine and the SV vaccine plus mucosal adjuvants is similar.     

Antigenicity and Predefined Specificities of the Multi-Epitope Vaccine in Candidate Consisting of Neutralizing Epitope and Mutated Epitopes Suggested a New Way against HIV-1 Mutation

A seven-amino acid epitope GPGRAFY located inside the V3 loop on envelope protein gp120 of HIV-1 is the principal neutralizing epitope (PNE), and a subset of anti-V3 antibodies specific for this epitope show a broad range of neutralizing activity. But this epitope undergoes restricted mutation. In this study, three epitope peptides [C-(GPGRAFY)2, C-(GPGQTFY)2 and C-(GPGQAWY)2] that contain neutralizing epitope GPGRAFY and its two mutated epitope GPGQTFY and GPGQAWY, were synthesized and then conjugated to carrier protein KLH (keyhole limpet hemocyanin). the epitope-vaccines C-(GPGRAFY)2-KLH, C-(GPGQTFY)2-KLH and C-(GPGQAWY)2-KLH induced high levels of antibodies to three V3 loop peptides that contain these epitopes respectively, and the antibody response induced by each epitope-vaccine showed predefined epitope-specific. When these three epitope-peptides mixed together and conjugated to carrier protein, or conjugated to carrier protein separately and then mixed together, high levels of epitope-specific antibodies which respectively recognized these epitopes on V3 loop peptide and both mutated peptides all can be induced by both of them. In blotting assay, these epitope-specific antibodies all recognized the neutralizing epitope and mutated epitopes on peptides respectively. In addition, the reactivity of the antibodies with whole gp120 molecule which contained the epitope GPGRAFY was tested. Only the GPGRAFY-epitope-specific antibodies but not the other antibodies recognized the gp120 molecule. These results provide experimental evidence that the candidate multi-epitope-vaccine containing neutralizing epitope and mutated epitopes may bring new hope against viral mutation resulting in HIV-1 immune evasion and may be developed as an effective vaccine with a broad neutralizing activity against HIV-1 infection.     

Development of subtype-specific and heterosubtypic antibodies to the influenza A virus hemagglutinin after primary infection in children

Children undergoing primary infection with an H1N1 or H3N2 influenza A virus developed subtype-specific hemagglutination inhibition antibodies and enzyme-linked immunosorbent assay antibodies to purified hemagglutinin (HA) of the infecting virus subtype. They also developed lower titered ELISA antibodies to the noninfecting H1 or H3 HA and to H8 (an avian strain) HA. Thus, after primary infection with an influenza A virus, children develop enzyme-linked immunosorbent assay, but not hemagglutination inhibition, antibodies reactive with heterosubtypic HAs. These heterosubtypic antibodies could influence the response to infection with other wild-type or attenuated vaccine strains of influenza A virus.