Ⅰ型人类免疫缺陷病毒包膜gp120与乙型肝炎病毒表面抗原免疫原性比较

目的 比较Ⅰ型人类免疫缺陷病毒(HIV-1)及乙型肝炎病毒(HBV)包膜蛋白初次免疫及加强免疫后诱导产生抗体的规律,为提高HIV-1包膜蛋白诱导保护性抗体产生能力提供创新思路.方法 以10周龄雌性C57BL/6小鼠为动物模型,分别用HIV-1 06044毒株gp120三聚体(gp120T)、HBV表面抗原(HBsAg)蛋白与AddaVax佐剂免疫小鼠,背部皮下注射,共免疫3次,每次免疫间隔3周,第一、第二次免疫后7d和第三次免疫后3d、7d取血;第一次免疫后7d、第三次免疫后3d、7d取脾组织.用酶联免疫吸附实验(ELISA)及酶联免疫斑点实验(ELISpot)方法检测免疫小鼠血浆特异性结合抗体滴度及抗体分泌细胞(ASC)数量.结果 gp120T和HBsAg两种蛋白初次免疫后,动物均未产生明显的特异性抗体.两种蛋白加强免疫后特异性抗体水平明显升高,gp120T一次加强免疫及两次加强特异性抗体滴度逐渐升高,而HBsAg一次加强抗体滴度已经接近两次加强的水平.二次加强免疫后,gp120T和HBsAg免疫鼠脾脏特异性ASC数量差异不显著.结论 HIV-1包膜gp120T加强免疫诱导抗体水平达到高峰慢于HBsAg加强免疫,即加强免疫后gp120T诱导的回忆反应慢于HBsAg.     

Construction and characterization of recombinant fowlpox virus coexpressing HIV-1(CN) gp120 and IL-2

The acquired immunodeficiency syndrome (AIDS) has now become one of the most serious infectious disease in the world, the safe and effective AIDS vaccines would be the best tools to control the disease. Fowlpox virus has been studied recently as a potential vector for the delivery of heterologous vaccine antigen. In this study, a recombinant fowlpox virus coexpressing gp120 of Chinese prevalent HIV-1 strain and IL-2 was constructed and the cellular immune responses against HIV-1 gp120 in BALB/c mice were evaluated. Chinese vaccine strain 282E4 of fowlpox virus was used as the vector to construct the recombinant fowlpox virus (rFPV) coexpressing HIV-1 gp120 and IL-2 via homologous recombination, and the recombinant virus was identified by PCR and Western blotting. The specific DNA fragment was amplified by PCR from the genomes of rFPV. Western blotting analysis showed that HIV-1 gp120 and IL-2 was expressed not only in chicken embryo fibroblast (CEF) cells infected by rFPV, but also in mammalian cells infected by rFPV. After the recombinant fowlpox virus was inoculated into BALB/c mice, specific CTL activities in the immunized mice were detected in the spleen. The results demonstrated that rFPV had good immunogenicity and could induce BALB/c mice to produce specific cellular immunity. IL-2 played a role of immunoadjuvant and enhanced the cellular immune response. The study provides the basis for the preparation of a Chinese vaccine candidate against HIV-1.     

原代HIV-1包膜糖蛋白修饰体诱导小鼠产生高滴度gp120抗体

目的 探索1型人类免疫缺陷病毒(HIV-1)包膜糖蛋白修饰对包膜免疫原性的的影响.方法 通过PCR扩增获得原代HIV-1 06044株包膜gp120基因及其突变体gp120/W427S基因,并构建gp120三聚体蛋白真核表达载体pcT-gp120和pcT-gp120/W427S,重组表达载体体外瞬时转染人胚肾HEK2...     

Improved Humoral and Cellular Immune Responses Against the gp120 V3 Loop of HIV-1 Following Genetic Immunization with a Chimeric DNA Vaccine Encoding the V3 Inserted into the Hepatitis B Surface Antigen

The gp120-derived V3 loop of HIV-1 is involved in co-receptor interaction, it guides cell tropism, and contains an epitope for antibody neutralization. Thus, HIV-1 V3 is an attractive vaccine candidate. The V3 of the MN strain (MN V3) contains both B- and T-cell epitopes, including a known mouse H-2^d-restricted cytotoxic T lymphocyte (CTL) epitope. In an attempt to improve the immunogenicity of V3 in DNA vaccines, a plasmid expressing MN V3 as a fusion protein with the highly immunogenic middle (pre-S2 + S) surface antigen of hepatitis B virus (HBsAg) was constructed. Epidermal inoculation by gene gun was used for genetic immunization in a mouse model. Antibody and CTL responses to MN V3 and HBsAg were measured and compared with the immune responses obtained after vaccination with plasmids encoding the complete HIV-1 MN gp160 and HBsAg (pre-S2 + S), respectively. DNA vaccination with the HIV MN gp160 envelope plasmid induced a slow and low titred anti-MN V3 antibody response at 12 weeks post-inoculation (p.i.) and a late appearing (7 weeks), weak and variable CTL response. In contrast, DNA vaccination with the HBsAg-encoding plasmid induced a rapid and high titred anti-HBsAg antibody response and a uniform strong anti-HBs CTL response already 1 week p.i. in all mice. DNA vaccination with the chimeric MN V3/HBsAg plasmid elicited humoral responses against both viruses within 3–6 weeks which peaked at 6–12 weeks and remained stable for at least 25 weeks. In addition, specific CTL responses were induced in all mice against both MN V3 and HBsAg already within the first 3 weeks, lasting at least 11 weeks. Thus, HBsAg acts as a ‘genetic vaccine adjuvant’ augmenting and accelerating the cellular and humoral immune response against the inserted MN V3 loop. Such chimeric HIV–HBsAg plasmid constructs may be useful in DNA immunizations as a ‘carrier’ of protein regions or minimal epitopes which are less exposed or poorly immunogenic.     

Characterization of neutralization epitopes of simian immunodeficiency virus (SIV) recognized by rhesus monoclonal antibodies derived from monkeys infected with an attenuated SIV strain

A major limitation in the simian immunodeficiency virus (SIV) system has been the lack of reagents with which to identify the antigenic determinants that are responsible for eliciting neutralizing antibody responses in macaques infected with attenuated SIV. Most of our information on SIV neutralization determinants has come from studies with murine monoclonal antibodies (MAbs) produced in response to purified or recombinant SIV envelope proteins or intact SIV-infected cells for relatively short periods of time. While these studies provide some basic information on the potential immunogenic determinants of SIV envelope proteins, it is unclear whether these murine MAbs identify epitopes relevant to antibody responses elicited in monkeys during infection with either wild-type or attenuated SIV strains. To accomplish maximum biological relevance, we developed a reliable method for the production of rhesus monoclonal antibodies. In the present study, we report on the production and characterization of a unique panel of monoclonal antibodies derived from four individual monkeys inoculated with SIV/17E-CL as an attenuated virus strain at a time when protective immunity from pathogenic challenge was evident. Results from these studies identified at least nine binding domains on the surface envelope glycoprotein; these included linear determinants in the V1, V2, cysteine loop (analogous to the V3 loop in human immunodeficiency virus type 1), and C5 regions, as well as conformational epitopes represented by antibodies that bind the C-terminal half of gp120 and those sensitive to defined mutations in the V4 region. More importantly, three groups of antibodies that recognize closely related, conformational epitopes exhibited potent neutralizing activity against the vaccine strain. Identification of the epitopes recognized by these neutralizing antibodies will provide insight into the antigenic determinants responsible for eliciting neutralizing antibodies in vivo that can be used in the design of effective vaccine strategies.     

Induction of cytotoxic and helper T cell responses by modified simian immunodeficiency virus hypervariable epitope constructs.

We previously reported the broad humoral immunogenicity of peptides synthesized according to the cumulative variability of an epitope (1,16). These peptides, hypervariable epitope constructs (HECs), are designed to represent the envelope glycoproteins of several isolates of the simian immunodeficiency virus (SIV). When HEC peptides were conjugated to palmitic acid and palmitic acid ester (lipoHECs), they promoted the induction of cellular immune responses. SIV envelope lipoHEC immunization of BALB/c and ICR mice resulted in up to 80% cytotoxic T lymphocyte (CTL) lysis of SIV envelope-expressing target cells and SIV envelope-specific delayed type hypersensitivity (DTH). This DTH response was significantly higher than that of single peptide controls, and the response peaked at 24 hours. Strong SIV envelope-specific T-cell proliferative responses were also induced in mice with stimulation indexes higher than 20 for spleen cells and higher than 10 for lymph node cells. Overall, our results demonstrate that conjugation of these variable synthetic peptides to a lipid moiety results in an immunogen capable of inducing strong and cross-reactive cellular immune responses.     

DNA vaccines expressing soluble CD4-envelope proteins fused to C3d elicit cross-reactive neutralizing antibodies to HIV-1

DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, DNA vaccines were constructed to express a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope. To enhance the immunogenicity of the expressed fusion protein, three copies of the murine C3d (mC3d3) were added to the carboxyl terminus of the complex. Monoclonal antibodies that recognize CD4-induced epitopes on gp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d3. In addition, both sCD4-gp120 and sCD4-gp120-mC3d3 bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. Mice (BALB/c) vaccinated with DNA vaccines expressing either gp120-mC3d3 or sCD4-gp120-mC3d3 elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d3-DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d3 had antibodies that elicited cross-protective neutralizing antibodies. The fusion of sCD4 to the HIV-1 envelope exposes neutralizing epitopes that elicit broad protective immunity when the fusion complex is coupled with the molecular adjuvant, C3d.     

Identification of HLA-A*3101-restricted cytotoxic T-lymphocyte response to human immunodeficiency virus type 1 (HIV-1) in patients with chronic HIV-1 infection

Virus-specific cytotoxic T-lymphocyte (CTL) responses are critical in the control of human immunodeficiency virus type 1 (HIV-1) infections. As several HIV-1 CTL epitopes restricted to many HLA types are already known, we aimed at identifying the CTL epitopes restricted by HLA-A*3101 in an effort to expand the epitope repertoire available for the development of potential T cell-mediated therapeutic measures and protective vaccines. Scanning of HIV-1 clade B SF2 strain proteins for the presence of peptides containing HLA-A*3101-binding motifs revealed 88 nine- to 11-mer peptides that had been synthesized and assayed for binding to HLA-A*3101 molecules. Peptides with medium to high HLA-binding affinity were tested for their ability to stimulate a CTL response in the peripheral blood mononuclear cells (PBMCs) from selected HIV-1-infected patients. Two of these binding peptides, Env769-779 (RLRDLLLIAAR) and Nef192-200 (KLAFHHMAR), induced peptide-specific CTLs in PBMCs from at least two of five HIV-1-seropositive individuals. CTL clones specific for the two peptides killed HLA-A*3101-expressing target cells infected with HIV-1 recombinant vaccinia virus, indicating that these peptides were naturally processed HLA-A*3101-restricted CTL epitopes. Identification of T-cell epitopes on HIV-1 proteins will increase our understanding of the role of CD8+ T cells in HIV-1 infections and assist in the design of new protective strategies.     

Confronting the hypervariability of an immunodominant epitope eliciting virus neutralizing antibodies from the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1).

Antibody mediated and cell mediated immune responses to the envelope glycoproteins gp120 and gp41 of the human immunodeficiency virus (HIV-1) are considered important for protection against infection and for attenuation of disease symptoms after infection. Virus neutralizing antibodies are mostly subtype specific and primarily directed against epitopes on a hypervariable loop from the V3 region of HIV-1 gp120. Such epitopes are recognized by helper and cytotoxic T-cells suggesting that all protective immune responses to HIV-1 are predominantly subtype specific. The extraordinary primary sequence variability of gp120 indicates that a combination of subtype specific components will be required to design a broadly effective protective immunogen against HIV-1. Peptides from hypervariable loops of the V3 region of 21 distinct HIV-1 isolates (clones) were synthesized and used to raise rabbit antisera. The antisera contained high levels of antibodies recognizing the homologous peptides and the parent gp120 sequence. The serological cross-reactivity between the distinct peptides was evaluated and related to amino acid divergence. The corresponding relationship approximated a linear regression with a correlation coefficient r = 0.718. The 21 peptides were combined into a single immunogen which elicited broadly reactive antibodies recognizing all 21 peptides as well as gp120 from the only isolate tested, HIV-1 IIIB. The results suggest the possibility of developing broadly protective HIV-1 immunogens by combining judiciously selected subtype specific peptides derived from envelope glycoproteins of divergent virus isolates.     

HBsAg/HLA-A2 transgenic mice: a model for T cell tolerance to hepatitis B surface antigen in chronic hepatitis B virus infection

A humanized murine model was developed to study T cell tolerance to the hepatitis B surface antigen (HBsAg) that is present in sera of hepatitis B virus chronic carriers. The HBsAg/HLA-A2 double-transgenic mice express a chimeric HLA-A2 MHC class I molecule and a high amount of the HBsAg in the liver that is secreted and present in sera during the animal's lifetime. In these mice, injection of plasmid DNA encoding HBsAg induced a high frequency of CD8(+) T cells secreting IFN-gamma in the periphery, with in vitro cytolytic activity and specificity for two dominant HBs-specific HLA-A2-restricted epitopes. Nevertheless, the DNA-based immunization elicited neither T(h)1 nor T(h)2 CD4(+) T cell responses. Despite a high concentration of HBsAg in sera, these mice developed an immunocompetent CD8(+) T cell repertoire towards the viral self-antigen, whereas the CD4(+) T cell repertoire was tolerized. In the absence of a CD4(+) T cell response, the IFN-gamma-secreting CD8(+) T cells primed by DNA-based immunization were unable to exert their antiviral functions in vivo on liver cells expressing the transgene product. However, when pro-inflammatory stimuli were given before or after DNA-based immunization, the HBsAg was cleared from the serum. This effect was antibody dependent and associated with the detection of an HBs-specific T(h)1 CD4(+) T cell response in the periphery. This model provides evidence that HBsAg displayed a strong tolerogenic effect on the CD4(+) T cell compartment that is associated with a defect in CD8(+) T cell effector functions in vivo.     

Sequence conservation of subdominant HLA-A2-binding CTL epitopes in HIV-1 clinical isolates and CD8+ T-lymphocyte cross-recognition may explain the immune reaction in infected individuals

Cytotoxic T-lymphocytes (CTL) are critical for immune control of infection with human immunodeficiency virus type-1 (HIV-1) and searches for relevant CTL epitopes for immune therapy are ongoing. Recently, we identified 28 HLA-A2-binding HIV-1 CTL epitopes (1). In this follow-up study we fully genome sequenced HIV-1 from 11 HLA-A2(+) patients to examine the sequence variation of these natural epitopes and compared them with the patient's CD8(+) T-cell recall response. Often the epitope was conserved but only a few patients showed a CD8(+) T-cell recall response. This infrequent targeting may be explained by immune subdominance. CD8(+) T-cell recall response to a natural epitope could be measured despite sequence differences in the patient's virus. T-cell cross-reaction between such variants could be demonstrated in HLA-A2 transgenic mice. Nine infrequently targeted but conserved or cross-reacting epitopes were identified in seven HIV-1 proteins. More immunogenic anchor amino acid optimized immunogens were designed that induced T-cell cross-reaction with these natural epitopes. It is concluded that most of the new CTL epitopes are conserved but subdominant during the infection. It is suggested that T-cell promiscuity may explain the observed CD8(+) T-cell reaction to epitope variants and it may be possible to use the selected immune optimized epitope peptides for therapeutic vaccination.     

In VivoInduction of Specific Cytotoxic T Lymphocytes in Mice and Rhesus Macaques Immunized with DNA Vector Encoding an HIV Epitope Fused with Hepatitis B Surface Antigen

DNA immunization offers a novel means to induce humoral and cellular immunity in inbred or in outbred animals. Here we have tested the efficiency of genetic immunization with hepatitis B virus (HBV) envelope-based vectors. In naive primates, injection of a plasmid DNA encoding HBV envelope proteins induced an HBV-specific cytotoxic response and appearance of potentially protective anti-HBs antibodies. Moreover, intramuscular and intradermal injections of a DNA expression vector encoding an epitope of the human immunodeficiency virus envelope fused to the surface protein of the hepatitis B virus (HBsAg) induced strong humoral and cytotoxic responses to antigenic determinants of both viruses in mice and nonhuman primates alike. In addition, in protein-primed Rhesus monkeys B-cell memory was successfully boosted by DNA injection of hybrid vectors and animals subsequently developed a multispecific cellular response. This suggests that DNA-based immunization could be used to boost efficiently and broaden the immune response in individuals immunized with conventional vaccines, regardless of their genetic variability. These results also indicate that it might be possible to rationally design HBsAg-based expression vectors to induce multispecific immune responses for vaccination against hepatitis B and other pathogens.     

Use of retroviral vectors for the analysis of SIV/HIV-specific CD8 T cell responses

CD8+ T cell responses and particularly cytotoxic T lymphocyte (CTL) activity are critical factors in controlling SHIV, SIV or HIV replication during natural infection and represent key parameters which need to be monitored during vaccine development. In order to improve the methodology for measuring CD8+ T cell responses, retroviral vectors expressing the full-length SIV-Gag or HIV-Env proteins were constructed and used to transduce B lymphoblastoid cell lines (BLCL) from cynomolgus monkeys infected with SHIV89.6P. Continuous expression of Gag and Env proteins was detected in stably transduced BLCL, which induced Gag- or Env-specific T cell responses, as measured by both IFNgamma-ELISPOT and chromium release assays, upon in vitro stimulation of PBMC from the SHIV89.6P-infected monkeys. Moreover, induction of Gag-specific CTL using BLCL transduced with retroviral vector expressing the SIV-Gag protein was more efficient and specific compared to that obtained using BLCL infected with a recombinant vaccinia virus (rVV) encoding for the same antigen. Assays on purified CD4+ and CD8+ T cells indicated that both populations specifically produced IFNgamma, but only the CD8+ T cells mediated Gag- and Env-specific cytotoxicity, indicating preferential expansion of these effector cells. Thus, this method represents an alternative tool for the analysis of CTL responses during vaccination protocols in those animal models where little information is available on MHC class I alleles or CTL epitopes.     

Expression of hepatitis B virus antigens in attenuated Salmonellae for oral immunization

The aim of our work is to identify hepatitis B virus antigens that can be stably expressed in attenuated Salmonellae and elicit protective immune responses as live oral route vaccines. As a first carrier system, we expressed T-cell and B-cell epitopes of hepatitis B virus as fusion proteins with the non-toxic subunit B (LT-B) in attenuated Salmonellae. These recombinant Salmonellae elicited anti-LT-B T- and B-cell immune responses and anti-HBV nucleocapsid antigen (HBcAg) T-cell responses when fed to mice. To combine the protective potential and the high immunogenicity of HBc with the induction of virus neutralizing antibodies to HBV surface antigen, we constructed vectors expressing hybrid HBc/pre-S particles in which the pre-S epitopes were surface-exposed. With one of these vectors, stable constitutive high level expression of hybrid HBc/pre-S2 particles was achieved in several attenuated Salmonella strains. When recombinant Salmonellae expressing such hybrid HBc/pre-S2 fusion proteins were fed to mice, the animals developed high titres of anti-HBcAg-specific serum IgG after a single or multiple oral immunizations, depending on the strain used as a carrier. In addition, lower titered antibodies against the pre-S2 antibody-binding sites were elicited. This is the first HBV antigen eliciting high-titered immune responses after a single oral immunization in recombinant Salmonellae. The immunogenicity of periplasmic LT-B and cytoplasmic HBc/pre-S2 shows that surface exposure of a foreign antigen is not a prerequisite for its immunogenicity in live attenuated Salmonellae.     

Cytotoxic T lymphocyte responses to vaccinia virus antigens but not HIV-1 subtype E envelope protein seen in HIV-1 seronegative Thais

The HIV-1 prime boost phase I/II vaccine trial using a recombinant canarypox vector, vCP1521, containing subtype E env (gp120), and subtype B env (gp41), gag and protease has started in Thailand. We have demonstrated that although 4 from 15 human immunodeficiency virus type 1 (HIV-1) seronegative Individuals showed cytotoxic T lymphocyte (CTL) responses to vaccinia virus antigens, none of them showed specific CTL responses to subtype E Env after in vitro stimulation. This preliminary study suggests that specific CTL responses to subtype E envelope detected in HIV-1 seronegative Individuals after vaccination should be considered as specific responses to the immunization.     

Immunogenicity and ability of variable loop-deleted human immunodeficiency virus type 1 envelope glycoproteins to elicit neutralizing antibodies

It has been extremely difficult to elicit broadly cross-reactive neutralizing antibodies (Nabs) against human immunodeficiency virus type 1 (HIV-1). In this study, we compared the immunogenic properties of the wild-type and variable loop-deleted HIV-1 envelope glycoproteins. Mice were immunized with recombinant vaccinia viruses expressing either the wild-type or the variable loop-deleted (V1-2, V3, V4, and V1-3) HIV-1(DH12) gp160s. The animals were subsequently boosted with respective recombinant gp120s. All envelope constructs elicited similar levels of gp120-binding antibodies when analyzed by enzyme-linked immunosorbent assay (ELISA). However, the highest neutralizing activity was observed in sera from animals immunized with the wild-type envelope protein, followed by those immunized with DeltaV4 and DeltaV1-2. No neutralizing activity was detected in sera from animals immunized with DeltaV3 or DeltaV1-3. To identify immunogenic epitopes, ELISA was performed with overlapping 15-mer peptides that cover the entire length of gp120. For the wild-type gp120, the immunogenic epitopes mapped primarily to the variable loops V1-2 and to the conserved regions C1 and C5. When they were plotted onto known coordinates of gp120 core crystal structure, the epitopes in the conserved regions mapped predominantly to the inner domain of the protein. By immunizing with variable loop-deleted envelopes, the immune responses could be redirected to other regions of the protein. However, the newly targeted epitopes were neither on the exposed surface of the protein nor on the receptor binding regions. Interestingly, the removal of the V3 loop resulted in loss of immunoreactivity for both V3 and V1/V2 loops, suggesting structural interaction between the two regions. Our results suggest that obtaining broadly reactive Nabs may not be achieved simply by deleting the variable loops of gp120. However, the observation that the immune responses could be redirected by altering the protein composition might allow us to explore alternative strategies for modifying the antigenic properties of HIV-1 envelope glycoprotein.     

Detection of Human Immunodeficiency Virus Type 1 Envelope Peptide- Stimulated T-helper Cell Responses and Variations in the Corresponding Regions of Viral Isolates among Vertically Infected Children

Although human immunodeficiency virus type 1 (HIV-1) specific CD4 T-helper cells are vital in mediating antiviral defence, little is known concerning the influence of HIV-1 antigenic variation on CD4 T-cell responses. In this study, the amino acid sequences of 5 synthetic HIV-1 envelope peptides used for in vitro stimulation (T2, P18 MN, P18 IIIB, T1 and TH4.1) were compared to the corresponding amino acid sequences of the gp 160 region of viruses isolated from HIV-1 infected children to determine whether variation in the envelope region of HIV-1 was associated with the ability to detect Env-specific T-helper cell responses. Although the T2 region appeared to offer some evidence as to the role antigenic variation may have played in class II-restricted CD4 T-cell responses between those children who showed a detectable Env-stimulated T-helper cell response and those who did not, the other regions studied showed no evidence of being more conserved among those children who showed detectable responses. The combined amino acid variation across the specific peptide regions studied was not associated with peripheral levels of HIV-1, nor did the degree of amino acid variation dictate the clinical category into which the children had been classified, although there was a tendency towards HIV-1 isolates from the younger children showing a greater degree of amino acid variation than isolates from the older children. These results suggest that HIV-1 specific CD4 responses may be somewhat tolerant of viral variation, although further studies are required to fully elucidate the effect of antigenic variation on immune recognition.     

Oral Immunization with Recombinant Vaccinia Virus Prime and Intramuscular Protein Boost Provides Protection against Intrarectal Simian-Human Immunodeficiency Virus Challenge in Macaques

Human immunodeficiency virus type 1 (HIV-1) acquisition occurs predominantly through mucosal transmission. We hypothesized that greater mucosal immune responses and protective efficacy against mucosal HIV-1 infection may be achieved by prime-boost immunization at mucosal sites. We used a macaque model to determine the safety, immunogenicity, and protective efficacy of orally delivered, replication-competent but attenuated recombinant vaccinia viruses expressing full-length HIV-1 SF162 envelope (Env) or simian immunodeficiency virus (SIV) Gag-Pol proteins. We examined the dose and route that are suitable for oral immunization with recombinant vaccinia viruses. We showed that sublingual inoculation of two vaccinia virus-naive pigtailed macaques with 5 × 10⁸ PFU of recombinant vaccinia viruses was safe. However, sublingual inoculation with a higher dose or tonsillar inoculation resulted in secondary oral lesions, indicating the need to optimize the dose and route for oral immunization with replication-competent vaccinia virus vectors. Oral priming alone elicited antibody responses to vaccinia virus and to the SF162 Env protein. Intramuscular immunization with the SF162 gp120 protein at either 20 or 21 weeks postpriming resulted in a significant boost in antibody responses in both systemic and mucosal compartments. Furthermore, we showed that immune responses induced by recombinant vaccinia virus priming and intramuscular protein boosting provided protection against intrarectal challenge with the simian-human immunodeficiency virus SHIV-SF162-P4.     

表达HIV-1 gag-pol△和gp 140 TM蛋白复制缺陷型重组腺病毒的构建及免疫效果研究

目的构建表达人免疫缺陷病毒Ⅰ型gag-pol△和gp140TM基因的非复制型重组腺病毒.方法首先将HIV-1的gag-pol△和gp140TM基因插入穿梭载体,再与腺病毒骨架质粒pAdEasy-1共转化E.coli BJ5183,获得重组子.转化293细胞后获得重组病毒.重组腺病毒与DNA疫苗联合免疫小鼠,ELISA检测小鼠血清中的抗体.结果获得两株重组腺病毒vAd-gag-pol△和vAd-gp140TM,能正确表达Gp140TM、Gag蛋白以及经剪切加工的P24蛋白,与DNA疫苗联合免疫小鼠后能产生高滴度的HIV-1特异性的抗体.结论成功构建了表达HIV-1结构基因的重组腺病毒,能有效诱导HIV-1特异性抗体.