Recombinant vaccinia virus primes and stimulates influenza haemagglutinin-specific cytotoxic T cells

The ability of vaccinia virus to accept and express cloned genes encoding immunologically important proteins of unrelated viruses and malarial parasites has suggested a novel approach to the development of live vaccines. Vaccinia virus recombinants retain infectivity and stimulate synthesis of specific antibodies to the cloned gene products in vaccinated animals. Moreover, animals inoculated with recombinants expressing the influenza virus haemagglutinin (HA), the hepatitis B virus surface antigen, and type 1 herpesvirus glycoprotein D were protected against subsequent challenge with the corresponding virus. For maximal effectiveness, vaccines should produce cellular as well as humoral immunity. We now report that a vaccinia virus recombinant, expressing the influenza HA, primes and stimulates a specific murine cytotoxic T-lymphocyte (CTL) response. Histocompatible cells infected with this recombinant also serve as targets for CTLs. These properties make vaccinia virus a unique tool for studying cell-mediated immunity and enhance the attractiveness of this vector for production of live vaccines.     

Eventual AIDS vaccine failure in a rhesus monkey by viral escape from cytotoxic T lymphocytes.

Potent virus-specific cytotoxic T lymphocyte (CTL) responses elicited by candidate AIDS vaccines have recently been shown to control viral replication and prevent clinical disease progression after pathogenic viral challenges in rhesus monkeys. Here we show that viral escape from CTL recognition can result in the eventual failure of this partial immune protection. Viral mutations that escape from CTL recognition have been previously described in humans infected with human immunodeficiency virus (HIV) and monkeys infected with simian immunodeficiency virus (SIV). In a cohort of rhesus monkeys that were vaccinated and subsequently infected with a pathogenic hybrid simian-human immunodeficiency virus (SHIV), the frequency of viral sequence mutations within CTL epitopes correlated with the level of viral replication. A single nucleotide mutation within an immunodominant Gag CTL epitope in an animal with undetectable plasma viral RNA resulted in viral escape from CTLs, a burst of viral replication, clinical disease progression, and death from AIDS-related complications. These data indicate that viral escape from CTL recognition may be a major limitation of the CTL-based AIDS vaccines that are likely to be administered to large human populations over the next several years.     

Induction of CD8+ cytotoxic T cells by immunization with purified HIV-1 envelope protein in ISCOMs

To reduce the risks of immunization with killed or live attenuated virus vaccines, it may be advantageous to use a pure, defined antigen that contains determinants for both humoral and cellular immunity. However, although most non-living intact protein preparations induce antibodies and CD4+ major histocompatibility complex (MHC) class II-restricted helper and/or cytotoxic T lymphocytes (CTL), they do not elicit CD8+ MHC class I restricted CTL. Indeed, with a few exceptions, it has not so far been possible to induce CD8+ CTL by immunizing with intact soluble proteins. We show here that a single subcutaneous immunization in mice with immunostimulating complexes containing either purified intact gp160 envelope glycoprotein of the human immunodeficiency virus (HIV)-1 or influenza haemagglutinin results in reproducible and long-lasting priming of HIV specific or influenza-specific CD8+, MHC class I restricted CTL.     

T-cell responses to human AIDS virus in macaques immunized with recombinant vaccinia viruses

There is much interest in developing vaccines against acquired immune deficiency syndrome (AIDS), which is caused by a retrovirus termed human immunodeficiency virus (HIV). Isolates of this virus include human T-lymphotropic virus type III (HTLV-III), lymphadenopathy-associated virus (LAV), and AIDS-associated retrovirus (ARV). Several approaches towards the development of an AIDS vaccine result in the production of antibodies in subprimates. These methods involve the use of: antigens isolated from the AIDS virus; viral antigens expressed by transfected cells or by recombinant vaccinia viruses; and particular synthetic peptides of viral antigens. Because T-cell-mediated immunity (in addition to antibodies) is involved in resistance to diseases and death caused by various enveloped viruses, we sought to determine whether potential AIDS vaccines can induce T-cell responses against the AIDS virus. Here we report that immunization of non-human primates, Macaca fascicularis (macaques), with recombinant vaccinia viruses that express LAV envelope glycoproteins gp41 and gp110 results not only in the production of antibodies against the LAV envelope antigens but also in the generation of T-cells that proliferate and produce the lymphokine interleukin-2 (IL-2), in response to stimulation with purified LAV. We believe this is the first report demonstrating T-cell-mediated immunity to the virus that causes AIDS.     

Tat-specific cytotoxic T lymphocytes select for SIV escape variants during resolution of primary viraemia

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections are characterized by early peaks of viraemia that decline as strong cellular immune responses develop. Although it has been shown that virus-specific CD8-positive cytotoxic T lymphocytes (CTLs) exert selective pressure during HIV and SIV infection, the data have been controversial. Here we show that Tat-specific CD8-positive T-lymphocyte responses select for new viral escape variants during the acute phase of infection. We sequenced the entire virus immediately after the acute phase, and found that amino-acid replacements accumulated primarily in Tat CTL epitopes. This implies that Tat-specific CTLs may be significantly involved in controlling wild-type virus replication, and suggests that responses against viral proteins that are expressed early during the viral life cycle might be attractive targets for HIV vaccine development.     

Different H-2 subregions influence immunization against retrovirus and immunosuppression

Friend murine leukaemia virus complex (FV) causes an immunosuppressive retrovirus-induced disease. In certain mouse strains, FV shows striking similarities to human immunodeficiency virus (HIV) infection in man in that infected mice have severe T-cell immunosuppression but also develop virus-neutralizing antibodies incapable of eliminating infected cells. Previously we noted the influence of mouse major histocompatibility complex (H-2) genes on both FV-induced immunosuppression and on ability to protect mice against FV by immunizing with a vaccinia-Friend murine leukaemia helper virus (F-MuLV) envelope (env) recombinant virus. Here we show that different subregions of H-2 are involved in susceptibility to virus-induced immunosuppression (H-2D subregion) and protective immunization with a recombinant vaccinia virus (H-2K or I-A subregions). Thus, susceptibility to virus-induced immunosuppression does not preclude protection by vaccinia-Friend immunization. The mechanism of protection seems to involve priming of immune T cells, and not initial induction of neutralizing antibodies or cytotoxic T lymphocytes (CTL) (ref.2). Subsequent virus challenge generates a secondary response, resulting in appearance of IgG antibodies and CTL. In human HIV infection there could also be host genetic influences on elements of disease pathogenesis, such as immunosuppression, and on the success of T-cell priming by potential protective vaccines.     

Precise prediction of a dominant class I MHC-restricted epitope of Listeria monocytogenes.

Listeria monocytogenes is a Gram-positive bacterium which grows in the cytoplasm of eukaryotic cells and can cause severe disease in immunocompromised individuals. In murine systems CD8+ T lymphocytes have been shown to be important effectors of acquired protective immunity against L. monocytogenes. Class I MHC-restricted CD8+ cytotoxic T lymphocytes (CTL), which lyse J774 macrophage-like targets infected with L. monocytogenes, are induced following in vivo injection of live organisms. Natural peptide epitopes derived from L. monocytogenes can be acid-extracted from heavily infected BALB/c spleens and detected by CTL. A CTL clone, B9, derived from a (BALB/c x C57BL/6)F1 (H-2dxb) mouse, recognizes one of these natural epitopes in an H-2Kd-restricted fashion. B9 also recognizes P815 (H-2d) mastocytoma cells transfected with the listeriolysin gene. To identify the region of the listeriolysin recognized by CTL we used the H-2Kd peptide-binding motif described by Rammensee and colleagues to synthesize 11 nonamer peptides. One of these peptides, listeriolysin 91-99, was recognized very efficiently by B9. This represents the first identified class I MHC-restricted epitope of bacteria and demonstrates the utility of the allele-specific motif for predicting CTL epitopes.     

A nucleoprotein peptide of influenza A virus stimulates assembly of HLA-B27 class I heavy chains and beta 2-microglobulin translated in vitro

Most cytotoxic T lymphocytes (CTL) recognize epitopes of foreign viral proteins in association with class I major histocompatibility complex (MHC) molecules. Viral proteins synthesized in the cytoplasm require intracellular fragmentation and exposure to the class I antigens for the development of CTL responses. Although indirect evidence for binding of peptides to class I antigens has accumulated, direct binding has only been shown recently. The formation of complexes between peptide and class I antigen may occur in the endoplasmic reticulum (ER) and peptides have been shown to induce assembly of the class I complex. We have translated the messenger RNAs encoding HLA-B27 (subtype 2705) and beta 2-microglobulin in a rabbit reticulocyte lysate supplemented with human microsomal membranes (to mimic ER membranes), in the absence and presence of a peptide derived from the nucleoprotein (residues 384-394) of influenza A virus. This peptide induces CTL activity against target cells expressing the HLA-B27 antigen. Here we report direct evidence that the nucleoprotein peptide promotes assembly of the HLA-B27 heavy chain and beta 2-microglobulin, and that this can occur in the ER immediately after synthesis of the two proteins.     

Cytotoxic T cells specific for the circumsporozoite protein of Plasmodium falciparum

Malaria is initiated by the inoculation of a susceptible host with sporozoites from an infected mosquito. The sporozoites enter hepatocytes and develop for a period as exoerythrocyte or hepatic stage parasites. Vaccination with irradiated sporozoites can provide protective immunity and a recent study shows that this can also be conferred by immunization with a recombinant salmonella expressing only the circumsporozoite protein that normally covers the sporozoites. Protection against infection is likely to be mediated by cytotoxic CD8+ cells, as depletion of CD8+ T cells in a sporozoite-immunized animal can completely abrogate immunity. Here we demonstrate directly the existence of CD8+ cytotoxic T lymphocytes (CTL) that recognize the circumsporozoite protein. B10.BR mice immunized with sporozoites or with recombinant vaccinia virus expressing the CS protein of Plasmodium falciparum contain CTL that specifically kill L cell fibroblasts transfected with the gene encoding the same CS protein. The peptide epitope from the CS protein that is recognized by CTL from this strain of mice is from a variant region of the protein.     

Cytotoxic T lymphocytes recognize a fragment of influenza virus matrix protein in association with HLA-A2

Both human and murine cytotoxic T cells (CTL) elicited in response to infection with influenza A viruses have been shown to be specific for internal viral proteins, such as the matrix and nucleoprotein. Individual CTL epitopes have been identified in the nucleoprotein by successfully substituting short synthetic peptides for the intact virus in the preparation of target cells in cytotoxicity assays. The defined peptide epitopes have each been recognized by CTL in association with individual class I major histocompatibility complex (MHC) proteins, H-2Db, H-2Kk, H-2Kd (Taylor, P. et al., unpublished data) and HLA-B37. A logical strategy to investigate the molecular details of the interaction between antigen and MHC class I proteins would be to define an epitope recognized by the MHC class I molecule HLA-A2. This is because the amino-acid sequence is known, several variants of A2 have been characterized and the protein has been purified and crystallized. Here we describe a peptide derived from the influenza matrix protein that is recognized by human CTL in association with the HLA-A2 molecule.     

Non-responsiveness to a foot-and-mouth disease virus peptide overcome by addition of foreign helper T-cell determinants

Study of the immune response to synthetic antigens has shown that uncoupled peptides can realize their potential as vaccines only if they contain domains that react with helper T-cell receptors and Ia antigens in addition to antibody binding sites. Here we consider whether genetically restricted non-responsiveness to an uncoupled peptide could be overcome by synthesizing a peptide with an additional helper T-cell epitope from a different protein. We demonstrate that H-2d mice, which are non-responders to the 141-160 VP1 peptide of foot-and-mouth disease virus (FMDV), can be converted into responders by immunization with peptides containing the FMDV sequence with defined 'foreign' helper T-cell determinants from ovalbumin or sperm whale myoglobin. Furthermore, the virus-neutralizing activity of the antibody raised against peptide was dependent on the determinant used. Thus, FMDV peptides with the added sequences 323-339 from ovalbumin and 132-148 from sperm-whale myoglobin elicited a high degree of neutralizing activity in B10.D2 mice. The sera from mice which received the peptide with the added sequence 105-121 from sperm whale myoglobin did not neutralize the virus, although they had high levels of anti-141-160 FMDV peptide activity. Our data indicate that the T-cell help given by the 'foreign' epitopes is B-cell clone specific. These results are likely to have important implications for the design of peptide vaccines.     

T细胞功能学与结构免疫学结合方法鉴定HLA-A2限制性的细胞毒性T淋巴细胞表位富集区

 T细胞表位在抗病毒的T细胞免疫中发挥核心作用,目前已在多种病原微生物的蛋白序列上发现存在T细胞表位的聚集现象。本文建立了一套功能学与结构学结合的策略鉴定病原体上细胞毒性T细胞(Cytotoxic T Lymphocyte,CTL)表位富集区的方法,并以严重急性呼吸道综合征(SARS)相关冠状病毒(SARS-CoV)的M蛋白为例,成功地鉴定了一个HLA-A2限制性的表位富集区。首先通过生物信息学的方法预测并合成M蛋白跨膜区的HLA-A2潜在结合多肽,通过体外复性实验和T2细胞结合实验验证多肽与HLA-A2的结合力;然后在HLA-A2.1/Kb转基因小鼠中检测这些多肽的免疫原性;最后通过X射线衍射技术,成功解析了其中一条多肽与HLA-A^*0201的复合物结构,其结构显示该多肽具有典型的HLA-A^*0201表位的结构特点,但却呈现出与以往鉴定多肽不同的构象和锚定残基。本文对于理解机体对SARS-CoV等病原体产生的T细胞免疫反应,以及为更广泛的人群设计T细胞疫苗具有重要意义。     

Anti-HLA-A2 antibody-enhancement of peptide association with HLA-A2 as detected by cytotoxic T lymphocytes

Most cytotoxic T lymphocytes (CTL) not only recognize epitopes of viral or other foreign proteins in association with class I major histocompatibility complex (MHC) molecules, but also recognize target cells sensitized with short synthetic peptides representing the epitopes. There is increasing evidence that these synthetic peptides associate with the class I molecule both at the cell surface and intracellularly. We have now investigated the effect of a monoclonal antibody specific for HLA-A2 and HLA-B17 (B57/58) molecules (antibody MA2.1)3 on the sensitization of target cells with peptide for lysis by HLA-A2-restricted CTL. Previously, anti-HLA class I monoclonal antibodies have been shown to inhibit the recognition of target cells, infected with influenza A virus, by virus-specific CTL. We find, however, that target cells treated with MA2.1 antibody can be sensitized with peptide for CTL lysis much more rapidly than untreated cells, or at greater than 100-fold lower peptide concentration than that required for sensitization of untreated cells. This implies that the antibody, which is believed to bind to one side of the peptide-binding groove, directly affects the binding of peptide to the HLA-A2 molecule at the cell surface.     

Three Candidate Epitope—Vaccines in Combination Inducing High Levels of Multiantibodies Against HIV—1

HIV-1 mutation results in immune evasion, which presents a serious challenge for conventional strategies for developing effective vaccines. So far, much experimental evidence indicates that HIV-1 particles in the blood of patients can be cleaned principally by neutralizing antibodies. Based on these facts, we prepared triple combination of epitope-vaccines with the objective of inducing antibodies with predefined multi-epitope-specificity against HW-1. According to the sequences of three neutralizing epitopes (RILAVERYLKD, ELDKWA and GPGRAFY, designated El, E2, and E3, respectively) on HIV-1 envelope proteins, three epitope-peptides ((E1)2: C-(RILAVERYLKDG)2; (E2)4: C-(ELDKWAG)4; and (E3)2:C-(GPGRAFY)2) were synthesized and then conjugated with carrier protein keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA), and used for immunizing rabbits. After the vaccine course, the triple combination of epitope-vaccines induced high levels of predefined multi-epitope-specific antibodies. An immunoblotting-analysis demonstrated that the antibodies could recognize the native epitopes on both gp41 protein and V3 loop peptide. Furthermore, we compared the immune responses of three doses of epitope-peptides in the candidate epitope-vaccine. Strong antibody responses to three epitopes were observed in a dose dependent manner, with increasing dose raising the immune response. This result indicated that immunotolerance did not occur using an epitope vaccine dose of 80 ~tg. Thus, our results demonstrate that epitope-vaccines in combination can synchronously induce high levels of antibodies with predefined multi-epitope-specificity against HIV-1, and may be used to develop effective vaccines against HIV as a new strategy.     

Combined immunity of DNA vector and recombinant vaccinia virus expressing Gag proteins of equine infectious anemia virus

In order to develop a new vaccine candidate for equine infectious anemia virus (EIAV), gag gene of Chinese donkey leukocyte attenuated strain (EIAV DLV) and its parental virulent strain (EIAV LN) were inserted respectively into the TK region of the Tiantan strain (VV) of vaccinia virus by homologous recombination and the positive clone was confirmed by blue plaque assay. Protein expression was examined by Western blot. Prime and prime-boost procedures were used to immunize mice with two DNA vectors and two recombinant vaccinia viruses expressing EIAV Gag proteins. The results showed that the specific lysis of CTL responses in the DNA rVV groups was stronger than those in the DNA groups, amounting to 31%. Although the levels of specific antibodies were not significantly different, we could conclude that the recombinant vaccinia virus could boost the cellular responses following DNA vector priming. There was no detectable difference between the immune responses induced by DLV and LN Gag proteins. This data demonstrates that the combined immunity of DNA vector and recombinant vaccinia virus expressing EIAV gag proteins, utilizing the prime-boost procedure, can drive immunized mice to produce powerful cellular responses. These results lay an important foundation for the development of a new EIAV genetic engineering vaccine.     

HIV-specific cytotoxic T lymphocytes in seropositive individuals

Virus-specific cytotoxic T lymphocytes (CTL) which kill virus-infected cells are thought to be a major host defence against viral infections. Here we report the existence of human immunodeficiency virus (HIV)-specific CTL in persons infected with this virus, the aetiological agent of AIDS (acquired immunodeficiency syndrome). Recombinant HIV-vaccinia viruses were used to express HIV antigens in B-cell lines established from subjects seropositive for HIV and seronegative controls. Circulating lymphocytes capable of killing HIV env-expressing autologous B cells were detected in eight of eight seropositive subjects; in addition, at least three seropositive subjects demonstrated gag-specific cytotoxic responses. No HIV-specific cytotoxicity was observed in seronegative subjects. Selective inhibition of the env-specific cytotoxicity by a CD3-specific monoclonal antibody indicates that the effectors are T cells. This demonstration of a cytotoxic T-cell immune response to HIV in infected individuals should prove useful in investigating the immunopathogenesis of HIV infection further and in evaluating AIDS vaccine strategies.     

A group specific anamnestic immune reaction against HIV-1 induced by a candidate vaccine against AIDS

The first experimental immunization of humans against the AIDS retrovirus, HIV-1, was started in a series of HIV seronegative, healthy volunteers in November 1986. For the primary vaccination recombinant vaccinia virus (V25) expressing the complete gp160 env protein of the HTLV-IIIB strain of HIV-1 was introduced by scarification. This elicited a weak primary response which we subsequently attempted to enhance by additional immunizations (boosting), using four different immunization protocols. We report here that intravenous injection of paraformaldehyde-fixed autologous cells infected in vitro with V25 (individual D.Z.) gave the best results. This individual received second and third boosts of intramuscular gp160 derived from an HTLV-IIIB clone using the hybrid vaccinia virus/bacteriophage T7 expression system. An anamnestic humoral and cellular immune reaction was achieved for over one year after the original vaccination, with high levels of antibodies to the viral envelope, and neutralizing antibodies against divergent HIV-1 strains such as HTLV-IIIB and HTLV-IIIRF (also called HTLV-III HAT) after the first boost. In addition, group-specific cell-mediated immunity and cell-mediated cytotoxicity against infected T4 cells were obtained after the primary vaccine and enhanced by the boosts. Finally, skin tests showed both immediate and delayed hypersensitivity to gp160 in vivo. Although this protocol is not practical for a large scale vaccine trial, our results show for the first time that an immune state against HIV can be obtained in man.     

Cytotoxic T lymphocytes recognize influenza haemagglutinin that lacks a signal sequence

A surprising feature of most cytotoxic T lymphocytes (CTL) responding to influenza infection is that they recognize the unglycosylated (non-transmembrane) proteins of the virus, including the nucleoprotein. Recognition of cells that express nucleoprotein by CTL does not depend on a definite signal sequence within the protein, and the epitopes recognized can be defined with short synthetic peptides in vitro. Haemagglutinin (HA), the major transmembrane protein of the virus, is recognized by a minor population of CTL from infected mice. We have deleted the sequence coding for the N-terminal signal peptide from a complementary DNA encoding HA of the H1 subtype. The signal-deleted HA is detected with antibodies as a short-lived, unglycosylated, intracellular protein. However, CTL raised to the complete molecule recognize cells expressing the signal-deleted HA and vice versa. These results cast doubts on the assumption that CTL recognize the HA molecule only after its insertion into the plasma membrane.     

Screening of HLA-A2 restricted CTL epitopes using molecular simulation

Immunity is one of the functions a body has to iden-tify and eliminate foreign antigens. As one of the surfacemolecules of antigen presenting cells, major histocom-patibility complex (MHC) will exert essential effects dur-ing the antigen presenting procession, and the presenting of MHC-Ⅰ peptide complex is regarded as the prelude to activate the cytotoxic T lymphocytes (CTL) which play an important role in the anti-tumor and anti-virus responses. As we know, CTLs can only identify the co…     

HIV感染的治疗性DNA疫苗（综述）

DNA疫苗被称为继完速病原体疫苗和基因工程重组蛋白疫苗后的第3代疫苗,其兼有重组亚单位疫苗的安全性和减毒活疫苗的有效性。HIV感染的防治则以激发细胞免疫为主,以激发细胞免疫为主的DNA疫苗给HIV感染防治带来曙光。目前DNA疫苗在HIV感染防治研究方面已取得显著进展,其临床研究已证明具有明显的刺激机体细胞免疫的效果,尤其是一些细胞因子、趋化因子可明显增强HIV DNA疫苗的免疫反应。就DNA免疫佐剂、HIV病毒基因DNA疫苗作用机制以及加强HIV DNA疫苗的各种调节因素方面的研究进展进行综述,以促进HIV DNA疫苗防治的研究。