Selective Increase in Lymphocyte Interferon Response to Vaccinia Antigen after Revaccination

Viral antigen prepared by heat inactivation of vaccinia virus stimulated production of interferon in association with transformation of sensitized human lymphocytes in vitro. Involvement of a macrophage-lymphocyte interaction in production of interferon stimulated by viral antigen was found in which macrophage greatly augmented the amount of interferon produced by lymphocytes. Reimmunization with live vaccinia virus resulted in a selective increase in the ability of lymphocytes to produce interferon in the presence of viral antigen 4-7 weeks later without a corresponding increase in the degree of already significant lymphocyte transformation. There was no correlation between the extent of lymphocyte transformation and the amount of interferon produced. The augmented interferon response after reimmunization described in this study may be a significant component of the protective effect of immunization with vaccinia against disease occurring after exposure to small-pox.     

Humoral and cellular immune responses to an envelope-associated antigen of herpes simplex virus.

The humoral and cellular immune responses of rabbits and guinea pigs to the envelope-associated antigen of herpes simplex virus type I were studied. Neutralizing antibody (at high titer) and lymphocytes reactive to herpes simplex virus were detected in both guinea pigs and rabbits after immunization with the antigen. In a standard assay of cellular immunity to herpes simplex virus, the antigen stimulated blast transformation of herpes simplex virus-reactive splenic lymphocytes in vitro. Furthermore, immunization of rabbits with the envelope-associated antigen protected the animals from a lethal dose of live herpes simplex virus. Thus an antigen of herpes simplex virus can be prepared which contains neither infectious nor noninfectious viral particles and which stimulates immunity to the virus in laboratory animals.     

Abomasal lymph node responses to Haemonchus contortus intestinal antigens established in kid goats by infection or immunization with intestinal antigens

Immune responses to Haemonchus contortus intestinal antigens were evaluated using abomasal lymph node (ALN) lymphocytes from kid goats protected against challenge infection by immunization with parasite intestinal antigen, and from kids that were challenged after immunization with ovalbumin. ALN lymphocytes from the intestinal antigen-immunized group produced significantly higher antibody levels against intestinal antigens than the ovalbumin group, supporting the theory that immunization contributed to that ALN response. In contrast, intestinal lysates and membrane enriched preparations from intestinal cells stimulated significant proliferation of ALN lymphocytes in both groups. The proliferation was antigen-dependent, since intestinal antigens failed to stimulate proliferation in ALN lymphocytes from unimmunized and uninfected kids. For both the intestinal antigen and ovalbumin immunized groups, CD4+ T lymphocytes predominated in ALN lymphocytes that were stimulated to proliferate by intestinal antigens. The results indicate that H. contortus infection alone can induce ALN lymphocyte responses to intestinal antigens. In contrast to ALN lymphocyte responses, serum antibody against intestinal antigens was generally low to undetectable in ovalbumin-immunized kids following infection. Abomasal mucus from an H. contortus infected lamb was probed with a monoclonal antibody that binds to a periodate sensitive determinant on numerous H. contortus intestinal membrane and secreted proteins. Numerous bands of reactivity were detected, indicating that multiple parasite intestinal antigens were released into abomasal mucus during infection. The results, challenge the general concept that H. contortus intestinal antigens are 'hidden' from the host immune system during an infection. On the contrary, parasite intestinal proteins may be relatively abundant antigens presented to the host during infection. In addition, ALN T lymphocytes appear to provide a more sensitive measure than serum antibody to detect presentation of these antigens to the host immune system.     

Investigations about the immunobiology of m-proteins of Streptococcus pyogenes. IV. Cell-mediated immunity of rhesus monkeys (Macaca mulatta) against streptococcal antigens (author's transl)

Cell-mediated immunity was tested in monkeys (Macaca mulatta) immunized with highly purified M-proteins by means of the lymphocyte transformation reaction. Lymphocytes from immunized and nonimmunized monkeys were stimulated with PAL-M-Proteins. IC-M-proteins were completely free of mitogenicity and no lymphocyte stimulation was to be found after immunization with 1.6 mg. Erythrogenic toxins of strain NY5 were able to stimulate lymphocytes of monkeys unspecifically.     

Mucosal vaccination overcomes the barrier to recombinant vaccinia immunization caused by preexisting poxvirus immunity

Overcoming preexisting immunity to vaccinia virus in the adult population is a key requirement for development of otherwise potent recombinant vaccinia vaccines. Based on our observation that s.c. immunization with vaccinia induces cellular and antibody immunity to vaccinia only in systemic lymphoid tissue and not in mucosal sites, we hypothesized that the mucosal immune system remains naive to vaccinia and therefore amenable to immunization with recombinant vaccinia vectors despite earlier vaccinia exposure. We show that mucosal immunization of vaccinia-immune BALB/c mice with recombinant vaccinia expressing HIV gp160 induced specific serum antibody and strong HIV-specific cytotoxic T lymphocyte responses. These responses occurred not only in mucosal but also in systemic lymphoid tissue, whereas systemic immunization was ineffective under these circumstances. In this context, intrarectal immunization was more effective than intranasal immunization. Boosting with a second dose of recombinant vaccinia was also more effective via the mucosal route. The systemic HIV-specific cytotoxic T lymphocyte response was enhanced by coadministration of IL-12 at the mucosal site. These results also demonstrate the independent compartmentalization of the mucosal versus systemic immune systems and the asymmetric trafficking of lymphocytes between them. This approach to circumvent previous vaccinia immunity may be useful for induction of protective immunity against infectious diseases and cancer in the sizable populations with preexisting immunity to vaccinia from smallpox vaccination.     

Induction of immune responses to SIV antigens by mucosally administered vaccines.

In an attempt to develop an immunization strategy to induce mucosal and circulatory antibodies against SIV antigens, we have investigated the potential of attenuated recombinant vaccinia virus to deliver SIV antigens (gp160 of SIVmac239) to mucosal surfaces of mice. After systemic or mucosal (intragastric, intranasal, or intrarectal) immunization with vaccinia virus-SIV Env recombinants the immune responses against the envelope glycoprotein of SIV, as well as against vaccinia virus antigens, were assessed by ELISA of serum, saliva, and intestinal and vaginal secretions. All immunization routes induced specific antibody titers against gp160 in both serum and external secretions. Recall responses against SIV were found to be acquired after administration of SIVmac239 Env and Gag antigens in a virus-like particle (VLP) form by the same mucosal routes as those used for the priming with recombinant vaccinia virus. The results obtained demonstrate the potential of vaccinia virus recombinants to elicit a primary immune response at mucosal surfaces, which could be enhanced by delivering the same antigen in the form of VLPs.     

An immunosuppressive factor in serum of rabbits lethally infected with the herpesvirus of bovine malignant catarrhal fever.

Virus-neutralizing antibody does not protect animals challenged with the herpesvirus of bovine malignant catarrh; therefore, other parameters of the immune response were investigated. In vitro transformation of peripheral blood lymphocytes with mitogens and specific antigens was monitored throughout the course of disease in lethally infected New Zealand white rabbits. The whole-blood culture method was used. Transformation with mitogens was normal throughout the incubation period but fell precipitously from the day of disease onset until death. Specific stimulation with viral antigens was not detected at any time following challenge. Purified and washed lymphocytes, however, reesponded normally to mitogens and were transformed in the presence of viral antigens. Acute-phase serum from rabbits inhibited transformation of normal rabbit lymphocytes, but this inhibition was reversible by washing. It is proposed that the development of a serum suppressive factor(s) is important in the determination of whether an animal is capable of controlling infection with this virus.     

Vaccination with a recombinant vaccinia virus encoding a "self" antigen induces autoimmune vitiligo and tumor cell destruction in mice: requirement for CD4(+) T lymphocytes

Many human and mouse tumor antigens are normal, nonmutated tissue differentiation antigens. Consequently, immunization with these "self" antigens could induce autoimmunity. When we tried to induce immune responses to five mouse melanocyte differentiation antigens, gp100, MART-1, tyrosinase, and tyrosinase-related proteins (TRP) 1 and TRP-2, we observed striking depigmentation and melanocyte destruction only in the skin of mice inoculated with a vaccinia virus encoding mouse TRP-1. These mice rejected a lethal challenge of B16 melanoma, indicating the immune response against TRP-1 could destroy both normal and malignant melanocytes. Cytotoxic T lymphocytes specific for TRP-1 could not be detected in depigmented mice, but high titers of IgG anti-TRP-1 antibodies were present. Experiments with knockout mice revealed an absolute dependence on major histocompatibility complex class II, but not major histocompatibility complex class I, for the induction of both vitiligo and tumor protection. Together, these results suggest that the deliberate induction of self-reactivity using a recombinant viral vector can lead to tumor destruction, and that in this model, CD4(+) T lymphocytes are an integral part of this process. Vaccine strategies targeting tissue differentiation antigens may be valuable in cancers arising from nonessential cells and organs such as melanocytes, prostate, testis, breast, and ovary.     

西尼罗病毒多表位基因免疫保护研究

目的探讨西尼罗病毒多表位基因诱导体液及细胞免疫的可行性及其免疫攻毒保护作用。方法将西尼罗病毒多抗原表位基因克隆到真核表达载体pcDNA3.1中,构建重组真核表达质粒pcDNA3.1-M。通过脂质体转染法将该多表位基因导入BHK细胞中,采用RT-PCR和IFA检测其在细胞内的瞬时表达。将重组质粒通过肌肉注射免疫BALB/c小鼠,通过间接免疫ELISA法、CTL杀伤功能检测和淋巴细胞增殖试验等,检测小鼠体内特异性体液免疫和细胞免疫的水平。在此基础上进行攻毒试验,分析重组质粒DNA对西尼罗病毒攻击的免疫保护作用。结果重组质粒pcDNA3.1-M能够在真核细胞内表达,采用IFA方法能在细胞内检测到西尼罗病毒特异性抗原。在抗原的刺激下,免疫后的小鼠脾淋巴细胞增殖显著,可诱导特异性CTL应答反应。攻毒试验显示,重组多表位基因能够对西尼罗病毒的攻击起到一定的保护作用,达到50%,中和抗体效价达到1∶50,应用免疫佐剂或应用重组蛋白加强免疫后,其保护率为62.5%,中和抗体效价达到1∶90。结论西尼罗病毒多表位基因可诱发特异性免疫应答,对西尼罗病毒感染具有保护作用,为其基因疫苗研制提供了一定的实验依据。     

Resistance to human immunodeficiency virus 1 infection of SCID mice reconstituted with peripheral blood leukocytes from donors vaccinated with vaccinia gp160 and recombinant gp160.

SCID mice reconstituted with adult human peripheral blood leukocytes (hu-PBL-SCID mice) make antigen-specific human antibody responses following secondary immunization and can be infected with human immunodeficiency virus 1 (HIV-1), suggesting that they might prove useful for evaluating protective immunity to HIV-1 following vaccination of PBL donors. HIV-seronegative volunteers were immunized with vaccinia expressing HIV-1LAV-1/Bru 160-kDa envelope glycoprotein (vaccinia gp160) and subsequently given booster injections of recombinant gp160 protein (rgp160). Their PBLs were used at intervals of 4-72 weeks after booster injections to construct hu-PBL-SCID mice, which were then challenged with 10(2)-10(3) minimal animal infectious doses of highly homologous HIV-1IIIB. Control hu-PBL-SCID mice were constructed from donors receiving vaccinia, alum, or hepatitis B vaccine. Protection against virus infection was defined as the absence of HIV-1 by culture and no detection of proviral genomes following PCR amplification. Control animals were highly susceptible to HIV infection. By contrast, hu-PBL-SCID mice reconstituted with cells from three of four donors immunized with vaccinia gp160 and recently injected with rgp160 showed no evidence of HIV-1 infection by culture or PCR assays. With increasing time after rgp160 injection, the ability of vaccine-derived hu-PBL-SCID mice to resist HIV-1 infection diminished. These results demonstrate that a potentially protective human immune response was stimulated by this HIV gp160 immunization protocol and show the utility of the hu-PBL-SCID model in the rapid evaluation of candidate vaccines.     

Cytotoxic T cell responses against hepatitis B virus polymerase induced by genetic immunization

BACKGROUND/AIMS: Individuals with chronic hepatitis B may benefit from genetic (DNA-based) immunization through induction of viral clearance by enhancement of suboptimal cellular immune responses. While marked cellular immune responses to hepatitis B virus (HBV) nucleocapsid and envelope proteins occur after genetic immunization in mice, it is unknown whether genetic immunization is capable of eliciting such responses to HBV polymerase. We wished to develop assays for the determination of HBV polymerase specific immune responses in mice and investigate whether genetic immunization may elicit humoral and cellular immune responses to HBV polymerase. METHODS: BALB/c (H-2d) mice were injected with a DNA expression construct for HBV polymerase. Humoral immune responses to HBV polymerase were analyzed with a newly established ELISA. Cellular immune responses were determined using recombinant vaccinia virus infected target cells expressing HBV polymerase at high levels. RESULTS: Assays for the detection of HBV polymerase-specific immune responses were developed. Immunized animals exhibited substantial polymerase-specific cytotoxic T lymphocyte responses. However, no humoral immune responses to HBV polymerase were detectable. CONCLUSIONS: Our study demonstrates that DNA-based immunization will generate substantial CTL responses to HBV polymerase and may be an important component of an immunotherapeutic strategy to combat chronic HBV infection.     

Inhibition of mitogen- and antigen-induced lymphocyte blastogenesis by herpes simplex virus.

Lymphocytes were separated from peripheral blood of adult human donors by Ficoll-Hypaque gradient centrifugation and cultured in the presence of nonspecific mitogens (phytohemagglutinin[PHA] and concanavalin A) or specific microbial anatigens (herpes simplex virus [HSV], mumps virus, streptococcal enzymes, and Candida albicans). Exposure of lymphocyte cultures to infectious HSV resulted in almost complete inhibition of blastogenesis ([3/H]thymidine uptake) induced by each of the mitogens and antigens, a finding which suggests that a common mechanism may underlie the inhibitory effect. Several characteristics of the effect of virus on blastogenesis were noted: (1) virus inactivated by heat or ultraviolet irradiation was ineffective; (2) inhibition (is greater than 90%) was greatest in cultures exposed to HSV on or before the addition of PHA; (3) lymphocyte preparations washed free of HSV continued to be refractory to stimulation, an observation indicating that the presence of unabsorbed virions or viral products was not essential; and (4) inhibition was independent of the cell donor's state of humoral immunity to HSV.     

Influenza A virus nucleoprotein is a major target antigen for cross-reactive anti-influenza A virus cytotoxic T lymphocytes.

Influenza A virus-specific cytotoxic T lymphocytes (CTL) capable of lysing cells infected with any influenza A virus ("cross-reactive CTL") constitute a major portion of the host CTL response to influenza. The viral nucleoprotein (NP), a major internal virion structural protein, has been implicated as a possible target antigen for cross-reactive CTL. To directly examine CTL recognition of NP, a vaccinia virus recombinant containing a DNA copy of an influenza A virus NP gene was constructed. We found that murine cells infected with this virus were efficiently lysed in a major histocompatibility complex-restricted manner by cross-reactive CTL populations obtained by immunization with a variety of influenza A virus subtypes. In addition, the recombinant vaccinia virus containing the PR8 NP gene was able to both stimulate and prime for a vigorous secondary cross-reactive CTL response. Significantly, splenocytes from mice primed by inoculation with the recombinant vaccinia virus containing the PR8 NP gene could be stimulated by influenza A viruses of all three major human subtypes. Finally, unlabeled target competition experiments suggest that NP is a major, but not the sole, viral target antigen recognized by cross-reactive CTL.     

Cell-mediated immune responses after immunization of healthy seronegative children with varicella vaccine: kinetics and specificity

Humoral and cell-mediated immune responses were determined in seronegative children immunized with live attenuated Oka strain varicella vaccine. At 2 weeks after immunization, 80% of children had detectable lymphocyte proliferation to varicella-zoster virus (VZV) antigens, while only 40% had antibodies to VZV as detected by ELISA. By 6 weeks after immunization, 97% of children seroconverted, and 95% of these responded to VZV antigens in the proliferation assay. A high proportion of immunized children also responded in the proliferation assay to purified glycoproteins I, II, and III of VZV. These results indicate that most children develop a broad cell-mediated immune response to VZV antigens within weeks after immunization with varicella vaccine.     

采用流式细胞仪检测艾滋病病毒1型膜蛋白小鼠免疫后IFN-γ的表达

目的通过流式细胞技术检测艾滋病病毒1型(HIV-1)Gp120膜蛋白免疫小鼠后的IFN-γ的表达。方法 构建表达HIV-1gp120基因的复制型重组痘苗病毒。与DNA疫苗联合免疫小鼠,用流式细胞仪检测小鼠脾淋巴细胞中IFN-γ的表达。结果 获得表达中国HIV-1流行株gp120基因的重组痘苗病毒rVVgp120、rVVM304,能正确表达Gp120蛋白。与DNA疫苗p120-VRC、pM304-VRC联合免疫小鼠后,用流式细胞仪检测到小鼠脾淋巴细胞能特异性的表达IFN-γ。结论 用流式细胞检测技术成功检测到免疫小鼠的脾淋巴细胞特异性的表达IFN-γ,用此方法可方便快捷地检测小鼠的细胞免疫效果。     

Cell-mediated immune proliferative responses to HIV-1 of chimpanzees vaccinated with different vaccinia recombinant viruses

The only animal that can be reproducibly infected with HIV, and that thus provides an experimental system for testing the effectiveness of prototype vaccines, is the chimpanzee. We compared proliferative responses to HIV and to vaccinia virus (VV) antigens of lymphocytes taken at various times from chimpanzees vaccinated with recombinant VV expressing different HIV genes. Animals were immunized with the original VV strain, as control, or with constructs expressing gp160 (VV160) given exclusively or in combination with one or two other constructs producing p25 (VV25), F/3'-orf (VVF), or the human interleukin-2 (IL-2) gene, which was included in an attempt to amplify immune responses. Irrespective of the HIV gene utilized, lymphocyte proliferation to HIV was usually weak and rapidly decreased after each inoculation, contrasting with strong and sustained responses to VV. Lack of adequate recall reactivity after challenge with fixed autologous lymphocytes expressing VV-produced HIV antigens indicated that vaccination resulted only in low levels of HIV-specific memory cell priming. The use of IL-2-producing VV did not lead to increased responsiveness. Reactivity to soluble purified gp160, but not to p25, could be detected in PBL from animals that had received both VV160 and VV25, while immunization with VVF resulted in a significant response to this protein in one of two animals. The transient nature of T cell reactivity to HIV might explain why, in similar studies, chimpanzees were not protected from infection with live HIV.     

Protective Role of Antigens from Peritoneal Exudates of Infected Mice against Toxoplasmosis

Background: Toxoplasma gondii is an obligate intracellular parasite that infects all mammalian cells. Several antigens such as excreted/secreted antigens have been identified as a potential vaccine candidate. Objective: To determine how excreted/secreted antigens from peritoneal exudates of infected mice (mESA) stimulate cell-mediated immune responses and induce protective immunity against toxoplasmosis in the murine model. Methods: The supernatants produced from the peritoneal fluids, were fractionated by precipitation in ammonium sulphate solution (30-80% saturated). For induction of cell-mediated immune responses, delayed type hypersensitivity was measured, in injected footpad. Response to purified antigen was measured by lymphocyte proliferation assay. Nitric oxide was measured by Griess method. For immunization, Balb/c mice were immunized 2 times with mESA, mESA-40% and Toxoplasma Lysate Antigen (TLA). The virulent RH strain of Toxoplasma gondii was used for challenging. Results: The pattern of lymphocyte responsiveness was dependent on the antigen employed. In sensitized mice, those received mESA-40% displayed higher lymphocyte response than mice stimulated by mESA (p<0.05). The highest amounts of nitric oxide were observed in macrophages, which received mESA-40% and mESA (p<0.05). Mice immunized with mESA-40% survived longer than those immunized with mESA and other antigens (p<0.05). Conclusion: As fraction 40% (mESA-40%) showed a good result in induction of cellmediated responses in the murine model, the purification and isolation of the mESA 40% is highly recommended for future study.     

Protection against lethal Sendai virus infection by in vivo priming of virus-specific cytotoxic T lymphocytes with a free synthetic peptide.

The only peptide of Sendai virus that is recognized by cytotoxic T lymphocytes (CTL) in B6 mice was found with (i) the use of recombinant vaccinia virus constructs containing separate genes of Sendai virus and (ii) a set of overlapping peptides completely spanning the identified nucleoprotein (NP) gene product. This immunodominant NP peptide is recognized by Sendai virus-specific CTL that are known to have therapeutic effects in vivo. By subcutaneous immunization, this peptide induced Sendai virus and NP peptide-specific CTL memory responses in vivo. Most importantly, mice that had been immunized with this peptide were protected against a lethal virus dose, indicating that viral peptides can be used as antiviral T-cell vaccines. The induction of T-cell memory by free peptide immunization potentially has wide applicability in biology and medicine, including protection against infectious disease.     

Virus dissemination via the lymphomyeloid complex

In the previous AIDS symposium organized by the Society, Witte and Witte (1) made a number of predictions, one of which was that in AIDS patients, "Lymph from the thoracic duct should be strongly positive for HIV." Though direct evidence for this is lacking, some early experiments of ours with vaccinia virus (2) are fully in accord with this prediction, to which they lend indirect support. In rabbits, nasally instilled vaccinia virus spreads via the lymphatic pathway (afferent peripheral lymph--deep cervical gland--efferent lymph--thoracic duct) in as short a time as nine hours. Virus is transported mainly in cells, for when the efferent lymph is centrifuged virus is found only in the cell sediment. It seems reasonable to assume that other viruses, including HIV, are similarly disseminated. Paradoxically, the lymphomyeloid complex both greatly facilitates the spread of virus, and at the same time, mounts the immunological defenses against the virus which it so effectively helps to disseminate. Whatever the portal of entry of the virus, its transport by migrating cells ensures its dissemination throughout the lymphomyeloid complex, including the bone marrow. The bone marrow is an integral part of the complex, as the prime source of B lymphocytes, T lymphocyte precursors, and many of the antigen-presenting cells as well as the granulocytes. There is some evidence concerning possible ways in which the bone marrow can contribute to the development of immune deficiency in AIDS patients. The bone marrow merits further study in this context.     

恶性疟原虫保护性抗原复合基因-痘苗病毒重组活疫苗株免疫动物后诱发的Th1细胞免疫反应

目的　测定恶性疟原虫 -痘苗病毒重组活疫苗候选株免疫动物后产生白细胞介素 - 2 (IL- 2 )和干扰素 (IFN)的生物学活性水平。　方法　用 MTT法测定了其诱导的保护性细胞免疫反应 (Th1细胞免疫反应 )。　结果　用重组痘苗病毒在免疫家兔及大白鼠 4～ 6 wk后血清中 IL- 2的生物学活性增强 ,免疫后 6 wk家兔、大白鼠及小白鼠 3种动物血清中 IFN的生物学活性水平比免疫前明显升高。　结论　恶性疟原虫 -痘苗病毒重组活疫苗候选株免疫动物后可诱发机体产生 Th1细胞免疫反应