Production and purification of melanoma gp100 antigen and polyclonal antibodies

gp100 is a melanoma-associated antigen found to carry immunogenic epitopes that can induce a CTL response against tumor cells. Production and purification of large quantities of this polypeptide may be important in the context of diagnosis and vaccinating against melanoma. To overcome the hydrophobic nature of gp100, we cloned and expressed only a part of the protein, and obtained a hydrophilic recombinant polypeptide (HR-gp100) that contained most of the immunogenic peptides. High yield was achieved in an Escherichia coli expression system. The protein was purified by AKTA Prime using anionic-columns. Polyclonal antibodies developed in chicken against HR-gp100 were efficient at detecting gp100 in melanoma cells, as determined by Western blot analysis and by immunohistochemistry. HR-gp100 can be used to develop a vaccine against melanoma. Antibodies to HR-gp100 may be used to detect tumors of melanocytic origin or to determine the level of gp100 expression in tumors prior to immunotherapy with the protein or one of its peptides.     

Delayed-type hypersensitivity and in vitro lymphocyte response in guinea pigs immunized with a live varicella vaccine

The relation of delayed type hypersensitivity (DTH) assessed by the Varicella-Zoster virus (VZV) skin test and lymphocyte transformation (LTF) with VZV antigen was investigated in guinea pigs immunized with live varicella vaccine virus, or heat-inactivated vaccine virus. Guinea pigs immunized with live varicella vaccine virus showed positive DTH and LTF responses to viral antigen as well as a neutralizing (NT) antibody response, while those immunized with heat-inactivated vaccine virus showed only an NT antibody response of the same degree as that to live vaccine virus. These results show the reliability of the skin test in assessing cell-mediated immunity (CMI) to VZV and the advantage of the live varicella vaccine over the inactivated one in immunizing guinea pigs.     

Chitosan Nanoparticle Encapsulated Hemagglutinin-Split Influenza Virus Mucosal Vaccine

Subunit/split influenza vaccines are less reactogenic compared with the whole virus vaccines. However, their immunogenicity is relatively low and thus required proper adjuvant and/or delivery vehicle for immunogenicity enhancement. Influenza vaccines administered intramuscularly induce minimum, if any, mucosal immunity at the respiratory mucosa which is the prime site of the infection. In this study, chitosan (CS) nanoparticles were prepared by ionic cross-linking of the CS with sodium tripolyphosphate (TPP) at the CS/TPP ratio of 1:0.6 using 2 h mixing time. The CS/TPP nanoparticles were used as delivery vehicle of an intranasal influenza vaccine made of hemagglutinin (HA)-split influenza virus product. Innocuousness, immunogenicity, and protective efficacy of the CS/TPP-HA vaccine were tested in influenza mouse model in comparison with the antigen alone vaccine. The CS/TPP-HA nanoparticles had required characteristics including nano-sizes, positive charges, and high antigen encapsulation efficiency. Mice that received two doses of the CS/TPP-HA vaccine intranasally showed no adverse symptoms indicating the vaccine innocuousness. The animals developed higher systemic and mucosal antibody responses than vaccine made of the HA-split influenza virus alone. The CS/TPP-HA vaccine could induce also a cell-mediated immune response shown as high numbers of IFN-γ-secreting cells in spleens while the HA vaccine alone could not. Besides, the CS nanoparticle encapsulated HA-split vaccine reduced markedly the influenza morbidity and also conferred 100% protective rate to the vaccinated mice against lethal influenza virus challenge. Overall results indicated that the CS nanoparticles invented in this study is an effective and safe delivery vehicle/adjuvant for the influenza vaccine.     

Stimulation of cynomolgus peripheral blood lymphocytes with tetanus toxoid and smallpox vaccine

The cynomolgus monkey was studied as an animal model to investigate the cell-mediated immunity induced by vaccines. Optimal conditions are described to isolate peripheral blood lymphocytes. Lymphocyte transformation tests were performed with tetanus toxoid and smallpox vaccine. Antigen-specific lymphocyte transformations with smallpox vaccine could only be demonstrated when lymphocytes were obtained from vaccinated monkeys. Tetanus toxoid appeared to be a weak antigen. However, after adsorption of the toxoid to aluminum phosphate, a significant antigen-specific lymphocyte transformation was observed.     

Transcutaneous immunisation with herpes simplex virus stimulates immunity in mice

Herpes simplex virus (HSV) is common throughout the world and is a target for vaccine development. Transcutaneous immunisation is a novel technique that uses the application of vaccine antigens in solution on the skin in the presence of cholera toxin (CT) as an adjuvant. This study investigated the potential of transcutaneous immunisation in C3H mice, using CT co-administered with whole inactivated HSV-1 (CT+HSVi) or HSV-1 antigens extracted from infected Vero cells (CT+HSVag) or a control protein (CT+BSA). The application of any of the three vaccines on to bare mouse skin resulted in the migration of Langerhans cells from the epidermis and in the production of serum antibodies to CT. Both HSV preparations generated serum and mucosal (faecal) antibodies to HSV, with the CT+HSVi vaccine being a more potent stimulator of humoral immunity. The CT+HSVag vaccine, however, was the more potent stimulator of cell-mediated immunity, giving rise to a strong delayed type hypersensitivity response and lymphocyte proliferation in vitro. When the mice were challenged by epidermal inoculation of HSV, the CT+HSVag vaccine induced a higher level of protection than the CT+HSVi vaccine, a result which may indicate that the efficacy of HSV vaccines depends on stimulation of cell-mediated rather than humoral responses. The success of topical vaccination suggests that the transcutaneous route may offer a promising potential for novel vaccine delivery which merits further investigation.     

Distinct Effects on Diversifying Selection by Two Mechanisms of Immunity against Streptococcus pneumoniae

Antigenic variation to evade host immunity has long been assumed to be a driving force of diversifying selection in pathogens. Colonization by Streptococcus pneumoniae, which is central to the organism''s transmission and therefore evolution, is limited by two arms of the immune system: antibody- and T cell- mediated immunity. In particular, the effector activity of CD4⁺ T_H17 cell mediated immunity has been shown to act in trans, clearing co-colonizing pneumococci that do not bear the relevant antigen. It is thus unclear whether T_H17 cell immunity allows benefit of antigenic variation and contributes to diversifying selection. Here we show that antigen-specific CD4⁺ T_H17 cell immunity almost equally reduces colonization by both an antigen-positive strain and a co-colonized, antigen-negative strain in a mouse model of pneumococcal carriage, thus potentially minimizing the advantage of escape from this type of immunity. Using a proteomic screening approach, we identified a list of candidate human CD4⁺ T_H17 cell antigens. Using this list and a previously published list of pneumococcal Antibody antigens, we bioinformatically assessed the signals of diversifying selection among the identified antigens compared to non-antigens. We found that Antibody antigen genes were significantly more likely to be under diversifying selection than the T_H17 cell antigen genes, which were indistinguishable from non-antigens. Within the Antibody antigens, epitopes recognized by human antibodies showed stronger evidence of diversifying selection. Taken together, the data suggest that T_H17 cell-mediated immunity, one form of T cell immunity that is important to limit carriage of antigen-positive pneumococcus, favors little diversifying selection in the targeted antigen. The results could provide new insight into pneumococcal vaccine design.     

CTL-dependent and -independent antitumor immunity is determined by the tumor not the vaccine

Previously, we compared the efficiency of direct injection with an adenovirus (Ad) expressing human gp100 (hgp100) to immunization with dendritic cells (DC) loaded with the same vector ex vivo. The DC vaccine provided the greatest protection against challenge with B16F10 melanoma, and antitumor immunity was found to be CD8(+) T cell-independent. In the current study, we sought to determine whether lack of CD8(+) T cell-mediated antitumor immunity was a function of the vaccine platform or the tumor line. Both Ad and DC/Ad vaccines elicited CD8(+) CTL reactive against hgp100 and provided protection against B16F10 engineered to express hgp100 demonstrating that both vaccination platforms can effectively generate protective CD8(+) T cell-mediated immunity. The hgp100-induced CTL cross-reacted with murine gp100 (mgp100) and lysed B16F10 cells pulsed with mgp100 peptide indicating that the resistance of B16F10 cells to CTL elicited by hgp100 vaccination may be due to a defect in processing of the endogenous mgp100. Indeed, introduction of the TAP-1 cDNA into B16F10 rendered the cells sensitive to lysis by gp100-specific CTL. Furthermore, gp100-immunized mice were protected from challenge with B16F10-TAP1 cells through a mechanism dependent upon CD8(+) T cells. These results demonstrate that tumor phenotype, not the vaccination platform, ultimately determines CD8(+) or CD4(+) T cell-mediated tumor clearance.     

Progression of pulmonary tuberculosis and efficiency of bacillus Calmette-Guérin vaccination are genetically controlled via a common sst1-mediated mechanism of innate immunity

Using a mouse model for genetic analysis of host resistance to virulent Mycobacterium tuberculosis, we have identified a genetic locus sst1 on mouse chromosome 1, which controls progression of pulmonary tuberculosis. In vitro, this locus had an effect on macrophage-mediated control of two intracellular bacterial pathogens, M. tuberculosis and Listeria monocytogenes. In this report, we investigated a specific function of the sst1 locus in antituberculosis immunity in vivo, especially its role in control of pulmonary tuberculosis. We found that the sst1 locus affected neither activation of Th1 cytokine-producing T lymphocytes, nor their migration to the lungs, but rather controlled an inducible NO synthase-independent mechanism of innate immunity. Although the sst1(S) macrophages responded to stimulation with IFN-gamma in vitro, their responsiveness to activation by T cells was impaired. Boosting T cell-mediated immunity by live attenuated vaccine Mycobacterium bovis bacillus Calmette-Guérin or the adoptive transfer of mycobacteria-activated CD4(+) T lymphocytes had positive systemic effect, but failed to improve control of tuberculosis infection specifically in the lungs of the sst1(S) animals. Thus, in the mouse model of tuberculosis, a common genetic mechanism of innate immunity mediated control of tuberculosis progression in the lungs and the efficiency of antituberculosis vaccine. Our data suggest that in immunocompetent humans the development of pulmonary tuberculosis and the failure of the existing vaccine to protect against it, in some cases, may be explained by a similar defect in a conserved inducible NO synthase-independent mechanism of innate immunity, either inherited or acquired.     

Cytokine GM-CSF genetic adjuvant facilitates prophylactic DNA vaccine against pseudorabies virus through enhanced immune responses

Granulocyte/macrophage colony-stimulatory factor (GM-CSF) is an attractive adjuvant for a DNA vaccine on account of its ability to recruit antigen-presenting cells (APCs) to the site of antigen synthesis as well as its ability to stimulate the maturation of dendritic cells (DCs). This study evaluated the utility of GM-CSF cDNA as a DNA vaccine adjuvant for glycoprotein B (gB) of pseudorabies virus (PrV) in a murine model. The co-injection of GM-CSF DNA enhanced the levels of serum PrV-specific IgG with a 1.5-to 2-fold increase. Moreover, GM-CSF co-injection inhibited the production of IgG2a isotype. However, it enhanced production of an IgG1 isotype resulting in humoral responses biased to the Th2-type against PrV antigen. In contrast, the co-administration of GM-CSF DNA enhanced the T cell-mediated immunity biased to the Th1-type, as judged by the significantly higher level of cytokine IL-2 and IFN-gamma production but not IL-4. When challenged with a lethal dose of PrV, the GM-CSF co-injection enhanced the resistance against a PrV infection. This suggests that co-inoculation with a vector expressing GM-CSF enhanced the protective immunity against a PrV infection. This immunity was caused by the induction of increased humoral and cellular immunity in response to PrV antigen.     

Induction of specific human primary immune responses to a Semliki Forest virus–based tumor vaccine in a Trimera mouse model

Recombinant Semliki Forest virus (rSFV) enables high-level, transient expression of heterologous proteins in vivo, and is believed to be a superior vector for genetic vaccination, compared with the conventional DNA plasmid. Nonetheless, the efficacy of rSFV-based vaccine in eliciting human immune responses has not been tested. We used a Trimera mouse model, consisting of lethally irradiated BALB/c host reconstituted with nonobese diabetes/severe combined immunodeficiency (NOD/SCID) bone marrow plus human peripheral blood mononuclear cells (PBMCs), to characterize the in vivo immune responses against rSFV-encoded human melanoma antigen MAGE-3. MAGE-3–specific antibody and cytotoxic T lymphocyte (CTL) activity were detected by ELISA and ⁵¹Cr-release assay, respectively, and the responses were compared with those induced by a plasmid DNA vaccine encoding the same antigen. The results showed that rSFV vaccine could elicit human MAGE-3–specific antibody and CTL response in the Trimera mice, and the antitumor responses were more potent than those by plasmid DNA vaccination. This is the first report to evaluate human immune responses to an rSFV-based tumor vaccine in the Trimera mouse model. Our data suggest that rSFV vector is better than DNA plasmid in inducing protective immunity, and the Trimera model may serve as a general tool to evaluate the efficacy of tumor vaccines in eliciting human primary immune response in vivo.     

Immunization with surface antigen vaccine alone and after treatment with 1-(2-fluoro-5-methyl-beta-L-arabinofuranosyl)-uracil (L-FMAU) breaks humoral and cell-mediated immune tolerance in chronic woodchuck hepatitis virus infection

Woodchucks chronically infected with the woodchuck hepatitis virus (WHV) were treated with the antiviral drug 1-(2-fluoro-5-methyl-beta-L-arabinofuranosyl)-uracil (L-FMAU) or placebo for 32 weeks. Half the woodchucks in each group then received four injections of surface antigen vaccine during the next 16 weeks. Vaccination alone elicited a low-level antibody response to surface antigen in most carriers but did not affect serum WHV DNA and surface antigen. Carriers treated first with L-FMAU to reduce serum WHV DNA and surface antigen and then vaccinated had a similar low-level antibody response to surface antigen. Following vaccinations, cell-mediated immunity to surface antigen was demonstrated in both groups, independent of serum viral and antigen load, but was significantly enhanced in woodchucks treated with L-FMAU and was broadened to include other viral antigens (core, e, and x antigens and selected core peptides). Cell-mediated immunity and antibody responses to surface antigen were observed after drug discontinuation in half of the carriers that received L-FMAU alone. Surface antigen vaccine alone or in combination with drug broke humoral and cell-mediated immune tolerance in chronic WHV infection, but the combination with drug was more effective. This suggested that a high viral and antigen load in carriers is important in maintaining immunologic tolerance during chronicity. The humoral and cellular immunity associated with the combination of L-FMAU and vaccine resembled that observed in self-limited WHV infection. Such combination therapy represents a potentially useful approach to the control of chronic hepatitis B virus infection in humans.     

Characterization of serologic and cell-mediated reactivity of a 38-kDa antigen isolated from Mycobacterium tuberculosis

An antigen of Mycobacterium tuberculosis with an m.w. of 38,000 has been isolated by affinity chromatography using a monoclonal antibody. This antibody bound only to an antigen found in M. tuberculosis and Mycobacterium bovis BCG. The specificity of the antigen was tested in a vertical study by immunodetection on western blots reacted with hyperimmune sera against M. tuberculosis, M. bovis, and 10 other Mycobacterium species. The antigen was detected only by antisera to M. tuberculosis and M. bovis. Specificity in cell-mediated immunity was tested by skin tests in guinea pigs sensitized with M. tuberculosis, Mycobacterium intracellulare, and Mycobacterium kansasii and by lymphocyte proliferation tests. The 38-kDa antigen induced positive skin test reactions regardless of the Mycobacterium species used to sensitize the animal. The ability of the 38-kDa antigen to sensitize for cell-mediated immunity was tested by injecting mice with the 38-kDa antigen and challenging their lymphocytes in vitro with various mycobacterial antigens. Lymphocyte proliferation was observed in the presence of 38-kDa antigen, M. tuberculosis sonicate antigen, and tuberculin purified protein derivative and to M. kansasii and M. intracellulare. The 38-kDa antigen may contain a specific epitope detected by serology, but also contains epitopes that are cross-reactive for cellular immunity.     

Anti-tumor effects of a human VEGFR-2-based DNA vaccine in mouse models

Background

Vascular endothelial growth factor (VEGF) and its receptor, VEGFR-2 (Flk-1/KDR), play a key role in tumor angiogenesis. Blocking the VEGF-VEGFR-2 pathway may inhibit tumor growth. Here, we used human VEGFR-2 as a model antigen to explore the feasibility of immunotherapy with a plasmid DNA vaccine based on a xenogeneic homologue of this receptor.

Methods

The protective effects and therapeutic anti-tumor immunity mediated by the DNA vaccine were investigated in mouse models. Anti-angiogenesis effects were detected by immunohistochemical staining and the alginate-encapsulate tumor cell assay. The mechanism of action of the DNA vaccine was primarily explored by detection of auto-antibodies and CTL activity.

Results

The DNA vaccine elicited a strong, protective and therapeutic anti-tumor immunity through an anti-angiogenesis mechanism in mouse models, mediated by the stimulation of an antigen-specific response against mFlk-1.

Conclusion

Our study shows that a DNA vaccine based on a xenogeneic homologue plasmid DNA induced autoimmunity against VEGFR-2, resulting in inhibition of tumor growth. Such vaccines may be clinically relevant for cancer immunotherapy.     

A review of the leishmanin skin test: A neglected test for a neglected disease

The leishmanin skin test (LST) has been used for decades to detect exposure and immunity to the parasite Leishmania, the causative agent of the neglected tropical disease leishmaniasis. In the LST, Leishmania antigen (leishmanin) is intradermally injected into the forearm. In an individual who has been previously infected, a delayed-type hypersensitivity (DTH) reaction results in a measurable induration at the site of the injection, indicating that previous exposure to Leishmania has resulted in the development of cell-mediated immunity. LST positivity is associated with long-lasting protective immunity against reinfection, most notably as reported for visceral leishmaniasis (VL). Despite efforts over the past few decades, leishmanin antigen is no longer produced under good manufacturing practice (GMP) conditions anywhere in the world. Consequently, the use of the LST in epidemiological studies has declined in favor of serological and molecular tests. In this review, we provide a historical overview of the LST and justification for the reintroduction of leishmanin. A GMP-grade leishmanin can be used to detect immunity in vivo by the LST and can be investigated for use in an interferon-γ release assay (IGRA), which may serve as an in vitro version of the LST. The LST will be a valuable tool for surveillance and epidemiological studies in support of the VL elimination programs and as a surrogate marker of immunity in vaccine clinical trials.MethodsA review of the literature was conducted using PubMed as the primary database, with MeSH terms “leishmanin skin test” OR “Montenegro test” OR “Montenegro skin test.” Articles written in English that describe the history or standardization of leishmanin, the use of leishmanin in an IGRA, or the use of the LST in epidemiological studies or vaccine trials were prioritized in our appraisal of the literature.     

The stability and immunogenicity of a dispersed-grown freeze-dried Pasteur BCG vaccine

The level of antituberculous immunity seems to be related to the number of memory T cells induced. This may vary as a function of the multiplication and persistence of BCG in host tissues. The most important requirements for a BCG vaccine are, therefore, the immunogenicity of the strain, the high proportion of live to dead bacilli, and adequate dispersion and low levels of soluble antigens. The surface-grown Pasteur BCG vaccine contains a very high proportion of bacilli killed by ball-milling and freeze-drying. It also contains clumps and soluble antigens, all factors influencing cell-mediated immune processes and viability control. Therefore, several batches of vaccine were prepared on an industrial scale using one of the most immunogenic strains (French 1173 P2) and grown as dispersed bacilli by a modified cell type culture method. This method provided fully viable, well-dispersed vaccines which have a viability and heat stability superior to that of the classical surface-grown BCG. The immunogenicity was checked by multiplication and persistence in mouse organs and the skin reactivity and tuberculin hypersensitivity in guinea-pigs showed results comparable to those obtained with classical vaccine. Small-scale tests in children showed superior immunogenicity of the dispersed as opposed to the classical vaccine and there was no suppurative adenitis.     

Expression of human prostate-specific antigen (PSA) in a mouse tumor cell line reduces tumorigenicity and elicits PSA-specific cytotoxic T lymphocytes

 Human prostate-specific antigen (PSA) has a highly restricted tissue distribution. Its expression is essentially limited to the epithelial cells of the prostate gland. Moreover, it continues to be synthesized by prostate carcinoma cells. This makes PSA an attractive candidate for use as a target antigen in the immunotherapy of prostate cancer. As a first step in characterizing the specific immune response to PSA and its potential use as a tumor-rejection antigen, we have incorporated PSA into a well-established mouse tumor model. Line 1, a mouse lung carcinoma, and P815, a mouse mastocytoma, have been transfected with the cDNA for human PSA. Immunization with a PSA-expressing tumor cell line demonstrated a memory response to PSA which protected against subsequent challenge with PSA-expressing, but not wild-type, tumors. Tumor-infiltrating lymphocytes could be isolated from PSA-expressing tumors grown in naive hosts and were specifically cytotoxic against a syngeneic cell line that expressed PSA. Immunization with tumor cells resulted in the generation of primary and memory cytotoxic T lymphocytes (CTL) specific for PSA. The isolation of PSA-specific CTL clones from immunized animals further demonstrated that PSA can serve as a target antigen for antitumor CTL. The immunogenicity studies carried out in this mouse tumor model provide a rationale for the design of methods to elicit PSA-specific cell-mediated immunity in humans. Received: 4 April 1996 / Accepted: 31 May 1996     

Genetic control of vaccine-induced immunity against a parasitic helminth, Schistosoma mansoni

Schistosoma mansoni is one of several species of trematode helminths responsible for schistosomiasis, a major parasitic infection of man. Genetic analysis in mice has revealed that the protective immunity induced against this parasite by an attenuated larval vaccine is strongly influenced by genes regulating the activation of macrophage effector cells. The latter finding suggests that the induction of cell-mediated immunity may be a successful strategy for a non-living vaccine against the human infection.     

嗜肺军团菌核酸疫苗研究进展

嗜肺军团菌通过气溶胶的形式感染人和动物,引起军团菌性肺炎,危害极其严重,但该病目前尚无有效的预防措施。军团菌病疫苗的研究经历了灭活疫苗、减毒活疫苗和蛋白亚单位疫苗不同阶段,但至今尚未能在临床得到应用。核酸疫苗作为一种新型疫苗,能有效诱导机体产生细胞免疫应答,符合兼胞内寄生菌嗜肺军团菌的预防需要,并且还具有诸多其他优点,引起了研究者们的兴趣。对嗜肺军团杆菌核酸疫苗的研究现状进行了简要介绍。     

Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. IV. Recognition and activation by cloned glycoproteins gB and gD

Results of studies in mice and clinical observations in man indicate that T cell-mediated immunity is important in resistance to herpes simplex virus (HSV) infections. This study was undertaken to elucidate the viral antigen specificity of human HSV-immune T cells. Purified HSV-1 glycoproteins gB-1 and gD-1, cloned and expressed in mammalian cells, were found to stimulate proliferation of, and interleukin 2 (IL 2) production by, peripheral blood lymphocytes (PBL) of HSV seropositive individuals, indicating the presence of memory T cells to gB-1 and gD-1 in individuals with serologic evidence of immunity to HSV. Second, T cell clones, generated by stimulation of PBL with HSV-1, were found to recognize gB-1 or gD-1, as evidenced by the ability of the clones to proliferate in response to stimulation with gB-1 or gD-1 in the absence of exogenous IL 2. Third, HSV-specific T cell clones, lytic for HSV-1 or both HSV-1- and HSV-2-infected autologous target cells, were generated after stimulation of PBL with purified cloned gB-1 or gD-1. Our findings, that human HSV-specific T cells can recognize and be activated by HSV subunit antigens gB-1 or gD-1, imply that these glycoproteins play a role in human T cell-mediated immunity to HSV and support the contention that a gB-1 or gD-1 subunit vaccine may be protective in man.