Potent Cellular Immune Responses Research Articles

Effective DNA vaccination of cattle with the mycobacterial antigens MPB83 and MPB70 does not compromise the specificity of the comparative intradermal tuberculin skin test

The current tuberculin test and slaughter strategy for the control of bovine tuberculosis in cattle has failed to prevent a sharp rise in cases over recent years, especially in the south-west of England. A recent scientific review has concluded that the development of a cattle vaccine holds the best prospect for tuberculosis control in British herds. In order to continue with test and slaughter-based control strategies, the development of TB vaccines that do not compromise the specificity of the tuberculin skin test are required. This report describes results of cattle vaccination experiments with TB DNA vaccines expressing the mycobacterial antigens MPB70, MPB83, and Ag85A and constitutes the first published vaccination study with DNA vaccines undertaken in a target host species. All calves vaccinated with the MPB83 expressing plasmid demonstrated potent cellular immune responses, characterised by CD4 + T cells producing interferon-gamma as well as humoral immunity characterised by IgG1 biased specific antibodies. Vaccination with MPB70 was less effective with immune responses only observed in half of the vaccinated animals, while vaccination with Ag85A did not result in vaccine-induced immune responses. Intramuscular vaccination was found to stimulate stronger cellular responses than intradermal immunisation. Significantly, the specificity of tuberculin skin testing was not compromised by DNA vaccination since none of the vaccinated calves showed positive skin test reactivity.

A truncated variant of the hepatitis C virus core induces a slow but potent immune response in mice following DNA immunization

Vaccination of BALB/c mice with pIDKCo, a plasmid containing the coding sequence for the first 176 amino acids of the hepatitis C virus (HCV) core protein, induced both humoral and cellular specific immune responses. Particularly, the level of anti-core antibodies increased slowly with time up to a mean value above 1:8000 that was generally superior than that found in anti-HCV positive individuals. Six out of nine anti-HCV positive human sera were able to inhibit at different extent the binding of mouse anti-core sera to a recombinant capsid protein. Our results show that it is possible to elicit a potent humoral and cellular immune response against the HCV core antigen in mice following DNA immunization.

Limitations of the C6/Wistar Rat Intracerebral Glioma Model: Implications for Evaluating Immunotherapy

Intracranial rat glioma models are a useful method for evaluating the efficacy and toxicity of novel therapies for malignant glioma. The C6/Wistar model has been used extensively as a reproducible in vivo model for studying primary brain tumors including anti-glioma immune responses. The objective of the present study is to provide in vivo evidence that the C6 rat glioma model is allogeneic within Wistar rats and is therefore inappropriate for evaluating immune responses. Growth patterns and immune responses of C6 cells implanted into the brain and flank of Wistar rats were analyzed and compared to an immunogenic syngeneic model (9L/Fischer). Wistar rats with C6 tumors developed a potent humoral and cellular immune response to the tumor. Wistar rats given simultaneous flank and intracerebral tumors had a survival rate of 100% compared to an 11% survival rate in control animals receiving only intracranial C6 cells. The C6 rat glioma induces a vigorous immune reaction that may mimic a specific anti-tumor response in Wistar rats. Efficacy of immunotherapy within this model must be cautiously interpreted.

Distribution of DNA vaccines determines their immunogenicity after intramuscular injection in mice.

Intramuscular injection of DNA vaccines elicits potent humoral and cellular immune responses in mice. However, DNA vaccines are less efficient in larger animal models and humans. To gain a better understanding of the factors limiting the efficacy of DNA vaccines, we used fluorescence-labeled plasmid DNA in mice to 1) define the macroscopic and microscopic distribution of DNA after injection into the tibialis anterior muscle, 2) characterize cellular uptake and expression of DNA in muscle and draining lymph nodes, and 3) determine the effect of modifying DNA distribution and cellular uptake by volume changes or electroporation on the magnitude of the immune response. Injection of a standard 50-microl dose resulted in the rapid dispersion of labeled DNA throughout the muscle. DNA was internalized within 5 min by muscle cells near the injection site and over several hours by cells that were located along muscle fibers and in the draining lymph nodes. Histochemical staining and analysis of mRNA expression in isolated cells by RT-PCR showed that the transgene was detectably expressed only by muscle cells, despite substantial DNA uptake by non-muscle cells. Reduction of the injection volume to 5 microl resulted in substantially less uptake and expression of DNA by muscle cells, and correspondingly lower immune responses against the transgene product. However, expression and immunogenicity were restored when the 5-microl injection was followed by electroporation in vivo. These findings indicate that distribution and cellular uptake significantly affect the immunogenicity of DNA vaccines.

Increased expression and immunogenicity of sequence-modified human immunodeficiency virus type 1 gag gene.

A major challenge for the next generation of human immunodeficiency virus (HIV) vaccines is the induction of potent, broad, and durable cellular immune responses. The structural protein Gag is highly conserved among the HIV type 1 (HIV-1) gene products and is believed to be an important target for the host cell-mediated immune control of the virus during natural infection. Expression of Gag proteins for vaccines has been hampered by the fact that its expression is dependent on the HIV Rev protein and the Rev-responsive element, the latter located on the env transcript. Moreover, the HIV genome employs suboptimal codon usage, which further contributes to the low expression efficiency of viral proteins. In order to achieve high-level Rev-independent expression of the Gag protein, the sequences encoding HIV-1(SF2) p55(Gag) were modified extensively. First, the viral codons were changed to conform to the codon usage of highly expressed human genes, and second, the residual inhibitory sequences were removed. The resulting modified gag gene showed increases in p55(Gag) protein expression to levels that ranged from 322- to 966-fold greater than that for the native gene after transient expression of 293 cells. Additional constructs that contained the modified gag in combination with modified protease coding sequences were made, and these showed high-level Rev-independent expression of p55(Gag) and its cleavage products. Density gradient analysis and electron microscopy further demonstrated that the modified gag and gag protease genes efficiently expressed particles with the density and morphology expected for HIV virus-like particles. Mice immunized with DNA plasmids containing the modified gag showed Gag-specific antibody and CD8(+) cytotoxic T-lymphocyte (CTL) responses that were inducible at doses of input DNA 100-fold lower than those associated with plasmids containing the native gag gene. Most importantly, four of four rhesus monkeys that received two or three immunizations with modified gag plasmid DNA demonstrated substantial Gag-specific CTL responses. These results highlight the useful application of modified gag expression cassettes for increasing the potency of DNA and other gene delivery vaccine approaches against HIV.

Eradication of helminthic infections may be essential for successful vaccination against HIV and tuberculosis.

The current epidemics of tuberculosis and acquired immunodeficiency syndrome (AIDS) (caused, respectively, by infection with Mj,cobacterium tuberculosis (MTB) and human immunodeficiency virus (HIV)) are a major cause for concern. No successful or effective anti-HIV vaccine has been yet developed, and Bacille Calmette-Guarin (13CG) has failed to confer protection against tuberculosis in developing countries. Nevertheless, apart from the use of social and educational measures, the only plausible way to overcome these epidemics is through mass vaccination. An effective protective vaccine against MTB or HIV infection should generate a potent cellular immune response, which is dependent on a dominant T-helper type 1 (TH1) cellular response, rather than a T-helper type 2 (TH2) humoral immune response. These two cch types cross-regulate each other and thus cytokines produced by one T-helper subset can suppress the production and/or activity of the other. Helminthic infections affect more than a third of the world's population, and have a similar geographical distribution to that of HIV and tuberculosis. In developing countries, children born in areas where intestinal nematodes are endemic harbour worms for most of their lives. Individuals with helminth infections are chronically immune-activated and have a very pronounced TH2 immune profile. We have hypothesized that the chronic immune activation and TH2 immune profile caused by helminthic infections make the host more susceptible to HIV infection and less able to cope with it once infected. This may play a major role in the pathogenesis of AIDS in Africa (Bentwich Z et al. Immunology Today, 1995, 16: 187-191) and account for the widespread tuberculosis in developing countries (]3entwich Z et al. Immunology Today, 1995, 201 485-487). Furthermore, intestinal helminth infections may also compromise the generation of protective immunity upon vaccination for both HIV and tuberculosis. This hypothesis is based on the following observations: several populations in Africa and South-east Asia have a pre-existent dominant TH2 cytokine profile and extremely high immune activation (e.g. Bentwich Z et al. Clinical and Experimental Immunology; 1996, 103: 239-243); lymphocytes isolated from Ethiopian immigrants recently arrived in Israel had very impaired signal transduction and anergy following stimulation; there was a clear inverse correlation between immune activation in these Ethiopian immigrants and the capacity of their lymphocytes to proliferate and secrete chemokines following stimulation with tuberculin purified protein derivative (PPD) (r= -0.58, P [is less than] 0.002); helminthinfected Ethiopian immigrants responded poorly to PPD skin test compared with such immigrants following deworming (P [is less than] 0.005) (Borkov G. et al. Journal of Clinical Investigation, in press). Why do the populations of developing countries exhibit such immune profiles? Though several factors could contribute to it, such as constant exposure to infectious diseases, poor hygiene and malnutrition, we have proposed that it is mainly a consequence ofhelminthic infections (e.g. Kalinkovich A et al. Clinical and Experimental Immunology, 1998,114: 414-421). This conclusion is largely based on the following evidence: the high prevalence of helminthic infections among the Ethiopian immigrants in Israel ([is greater than] 90% were infested with at least one parasite, while some had even 5 different parasitic infections); the immune profile of the Ethiopian immigrants returned to normal following the eradication of the helminthic infections; helminth eradication had also a clear effect on the HIV infection/ disease, i.e. once their helminth infections had been eradicated the response of the Ethiopian immigrants to highly active antiretroviral treatment was similar to that of other Israeli. inhabitants (Weisman Z et al. Journal of AIDS, 1999, 21: 157-163); preliminary results of a study carried out in Ethiopia show that eradication of helminthic infections in people infected with both these and HIV is associated with significant decreases in HIV plasma viral load. …

Histocompatibility Testing in Organ Transplantation

The human leukocyte antigen (HLA) system is crucial in lymphocyte activation, and HLA antigens can provoke potent humoral and cellular immune responses that mediate allograft rejection. Matching for HLA promotes the successful outcome of transplanting an organ such as a kidney or a heart. The role of the tissue-typing laboratory is to assess the degree of HLA compatibility between donor and recipient, and by serum screening and crossmatching to determine the presence of donor-specific antibodies that might be harmful to the organ transplant.

Differential methylation of Epstein-Barr virus latency promoters facilitates viral persistence in healthy seropositive individuals.

Epstein-Barr virus (EBV) establishes a life-long infection in humans, with distinct viral latency programs predominating during acute and chronic phases of infection. Only a subset of the EBV latency-associated antigens present during the acute phase of EBV infection are expressed in the latently infected memory B cells that serve as the long-term EBV reservoir. Since the EBV immortalization program elicits a potent cellular immune response, downregulation of viral gene expression in the long-term latency reservoir is likely to facilitate evasion of the immune response and persistence of EBV in the immunocompetent host. Tissue culture and tumor models of restricted EBV latency have consistently demonstrated a critical role for methylation of the viral genome in maintaining the restricted pattern of latency-associated gene expression. Here we extend these observations to demonstrate that the EBV genomes in the memory B-cell reservoir are also heavily and discretely methylated. This analysis reveals that methylation of the viral genome is a normal aspect of EBV infection in healthy immunocompetent individuals and is not restricted to the development of EBV-associated tumors. In addition, the pattern of methylation very likely accounts for the observed inhibition of the EBV immortalization program and the establishment and maintenance of a restricted latency program. Thus, EBV appears to be the first example of a parasite that usurps the host cell-directed methylation system to regulate pathogen gene expression and thereby establish a chronic infection.

Immunogenicity and protective efficacy of DNA vaccines encoding secreted and non-secreted forms of Mycobacterium tuberculosis Ag85A

Objective: To determine the efficacy of Ag85A-DNA against challenge with a highly virulent human clinical isolate of Mycobacterium tuberculosis (CSU37) and to compare the potencies of two types of Ag85A-DNA vaccines; those expressing secreted and non-secreted forms of the protein.Design: Ag85A-DNA vaccinated mice were challenged with a highly virulent clinical isolate of M. tuberculosis (CSU37) in order to compare the efficacy of these vaccines. In vitro studies were also performed.Results: Enhanced humoral and cellular responses were induced in mice vaccinated with the secreted Ag85A-DNA compared to the non-secreted Ag85A-DNA. In addition, secreted Ag85A-DNA conferred protective immunity against infection with M. tuberculosis (CSU37).Conclusions: DNA vaccines encodingM. tuberculosis Ag85A have been shown to induce potent humoral and cellular immune responses leading to protection fromM. tuberculosis (Erdman) challenge in mouse models.1In this study we demonstrate that Ag85A can confer protection in a rigorous challenge model using a highly virulent human clinical isolate of M. tuberculosis (CSU37). This challenge model appears able to discriminate between DNA vaccines of differing potencies, as the more immunogenic DNA construct encoding a secreted form of Ag85A was protective, whereas the less immunogenic DNA construct encoding a non-secreted form of Ag85A was not.

Enhancement of experimental allergic encephalomyelitis (EAE) by DNA immunization with myelin proteolipid protein (PLP) plasmid DNA.

Relapsing-remitting experimental allergic encephalomyelitis (R-EAE) is an animal model for multiple sclerosis (MS). Many potential immunomodulatory strategies for MS have been used first in EAE to assess their effectiveness. Recently, the injection of plasmid DNA has been shown to induce potent humoral and cellular immune responses. The primary aim of our experiments reported here was to determine if vaccination with cDNAs encoding myelin proteolipid protein (PLP) could prime for a PLP-specific immune response and affect subsequent R-EAE. We constructed cDNAs encoding whole PLP (pPLP(all)) or encephalitogenic epitopes PLP(139-151) (pPLP(139-151)) and PLP(178-191) (pPLP(178-191)). Following DNA injection, we induced R-EAE in SJL/J mice using PLP(139-151) or PLP(178-191) peptides in adjuvant. All 3 plasmid constructs enhanced R-EAE induced with PLP(139-151), and injection of mice with pPLP(all) increased R-EAE induced with PLP(178-191). DNA immunization induced higher PLP peptide-specific lymphoproliferative responses than did vector alone following R-EAE induction with IgG1 or IgG2b antibody responses. These data suggest that DNA immunization of PLP can modulate immune responses, leading to enhancement of R-EAE.

Specificity of helper T-cells generated from macaques infected with attenuated simian immunodeficiency virus.

Deletion of the simian immunodeficiency virus (SIV) nef gene leads to an attenuated virus phenotype in vivo. We have previously shown that these viruses induce a potent cellular immune response in macaques. To extend these studies, we established virus-specific short-term T-cell lines from four rhesus macaques infected with a nef deletion mutant of SIV. These T-cell lines proliferated upon restimulation with whole SIV or SIV gp140 antigen in vitro. The proliferating cells were characterized as CD4+ helper T-cells (TH) and their antigen recognition was MHC class II DR-restricted. After antigenic stimulation, they transcribed mRNA for various TH1- and TH2-like cytokines. Using these SIV-specific cell lines, a variety of helper T-cell epitopes in the SIV Env protein were determined with overlapping peptides. TH epitopes were identified throughout the whole SIV Env including both constant and variable regions. Although the recognition of TH epitopes was heterogeneous among different animals, five more broadly reactive T-cell epitopes were identified. As expected, recognition was associated with the MHC class II DRB background of the animals. This is the first report on helper T-cell epitopes in SIV-infected monkeys. Such studies should be of considerable significance for AIDS/ vaccine research.

Gene vaccination with naked plasmid DNA: mechanism of CTL priming.

The injection of naked plasmid DNA directly into the muscle cells of mice has been shown to induce potent humoral and cellular immune responses. The generation of a cytotoxic T lymphocyte (CTL) response after plasmid DNA injection may involve the presentation of the expressed antigen in the context of the injected myocytes' endogenous major histocompatibility (MHC)-encoded class I molecules or may use the MHC molecules of bone marrow-derived antigen presenting cells (APC) which are capable of providing co-stimulation as well. To resolve which cell type provides the specific restricting element for this method of vaccination we generated parent-->F1 bone marrow chimeras in which H-2bxd recipient mice received bone marrow that expressed only H-2b or H-2d MHC molecules. These mice were injected intramuscularly with naked plasmid DNA that encoded the nucleoprotein from the A/PR/8/34 influenza strain, which as a single antigen has epitopes for both H-2Db and H-2Kd. The resulting CTL responses were restricted to the MHC haplotype of the bone marrow alone and not to the second haplotype expressed by the recipient's myocytes. The role of somatic tissues that express protein from injected plasmids may be to serve as a reservoir for that antigen which is then transferred to the APC. Consequently, our data show that the mechanism of priming in this novel method for vaccination uses the MHC from bone marrow-derived APC, which are efficient at providing all of the necessary signals for priming the T cell.

Particle-mediated nucleic acid immunization

Nucleic acid immunization involves the direct in vivo administration of antigen-encoding plasmid DNA molecules that results in the de novo production of correctly folded microbial antigens at the site of DNA delivery. While this process can lead to the development of neutralizing antibody responses recognizing authentic protein conformations, in vivo antigen production also results in epitope presentation via the MHC class I antigen processing pathway, leading to the elicitation of cytotoxic cellular immune responses. Recent efforts in the authors' laboratories have focused on use of the Accell ® gene delivery system (gene gun) to achieve the direct, intracellular delivery of small quantities of DNA into cells of the epidermis. The gene gun approach to nucleic acid vaccination capitalizes on the synergistic combination of an effective DNA delivery system and a target tissue that serves as a major immunological inductive site. Experimental gene gun-based nucleic acid vaccines can achieve potent humoral and cytotoxic cellular immune responses in rodent models following immunization with as little as 16 ng of DNA. Equally strong responses have also been elicited in larger animals, such as pigs and monkeys, following epidermal immunization with as little as 2 to 4 μg of DNA.

Immunization with plasmid DNA using a pneumatic gun

We characterize a method by which the Med-E-Jet pneumatic vaccination gun can be used to propel intact, supercoiled plasmid DNA through skin and into skeletal muscles of mice. Intramuscular injection of plasmids containing the firefly luciferase gene linked to the human cytomegalovirus promoter resulted in the expression of several hundred picograms of luciferase enzyme in quadriceps muscles. Intramuscular injections of a plasmid containing the influenza A nuclear protein gene regulated by the same promoter resulted in the generation of potent and specific anti-nuclear protein humoral and cellular immune responses. This convenient and rapid injection method would be well-suited for genetic immunization of humans.

Oral Salmonella typhimurium vaccine expressing circumsporozoite protein protects against malaria.

Immunization with radiation-attenuated malaria sporozoites induces potent cellular immune responses, but the target antigens are unknown and have not previously been elicited by subunit vaccines prepared from the circumsporozoite (CS) protein. A method is described here for inducing protective cell-mediated immunity to sporozoites by immunization with attenuated Salmonella typhimurium transformed with the Plasmodium berghei CS gene. These transformants constitutively express CS antigens and, when used to immunize mice orally, colonize the liver, induce antigen-specific cell-mediated immunity, and protect mice against sporozoite challenge in the absence of antisporozoite antibodies. These data indicate that the CS protein contains T cell epitopes capable of inducing protective cell-mediated immunity, and emphasize the importance of proper antigen presentation in generating this response. Analogous, orally administered vaccines against human malaria might be feasible.