Lentiviral transduction of dendritic cells confers protective antiviral immunity in vivo

Control of a viral infection in vivo requires a rapid and efficient cytotoxic-T-lymphocyte response. We demonstrate that lentivirus-mediated introduction of antigen in dendritic cells confers a protective antiviral immunity in vivo in a lymphocytic choriomeningitis virus model. Therefore, lentiviral vectors may be excellent vaccine candidates for viral infections.     

Acute resolving woodchuck hepatitis virus (WHV) infection is associated with a strong cytotoxic T-lymphocyte response to a single WHV core peptide

Woodchucks infected with woodchuck hepatitis virus (WHV) are an excellent model for studying acute, self-limited and chronic hepadnaviral infections. Defects in the immunological response leading to chronicity are still unknown. Specific T-helper cell responses to WHV core and surface antigens (WHcAg and WHsAg, respectively) are associated with acute resolving infection; however, they are undetectable in chronic infection. Up to now, cytotoxic T-lymphocyte (CTL) responses could not be determined in the woodchuck. In the present study, we detected virus-specific CTL responses by a CD107a degranulation assay. The splenocytes of woodchucks in the postacute phase of WHV infection (18 months postinfection) were isolated and stimulated with overlapping peptides covering the whole WHcAg. After 6 days, the cells were restimulated and stained for CD3 and CD107a. One peptide (c96-110) turned out to be accountable for T-cell expansion and CD107a staining. Later, we applied the optimized degranulation assay to study the kinetics of the T-cell response in acute WHV infection. We found a vigorous T-cell response against peptide c96-110 with peripheral blood cells beginning at the peak of viral load (week 5) and lasting up to 15 weeks postinfection. In contrast, there was no T-cell response against peptide c96-110 detectable in chronically WHV-infected animals. Thus, with this newly established flow cytometric degranulation assay, we detected for the first time virus-specific CTLs and determined one immunodominant epitope of WHcAg in the woodchuck.     

A novel CD8-independent high-avidity cytotoxic T-lymphocyte response directed against an epitope in the phosphoprotein of the paramyxovirus simian virus 5

Adoptive transfer studies have shown that cytotoxic T lymphocytes (CTL) of high avidity, capable of recognizing low levels of peptide-MHC I molecules, are more efficient at reducing viral titers than are low-avidity CTL, thus establishing CTL avidity as a critical parameter for the ability of a CTL to clear virus in vivo. It has been well documented that CTL of high avidity are relatively CD8 independent, whereas low-avidity CTL require CD8 engagement in order to become activated. In this study we have analyzed the antiviral CTL response elicited following infection with the paramyxovirus simian virus 5 (SV5). We have identified the immunodominant and subdominant CTL responses and subsequently assessed the avidity of these responses by their CD8 dependence. This is the first study in which the relationship between immunodominance and CTL avidity has been investigated. The immunodominant response was directed against an epitope present in the viral M protein, and subdominant responses were directed against epitopes present in the P, F, and HN proteins. Similarly to other CTL responses we have analyzed, the immunodominant response and the subdominant F and HN responses were comprised of both high- and low-avidity CTL. However, the subdominant response directed against the epitope present in the P protein is novel, as it is exclusively high avidity. This high-avidity response is independent of both the route of infection and expression by recombinant SV5. A further understanding of the inherent properties of P that elicit only high-avidity CTL may allow for the design of more efficacious vaccine vectors that preferentially elicit high-avidity CTL in vivo.     

Molecular basis of viral persistence: a single amino acid change in the glycoprotein of lymphocytic choriomeningitis virus is associated with suppression of the antiviral cytotoxic T-lymphocyte response and establishment of persistence.

Isolates of lymphocytic choriomeningitis virus (LCMV) that elicit a cytotoxic T-lymphocyte response (CTL+) have been compared with isolates that suppress the CTL response (CTL-) in an effort to map this phenotype. A single amino acid change in the glycoprotein of the LCMV Armstrong (ARM) strain is consistently associated with the CTL- trait and the ability of the virus to persist (P+). The CTL+ P- parental strain spontaneously gives rise to CTL- P+ variants within lymphoid tissues of mice persistently infected from birth. To map the structural basis of the phenotype, the complete RNA sequence of LCMV ARM 53b (CTL+) was compared with that of its variant ARM clone 13 (CTL-). Differences in 5 of 10,600 nucleotides were found. Three changes are noted in the large L RNA segment, and two are noted in the small S RNA segment. Only two of the changes distinguishing CTL+ from CTL- isolates affect amino acid coding: lysine to glutamine at amino acid 1079 of the polymerase protein, and phenylalanine to leucine at amino acid 260 of the envelope glycoprotein (GP). We also analyzed two additional CTL- variants and four spontaneous CTL+ revertants. All three CTL- variants differ from the original CTL+ parental strain at GP amino acid 260, indicating that this amino acid change is consistently associated with the CTL- phenotype. By contrast the other four mutations in LCMV are not associated with the CTL- phenotype. Sequence analysis of the coding regions of four CTL+ revertants of ARM clone 13 did not reveal back mutations at the GP 260 locus. This finding indicates that the GP 260 mutation is necessary but not sufficient for a CTL- P+ phenotype and that the reversion to CTL+ P- is likely either due to secondary mutations in other regions of the viral genome or to quasispecies within the revertant population that make significant contributions to the phenotype.     

In vivo cytotoxic T lymphocyte induction with soluble proteins administered in liposomes.

The in vivo induction of a CTL response usually requires that Ag be endogenously synthesized so that appropriate processing can occur. In most of the few examples where successful CTL induction was reported with proteins and peptides, unacceptable adjuvants or means of Ag formulation were used. In the present report, liposomes were used to incorporate the soluble proteins OVA and beta-galactosidase. This simple and convenient to use approach, which requires minimal amounts of Ag, results in priming for a CD8+ CTL response and the establishment of immunologic memory. The liposome approach may not only prove a convenient means of inducing CTL responses in vivo but may also be useful to study the mechanisms of Ag processing.     

Generation of a cytotoxic T-lymphocyte response using a Salmonella antigen-delivery system

We have constructed a general-use vector for the cloning and stable expression of foreign genes in the chromosome of attenuated Salmonella typhimurium. Using this chromosomal expression vector (CEV), we expressed the circumsporozoite (CS) gene of the mouse malaria Plasmodium yoelii in an aroA S. typhimurium strain. Mice immunized with CS-expressing Salmonella recombinants mount a CS-specific cytotoxic T-lymphocyte (CTL) response. This is the first demonstration that attenuated Salmonella can elicit a specific CTL response to a foreign protein in mice. The ability to easily and stably express foreign genes from the Salmonella chromosome and the generation of specific CTL greatly expands the potential of Salmonella as an antigen-delivery system.     

The role of the cytotoxic T-lymphocyte response and virus cytopathogenicity in the virus decline during antiviral therapy

Although it is clear that HIV can lyse HIV-infected CD4 T cells, it is still controversial whether the depletion of CD4 T cells seen in HIV-infected patients after years of asymptomatic disease is caused by the direct cytopathic effects of the virus or is mediated by the immune response. Assuming the initial decline in viraemia during highly active antiretroviral therapy (HAART) is caused by the death of cells productively infected with HIV, I investigate how the rate of the virus decline is affected by the efficiency of the cytotoxic T-lymphocyte (CTL) response. I find that whether the stronger immune response causes a more rapid virus decline depends critically on how the virus is controlled by the CTL response (lytic versus non-lytic mechanisms). Moreover, variation in the efficiency of the immune response does not always cause variation in the rate of the virus decline (and, therefore, in the death rate of infected cells), implying that the constancy of the virus decline rate measured in different patients does not necessarily indicate that the virus is cytopathic. The potential problems associated with the model and the approach undertaken are also discussed.     

In vivo induction of cytotoxic T cell response by a free synthetic peptide requires CD4+ T cell help.

Most attempts to induce CTL responses by in vivo priming with free synthetic peptides have been unsuccessful so far. However, two separate studies have recently succeeded in inducing antiviral CTL responses by immunizing mice with unmodified free synthetic peptides derived from nucleoproteins from either lymphocytic choriomeningitis virus or Sendai virus. In the present study, we have analyzed the cellular mechanisms by which the lymphocytic choriomeningitis virus synthetic peptide induced CTL responses. We demonstrated that this peptide, which was previously shown to be recognized by CD8+ T cells, also contains a helper CD4+ T cell epitope. It stimulates in vivo both CD4+ T cell-mediated CTL response. The in vivo elimination of CD4+ T cells by treatment with a mAb was shown to strongly reduce the antipeptide CTL response. This study therefore demonstrates that to be able to induce CTL responses, a peptide has to stimulate both CD4+ and CD8+ T cell subset.     

DNA immunization: Effects of vehicle and route of administration on the induction of protective antiviral immunity

Abstract The effectiveness of DNA immunization has been demonstrated in several model systems, usually following intramuscular injection of DNA in saline, or topical administration to the skin. In this study we have compared DNA delivered by three routes (intramuscular, intravenous, and intraperitoneal) and, for each route, in two vehicles (cationic liposome complex and pH sensitive liposome). These two lipid vehicles were evaluated because they are frequently used in gene therapy studies, but their immunogenicity has not been extensively studied. Each of these six combinations has been evaluated not only by assay of marker gene expression in a variety of tissues, but also by measurement of biologically-relevant parameters of immunity induction of antibodies, cytotoxic T lymphocytes, and protection against viral challenge. By both criteria (marker gene expression and induced immunity), the outcomes vary markedly among the six combinations. The combination leading to maximal marker gene expression (DNA with cationic lipid, administered i.v.) also induces detectable antibodies and CTL, and is the only one of the six combinations to induce immune responses comparable to those seen following i.m. injection of DNA in saline. However, marker gene expression can be detected in other combinations in the absence of induced immunity thus the value of marker gene expression in predicting the protection induced by a microbial antigen is questionable suggesting that, when evaluating various promoter constructs, marker gene expression may not adequately replace the direct measurement of biological outcomes.     

An early, abundant cytotoxic T-lymphocyte response against Theiler's virus is critical for preventing viral persistence.

In genetically susceptible strains of mice, the DA strain of Theiler's virus, a picornavirus, causes a persistent infection of the white matter of the spinal cord associated with chronic demyelination. In resistant strains, on the other hand, the infection is cleared within 1 to 2 weeks. In this article, we show that Theiler's virus induces a rapid and abundant cytotoxic T lymphocyte (CTL) response in resistant C57BL/6 mice, while the response remains low throughout infection in susceptible SJL/J mice. This difference can be referred to a higher number of virus-specific CTL precursors in C57BL/6 mice. These observations indicate that the efficient induction of virus-specific CTL precursors is critical for avoiding the establishment of a persistent picornaviral infection.     

The cytotoxic T-lymphocyte response to Sendai virus is unimpaired in the absence of gamma interferon.

Sendai virus is eliminated from the respiratory tract of gamma interferon (IFN-gamma) -/- BALB/c mice with normal kinetics. The level of virus-specific cytotoxic T-lymphocyte (CTL) activity in the cell population recovered by bronchoalveolar lavage is unimpaired, the prevalence of interleukin-4 (IL-4)-producing cells is increased, and the titers of virus-specific immunoglobulins IgG1 and IgG2b are higher in the IFN-gamma -/- mice. The emergence of this T-helper 2 response profile in both lymphoid tissue and the pneumonic lung has no obvious deleterious consequences. Virus clearance is slightly delayed following depletion of the CD4+ subset, with the effect being similar in magnitude for IFN-gamma -/- and +/+ mice. However, the generation of CTL precursors (CTLp) is diminished in the IFN-gamma -/- (but not +/+) mice in the absence of concurrent CD4+ T help. Apparently the clonal expansion of the CTLp population can be promoted either by a cytokine (perhaps IL-2) produced by the IFN-gamma -/- CD4+ T cells or by IFN-gamma made by other cell types in the +/+ mice.     

Expression of interleukin-4 by recombinant respiratory syncytial virus is associated with accelerated inflammation and a nonfunctional cytotoxic T-lymphocyte response following primary infection but not following challenge with wild-type virus

The outcome of a viral infection or of immunization with a vaccine can be influenced by the local cytokine environment. In studies of experimental vaccines against respiratory syncytial virus (RSV), an increased stimulation of Th2 (T helper 2) lymphocytes was associated with increased immunopathology upon subsequent RSV infection. For this study, we investigated the effect of increased local expression of the Th2 cytokine interleukin-4 (IL-4) from the genome of a recombinant RSV following primary infection and after a challenge with wild-type (wt) RSV. Mice infected with RSV/IL-4 exhibited an accelerated pulmonary inflammatory response compared to those infected with wt RSV, although the wt RSV group caught up by day 8. In the first few days postinfection, RSV/IL-4 was associated with a small but significant acceleration in the expansion of pulmonary T lymphocytes specific for an RSV CD8(+) cytotoxic T-lymphocyte (CTL) epitope presented as a major histocompatibility complex class I tetramer. However, by day 7 the response of tetramer-positive T lymphocytes in the wt RSV group caught up and exceeded that of the RSV/IL-4 group. At all times, the CTL response of the RSV/IL-4 group was deficient in the production of gamma interferon and was nonfunctional for in vitro cell killing. The accelerated inflammatory response coincided with an accelerated accumulation and activation of pulmonary dendritic cells early in infection, but thereafter the dendritic cells were deficient in the expression of B7-1, which governs the acquisition of cytolytic activity by CTL. Following a challenge with wt RSV, there was an increase in Th2 cytokines in the animals that had previously been infected with RSV/IL-4 compared to those previously infected with wt RSV, but the CD8(+) CTL response and the amount of pulmonary inflammation were not significantly different. Thus, a strong Th2 environment during primary pulmonary immunization with live RSV resulted in early inflammation and a largely nonfunctional primary CTL response but had a minimal effect on the secondary response.     

Progress toward a malaria vaccine: efficient induction of protective anti-malaria immunity

Malaria can be a very severe disease, particularly in young children, pregnant women (mostly in primipara), and malaria na?ve adults, and currently ranks among the most prevalent infections in tropical and subtropical areas throughout the world. The widespread occurrence and the increased incidence of malaria in many countries, caused by drug-resistant parasites (Plasmodium falciparum and P. vivax) and insecticide-resistant vectors (Anopheles mosquitoes), indicate the need to develop new methods of controlling this disease. Experimental vaccination with irradiated sporozoites can protect animals and humans against the disease, demonstrating the feasibility of developing an effective malaria vaccine. However, developing a universally effective, long lasting vaccine against this parasitic disease has been a difficult task, due to several problems. One difficulty stems from the complexity of the parasite's life cycle. During their life cycle, malaria parasites change their residence within the host, thus avoiding being re-exposed to the same immunological environment. These parasites also possess some distinct antigens, present at different life stages of the parasite, the so-called stage-specific antigens. While some of the stage-specific antigens can induce protective immune responses in the host, these responses are usually genetically restricted, this being another reason for delaying the development of a universally effective vaccine. The stage-specific antigens must be used as immunogens and introduced into the host by using a delivery system that should efficiently induce protective responses against the respective stages. Here we review several research approaches aimed at inducing protective anti-malaria immunity, overcoming the difficulties described above.     

In vivo modulation of T cell responses and protective immunity by TCR antagonism during infection

Infectious agents are known to express altered peptide ligands that antagonize T cells in vitro; however, direct evidence of TCR antagonism during infection is still lacking, and its importance in the context of infection remains to be established. In this study, we used a murine model of infection with recombinant Listeria monocytogenes and addressed three issues that are critical for assessing the role of TCR antagonism in the modulation of the immune response. First, we demonstrated that the antagonist peptide efficiently inhibited the ability of the agonist to prime naive TCR-transgenic T cells in vivo. Second, we showed clonal memory T cells were antagonized during recall responses, resulting in loss of protective immunity. Lastly, we observed that even in the context of a polyclonal response, TCR antagonism greatly inhibits the agonist-specific response, leading to altered hierarchy of immunodominance and reduced T cell memory and protective immunity. These results provide direct evidence of clonal TCR antagonism of naive and memory CD8 T cells during infection and demonstrate the effect of TCR antagonism on protective immunity. Thus, agonist/antagonist interactions may play an important role in determining the immunodominance and repertoire of T cell targets, and evaluation of immune responses and vaccine strategies may require examination of not only agonists but also antagonists and their interactions during an infection.     

In vivo molecular imaging analysis of a nasal vaccine that induces protective immunity against botulism in nonhuman primates

Nasal administration is an effective route for a needle-free vaccine. However, nasally administered Ags have the potential to reach the CNS directly from the nasal cavity, thus raising safety concerns. In this study, we performed real-time quantitative tracking of a nasal vaccine candidate for botulism, which is a nontoxic subunit fragment of Clostridium botulinum type A neurotoxin (BoHc/A) effective in the induction of the toxin-neutralizing immune response, by using (18)F-labeled BoHc/A-positron-emission tomography, an in vivo molecular imaging method. This method provides results that are consistent with direct counting of [(18)F] radioactivity or the traditional [(111)In]-radiolabel method in dissected tissues of mice and nonhuman primates. We found no deposition of BoHc/A in the cerebrum or olfactory bulb after nasal administration of (18)F-labeled BoHc/A in both animals. We also established a real-time quantitative profile of elimination of this nasal vaccine candidate and demonstrated that it induces highly protective immunity against botulism in nonhuman primates. Our findings demonstrate the efficiency and safety of a nasal vaccine candidate against botulism in mice and nonhuman primates using in vivo molecular imaging.     

Complement activation is required for induction of a protective antibody response against West Nile virus infection

Infection with West Nile virus (WNV) causes a severe infection of the central nervous system (CNS) with higher levels of morbidity and mortality in the elderly and the immunocompromised. Experiments with mice have begun to define how the innate and adaptive immune responses function to limit infection. Here, we demonstrate that the complement system, a major component of innate immunity, controls WNV infection in vitro primarily in an antibody-dependent manner by neutralizing virus particles in solution and lysing WNV-infected cells. More decisively, mice that genetically lack the third component of complement or complement receptor 1 (CR1) and CR2 developed increased CNS virus burdens and were vulnerable to lethal infection at a low dose of WNV. Both C3-deficient and CR1- and CR2-deficient mice also had significant deficits in their humoral responses after infection with markedly reduced levels of specific anti-WNV immunoglobulin M (IgM) and IgG. Overall, these results suggest that complement controls WNV infection, in part through its ability to induce a protective antibody response.