Matthias Regner

Pivotal Role of Antibody and Subsidiary Contribution of CD8+ T Cells to Recovery from Infection in a Murine Model of Japanese Encephalitis

ABSTRACT The immunological correlates for recovery from primary Japanese encephalitis virus (JEV) infection in humans and experimental animals remain poorly defined. To investigate the relative importance of the adaptive immune responses, we have established a mouse model for Japanese encephalitis in which a low-dose virus inoculum was administered into the footpads of adult C57BL/6 mice. In this model, ∼60% of the mice developed a fatal encephalitis and a virus burden in the central nervous system (CNS). Using mice lacking B cells (μMT−/− mice) and immune B cell transfer to wild-type mice, we show a critically important role for humoral immunity in preventing virus spread to the CNS. T cell help played an essential part in the maintenance of an effective antibody response necessary to combat the infection, since mice lacking major histocompatibility complex class II showed truncated IgM and blunted IgG responses and uniformly high lethality. JEV infection resulted in extensive CD8+ T cell activation, judged by upregulation of surface markers CD69 and CD25 and cytokine production after stimulation with a JEV NS4B protein-derived H-2Db-binding peptide and trafficking of virus-immune CD8+ T cells into the CNS. However, no significant effect of CD8+ T cells on the survival phenotype was found, which was corroborated in knockout mice lacking key effector molecules (Fas receptor, perforin, or granzymes) of cytolytic pathways triggered by T lymphocytes. Accordingly, CD8+ T cells are mostly dispensable for recovery from infection with JEV. This finding highlights the conflicting role that CD8+ T cells play in the pathogenesis of JEV and closely related encephalitic flaviviruses such as West Nile virus.

MHC class I up-regulation by flaviviruses: Immune interaction with unknown advantage to host or pathogen.

In contrast to many other viruses that escape from cytotoxic T cell recognition by down‐regulating major histocompatibility complex class I‐restricted antigen presentation, flavivirus infection of mammalian cells up‐regulates cell surface expression of major histocompatibility complex class I molecules. Two putative mechanisms for flavivirus‐induced major histocompatibility complex class I up‐regulation, one via activation of the transcription factor NF‐κB, the second by augmentation of peptide import into the lumen of the endoplasmic reticulum, are reviewed, and the biological effect of the flavivirus‐mediated phenomenon on target cell recognition by natural killer and cytotoxic T cells is addressed. Finally, we speculate on the physiological role of flavivirus‐mediated modulation of major histocompatibility complex class I antigen presentation in the context of the biology of flavivirus transmission between the vertebrate host and arthropod vector and suggest that it may represent a strategy for immune evasion from the natural killer cell response or, alternatively, that up‐regulation of major histocompatibility complex class I is a by‐product of flavivirus replication without significance for virus growth.

Cytotoxic T Cells Are the Predominant Players Providing Cross-Protective Immunity Induced by γ-Irradiated Influenza A Viruses

ABSTRACT We previously demonstrated that a single dose of nonadjuvanted intranasal γ-irradiated influenza A virus can provide robust protection in mice against both homologous and heterosubtypic challenges, including challenge with an H5N1 avian virus strain. We investigated the mechanism behind the observed cross-protection to define which arms of the adaptive immune response are involved in mediating this protection. Studies with gene knockout mice showed the cross-protective immunity to be mediated mainly by T cells and to be dependent on the cytolytic effector molecule perforin. Adoptive transfer of memory T cells from immunized mice, but not of memory B cells, protected naïve recipients against lethal heterosubtypic influenza virus challenge. Furthermore, γ-irradiated influenza viruses induced cross-reactive Tc-cell responses but not cross-neutralizing or cross-protective antibodies. In addition, histological analysis showed reduced lung inflammation in vaccinated mice compared to that in unvaccinated controls following heterosubtypic challenge. This reduced inflammation was associated with enhanced early recruitment of T cells, both CD4+ and CD8+, and with early influenza virus-specific cytotoxic T-cell responses. Therefore, cross-protective immunity induced by vaccination with γ-irradiated influenza A virus is mediated mainly by Tc-cell responses.

Interferon type I responses in primary and secondary infections.

The mammalian host responds to a microbial infection with a rapid innate immune reaction that is dominated by type I interferon (IFN‐I) release. Most cells of vertebrates can respond to microbial attack with IFN‐I production, but the cell type responsible for most of the systemic IFN‐I release is thought to be plasmacytoid dendritic cells (pDCs). Besides its anti‐microbial and especially anti‐viral properties IFN‐I also exerts a regulatory role on many facets of the sequential adaptive immune response. One of these is being the recently described partial, systemic activation of the vast majority of B and T lymphocytes in mice, irrespective of antigen reactivity. The biological significance of this partial activation of lymphocytes is at present speculative. Secondary infections occurring within a short time span of a primary infection fail to elicit a similar lymphocyte activation response due to a refractory period in systemic IFN‐I production. This period of exhaustion in IFN‐I responses is associated with an increased susceptibility of the host to secondary infections. The latter correlates with well‐established clinical observations of heightened susceptibility of patients to secondary microbial infections after viral episodes.

Intranasal Flu Vaccine Protective against Seasonal and H5N1 Avian Influenza Infections

Background Influenza A (flu) virus causes significant morbidity and mortality worldwide, and current vaccines require annual updating to protect against the rapidly arising antigenic variations due to antigenic shift and drift. In fact, current subunit or split flu vaccines rely exclusively on antibody responses for protection and do not induce cytotoxic T (Tc) cell responses, which are broadly cross-reactive between virus strains. We have previously reported that γ-ray inactivated flu virus can induce cross-reactive Tc cell responses. Methodology/Principal Finding Here, we report that intranasal administration of purified γ-ray inactivated human influenza A virus preparations (γ-Flu) effectively induces heterotypic and cross-protective immunity. A single intranasal administration of γ-A/PR8[H1N1] protects mice against lethal H5N1 and other heterotypic infections. Conclusions/Significance Intranasal γ-Flu represents a unique approach for a cross-protective vaccine against both seasonal as well as possible future pandemic influenza A virus infections.

Type I Interferons Trigger Systemic, Partial Lymphocyte Activation in Response to Viral Infection

The vast majority of both T and B cells in mice were found to up-regulate cell surface expression of the early activation markers CD69 and CD86, but not CD25, within 24 h of infection with Semliki Forest virus. Kinetics and magnitude of activation marker expression was dependent on live virus, dose, and correlated with strain virulence. Activation marker expression declined to baseline levels over the next 96 h. This very early “activation” of such a high percentage of lymphocytes required the presence of type I IFN receptor genes, was inducible with poly(I:C), and correlated with IFN-I levels in serum. We conclude that virus-induced IFN-I release systemically affects most of the hosts T and B cells by triggering them rapidly and independently of Ag-reactivity into a semiactivated state.

Effect of inactivation method on the cross-protective immunity induced by whole 'killed' influenza A viruses and commercial vaccine preparations.

We have recently shown that intranasal (i.n.) administration of gamma-irradiated A/PR/8 [A/Puerto Rico/8/34 (H1N1)] protects mice against lethal avian influenza A/Vietnam/1203/2004 (H5N1) and other heterosubtypic influenza A infections. Here, we used gamma-irradiated, formalin- and UV-inactivated A/PC [A/Port Chalmers/1/73 (H3N2)] virus preparations and compared their ability to induce both homologous and heterosubtypic protective immunity. Our data show that, in contrast to i.n. vaccination with formalin- or UV-inactivated virus, or the present commercially available trivalent influenza vaccine, a single dose of gamma-ray-inactivated A/PC (gamma-A/PC) conferred significant protection in mice against both homologous and heterosubtypic virus challenges. A multiple immunization regime was required for formalin-inactivated virus preparations to induce protective immunity against a homotypic virus challenge, but did not induce influenza A strain cross-protective immunity. The highly immunogenic gamma-A/PC, but not formalin- or UV-inactivated A/PC, nor the currently available subvirion vaccine, elicited cytotoxic T-cell responses that are most likely responsible for the cross-protective and long-lasting immunity against highly lethal influenza A infections in mice. Finally, freeze-drying of gamma-A/PC did not affect the ability to induce cross-protective immunity.

Cutting Edge: Rapid and Efficient In Vivo Cytotoxicity by Cytotoxic T Cells Is Independent of Granzymes A and B

Cytotoxic T (Tc) cells lyse target cells via exocytosis of granules containing perforin (perf) and granzymes (gzm). In vitro, gzm delivery into the target cell cytosol results in apoptosis, and in the absence of gzm A and B the induction of apoptosis is severely impaired. However, using in vivo Tc cell killing assays, we find that virus-immune, gzm A × B-deficient (gzmA×B−/−) mice are competent to eliminate adoptively transferred target cells pulsed with an immunodominant Tc cell determinant as rapidly and completely as their wild-type counterparts. Specific target cell elimination occurred with similar kinetics in both spleen and lymph nodes. Thus, neither gzmA nor gzmB are required for rapid and efficient in vivo cytotoxicity by Tc cells.

Antiviral cytotoxic T cells cross-reactively recognize disparate peptide determinants from related viruses but ignore more similar self- and foreign determinants.

We have investigated the reactivities of cytotoxic T (Tc) cells against the two immunodominant, H-2Kk-restricted determinants from the Flavivirus Murray Valley encephalitis virus (MVE), MVE1785 (REHSGNEI) and MVE1971 (DEGEGRVI). The respective Tc cell populations cross-reactively lysed target cells pulsed with determinants from the MVE1785- and MVE1971-corresponding positions of six other flaviviruses, despite low sequence homology in some cases. Notably, anti-MVE1785 Tc cells recognized a determinant (TDGEERVI) that shares with the determinant used for stimulation only the carboxyl-terminal amino acid residue, one of two H-2Kk anchor residues. These reactivity patterns were also observed in peptide-dependent IFN-γ production and the requirements for in vitro restimulation of memory Tc cells. However, the broad cross-reactivity appeared to be limited to flavivirus-derived determinants, as none of a range of determinants from endogenous mouse-derived sequences, similar to the MVE-determinants, were recognized. Neither were cells infected with a number of unrelated viruses recognized. These results raise the paradox that virus-immune Tc cell responses, which are mostly directed against only a few “immunodominant” viral determinants, are remarkably peptide cross-reactive.

Cross-reactivity in T-cell antigen recognition.

The molecular interactions between the T‐cell receptor (TCR) and peptide‐MHC (pMHC) have been elucidated in recent years. Nevertheless, the fact that binding of only slightly different ligands by a TCR, or ligation of the same pMHC at different developmental stages of the T cell, can have opposing consequences, continues to pose intellectual challenges. Kinetic proofreading models, which have at their core the dissociation rates of pMHC from the TCR, are best suited to account for these observations. However, T cells can be triggered by peptides with often minimal homology to the primary immunogenic peptide. This cross‐reactivity of the TCR is manifest at several levels, from positive selection of immature thymocytes to homeostasis and antigen‐cross‐ reactive immune responses of mature peripheral T cells. The implications of the high cross‐reactivity of T‐cell antigen recognition for self‐tolerance and T‐cell memory are discussed.