Antigen kinetics determines immune reactivity.

A current paradigm in immunology is that the strength of T cell responses is governed by antigen dose, localization, and costimulatory signals. This study investigates the influence of antigen kinetics on CD8 T cell responses in mice. A fixed cumulative antigen dose was administered by different schedules to produce distinct dose-kinetics. Antigenic stimulation increasing exponentially over days was a stronger stimulus for CD8 T cells and antiviral immunity than a single dose or multiple dosing with daily equal doses. The same was observed for dendritic cell vaccination, with regard to T cell and anti-tumor responses, and for T cells stimulated in vitro. In conclusion, stimulation kinetics per se was shown to be a separate parameter of immunogenicity. These findings warrant a revision of current immunization models and have implications for vaccine development and immunotherapy.

Critical role for DNA vaccination frequency in induction of antigen-specific cytotoxic responses.

Since antigen-persistence plays a role for induction of immunity, we investigated the in vivo pharmacokinetic of a naked DNA vaccine at the site of its action, i.e., in the lymph node. After direct intralymphatic injection, naked DNA vaccine degraded within a few hours. In correlation with the short persistence of the DNA vaccine we found that the frequency of vaccination critically influenced the strength of the immune response. In mice vaccinated every 3 days, cytotoxic T-cell responses were enhanced compared to immunization in 6 or 9 days intervals. The results suggest that the so far disappointing efficiency of naked DNA vaccines in humans may be overcome by more frequent vaccination.

Improving the therapeutic index of CpG oligodeoxynucleotides by intralymphatic administration.

Signal transduction initiated by TLR such as TLR9, a natural receptor for unmethylated cytosine-guanine-rich motifs (CpG), results in activation of transcription factors, including NF-kappaB, with substantial impact on the innate and adaptive immunity. However, practical application of new adjuvants such as CpG oligodeoxynucleotides (ODN) remains a challenge, since prominent systemic activation of NF-kappaB may result in severe side effects reminiscent of septic shock, thus limiting their therapeutic index (TI). Low-dose administration of CpG ODN into lymph nodes has been evaluated as a means to reduce systemic side effects while retaining strong adjuvant properties. To this aim, a prototype immune-stimulating CpG ODN was used to enhance the antibody production against the antigen phospholipase A(2) and the CD8(+) T cell responses to ovalbumin in mice. When administered subcutaneously, high CpG ODN doses (>10 nmol) were required to enhance antibody and CD8(+) T cell responses. In contrast, when administered directly into a lymph node, much lower amounts of CpG (<0.1 nmol) were sufficient for a similar immune-enhancing effect. Systemic adverse reactions induced by CpG ODN were only detected at higher doses (1-10 nmol), independently of the route of administration. Finally, low-dose CpG ODN, administered in a targeted fashion to HLA-A2.1(+) transgenic mice, greatly elevated anti-tumor CD8(+) T cell immunity. Thus, intralymphatic administration of CpG ODN considerably improves the TI and may greatly enable a safe and effective use in the clinic.

Limited in vivo reactivity of polyclonal effector cytotoxic T cells towards altered peptide ligands.

T cell responses are regulated by the affinity/avidity of the T cell receptor for the MHC/peptide complex, available costimulation and duration of antigenic stimulation. Altered peptide ligands (APLs) are usually recognized with a reduced affinity/avidity by the T cell receptor and are often able to only partially activate T cells in vitro or may even function as antagonists. Here we assessed the ability of APLs derived from peptide p33 of lymphocytic choriomeningitis virus (LCMV) to mediate lysis of target cells in vivo, confer anti-viral protection and cause auto-immune disease. In general, in vitro cross-reactivity between APLs was rather limited, and even strongly cross-reactive cytotoxic T lymphocytes were only able to mediate moderate anti-viral protection. Partial protection was observed for infection with LCMV or low doses of recombinant vaccinia virus, while no reduced viral titers could be seen upon infection with high dose of vaccinia virus. In a transgenic mouse model expressing LCMV glycoprotein in the islets of the pancreas, APLs induced a transient insulitis but failed to induce autoimmune diabetes. Thus, effector functions induced by even highly homologous APLs are rather limited in vivo.

Virus-like particles as carriers for T-cell epitopes: limited inhibition of T-cell priming by carrier-specific antibodies.

Virus-like particles (VLPs) are able to induce cytotoxic T-cell responses in the absence of infection or replication. This makes VLPs promising candidates for the development of recombinant vaccines. However, VLPs are also potent inducers of B-cell responses, and it is generally assumed that such VLP-specific antibodies interfere with the induction of protective immune responses, a phenomenon summarized as carrier suppression. In this study, we investigated the impact of preexisting VLP-specific antibodies on the induction of specific cytotoxic T-cell and Th-cell responses in mice. The data show that VLP-specific antibodies did not measurably reduce antigen presentation in vitro or in vivo. Nevertheless, T-cell priming was slightly reduced by antigen-specific antibodies; however, the overall reduction was limited and vaccination with VLPs in the presence of VLP-specific antibodies still resulted in protective T-cell responses. Thus, carrier suppression is unlikely to be a limiting factor for VLP-based T-cell vaccines.

Immunity in response to particulate antigen-delivery systems.

Adjuvants and antigen-delivery systems are essential in inducing and modifying immune responses, and despite the variety of materials available for such use, mechanisms by which they support immunity appear to be little known. A common denominator for most antigen-delivery systems is their particulate nature. Together with a certain depot effect, it is the particulate nature that primarily decides whether the antigen-delivery system will be successful in inducing an immune response. If this first requirement is fulfilled, the chemical composition of the vaccine decides which type of immune response will develop, e.g. which isotype of antibodies the B cells will produce, and which cytokines the T cells will secrete, and can be controlled by combining the antigen with immunomodulatory or co-stimulatory molecules. It is our goal to provide an overview of the cellular and molecular factors involved in the induction of immunity and how such factors may influence the potency of an adjuvant or a vaccine. Such factors should then be implemented in the design of new vaccines or in tuning the properties of existing vaccines in order to reach the properties that are necessary for successful vaccination.

Loading of MHC class I and II presentation pathways by exogenous antigens: a quantitative in vivo comparison.

The MHC class I pathway is usually fueled by endogenous Ags, while exogenous Ags reach the MHC class II pathway. Although exogenous epitopes may also enter the MHC class I pathway, quantification of the efficiency of the process has remained a difficult task. In an attempt of such a quantification, we directly compared the amount of exogenous virus-like particles required for induction of cytotoxic T cell responses by cross-priming with the amount of virus-like particles required for induction of Th cell responses by the conventional route of MHC class II loading as an internal standard. Surprisingly, we found that cross-presentation of peptides derived from exogenous Ags on MHC class I molecules is of only marginally lower efficiency ( approximately 1- to 10-fold) than the classical MHC class II pathway in vitro and in vivo. Thus, Ag quantities required for cross-presentation and cross-priming are similar to those required for fueling the MHC class II pathway.

Influenza A vaccine based on the extracellular domain of M2: weak protection mediated via antibody-dependent NK cell activity.

Vaccination of mice with a peptide corresponding to the extracellular part of M2 protein coupled to the immunodominant domain of hepatitis B core can protect mice from a lethal challenge with influenza A virus. As the extracellular part of M2 protein is highly conserved in all known human influenza A strains, such a vaccine may protect against all human influenza A strains, which would represent a major advantage over current vaccine strategies. The present study demonstrates that protection is mediated exclusively by Abs, a very important feature of a successful preventive vaccine. However, these Abs neither bind efficiently to the free virus nor neutralize virus infection, but bind to M2 protein expressed on the surface of virus-infected cells. The presence of NK cells is important for protection, whereas complement is not, supposing that protection is mediated via Ab-dependent, cell-mediated cytotoxicity. The absence of neutralizing Abs results in much weaker protection than that achieved by vaccination with UV-inactivated influenza virus. Specifically, whereas neutralizing Abs completely eliminate signs of disease even at high viral challenge doses, M2-specific Abs cannot prevent infection, but merely reduce disease at low challenge doses. M2-specific Abs fail to protect from high challenge doses, as vaccinated mice undergo lethal infection under these conditions. In conclusion, protection mediated by M2-hepatitis B core vaccine would be insufficient during the yearly epidemics, for which full protection is desirable, and overall is clearly inferior to protection achieved by immunization with classical inactivated viral preparations.

Maintenance of memory CTL responses by T helper cells and CD40-CD40 ligand: antibodies provide the key.

Cytotoxic T lymphocytes (CTL) are essential for control of primary infections by many pathogens and in particular by non-cytopathic viruses. It has been proposed that long-term maintenance of CTL memory and control of lymphocytic choriomeningitis virus (LCMV) is dependent upon the presence of T helper cells and interaction of antigen-presenting cells and CTL via CD40 and its ligand CD40L. However, we demonstrate here that CD40-CD40L interaction maintains CTL memory by induction of virus-specific antibodies. In fact, loss of CTL memory responses and spread of virus in mice lacking CD40 or its ligand is prevented by repetitive therapeutic injections of LCMV-specific antibodies. This indicates that antibodies are essential for long-term control of non-cytopathic virus and to maintain protective memory. Transfer of neutralizing antibodies or induction of antibodies by therapeutic vaccination within weeks after infection may therefore prove beneficial for the treatment of chronic virus infections such as HIV, hepatitis B, and hepatitis C. See accompanying article http://dx.doi.org/10.1002/eji.200324844

Nonmethylated CG motifs packaged into virus-like particles induce protective cytotoxic T cell responses in the absence of systemic side effects.

DNA rich in nonmethylated CG motifs (CpGs) greatly facilitates induction of immune responses against coadministered Ags. CpGs are therefore among the most promising adjuvants known to date. Nevertheless, CpGs are characterized by two drawbacks. They have unfavorable pharmacokinetics and may exhibit systemic side effects, including splenomegaly. We show in this study that packaging CpGs into virus-like particles (VLPs) derived from the hepatitis B core Ag or the bacteriophage Qbeta is a simple and attractive method to reduce these two problems. CpGs packaged into VLPs are resistant to DNase I digestion, enhancing their stability. In addition, and in contrast to free CpGs, packaging CpGs prevents splenomegaly in mice, without affecting their immunostimulatory capacity. In fact, vaccination with CpG-loaded VLPs was able to induce high frequencies of peptide-specific CD8(+) T cells (4-14%), protected from infection with recombinant vaccinia viruses, and eradicated established solid fibrosarcoma tumors. Thus, packaging CpGs into VLPs improves both their immunogenicity and pharmacodynamics.

On the role of APC-activation for in vitro versus in vivo T cell priming.

Professional antigen-presenting cells take up antigens for processing and presentation in association with MHC class I and II molecules. When APCs receive the right stimuli, they undergo a maturation process and migrate to secondary lymphoid organs to trigger T cell activation. In this study, we compared side-by-side in vivo and in vitro activation of T cells. Transgenic CD8(+) T cells specific for the p33 epitope, derived from the lymphocytic choriomeningitis virus glycoprotein, were labeled with CFSE and injected into syngeneic mice or alternatively, co-cultured in vitro with APCs. The p33 epitope was delivered as free peptide or genetically fused to virus-like particles. Whereas proliferation of specific T cells was comparable in both systems, the production of IFN-gamma and the expression of CD25 showed important differences. Induction of effector function and expression of activation markers were strongly enhanced in vitro by both the free peptide and VLPs. Surprisingly, addition of CpG-containing immune-stimulating DNA for activation of APCs dramatically increased effector T cell differentiation in vitro, whereas no enhancement could be observed in vitro. Thus, activation of professional APCs was mandatory for induction of effector CD8(+) T cell responses in vivo, while this step was largely dispensable in vitro.

Innate immunity together with duration of antigen persistence regulate effector T cell induction.

Proliferation of T cells is important for the expansion of specific T cell clones during immune responses. In addition, for the establishment of protective immunity against viruses, bacteria, and tumors, the expanded T cells must differentiate into effector T cells. Here we show that effector T cell generation is driven by activation of APCs and duration of antigenic stimulation. Adoptively transferred TCR-transgenic T cells extensively proliferated upon immunization. However, these T cells failed to differentiate into effector cells and died within 1 wk after immunization unless antigenic peptides persisted for >1 day or were presented by activated APCs. The induction of protective immunity in a nontransgenic system was more stringent, since activation of APCs or prolonged Ag persistence alone was not sufficient to drive immunity. In contrast, Ag had to be presented for several days by activated APCs to trigger protective T cell responses. Thus, activation of APCs and duration of Ag presentation together regulate the induction of protective T cell responses.

Role of Toll-like receptors in costimulating cytotoxic T cell responses.

Stimulation of Toll-like receptors (TLR) by pathogen-derived compounds leads to activation of APC, facilitating the induction of protective immunity. This phenomenon is the basis of most adjuvant formulations currently in development. Here, we tested the ability of TLR2, 3, 4, 5, 7 and 9 signaling to enhance CTL responses upon vaccination with virus-like particles. Stimulation of TLR2 and 4 failed to increase CTL responses, whereas ligands for TLR3, 5 and 7 exhibited moderate adjuvant function. In contrast, stimulation of TLR9 dramatically increased CTL responses, indicating that ligands for TLR9 are likely to be the most promising candidates for the development of novel adjuvant formulations for stimulating CTL responses.

A molecular assembly system that renders antigens of choice highly repetitive for induction of protective B cell responses.

Virus like particles (VLPs) are known to induce potent B cell responses in the absence of adjuvants. Moreover, epitope-specific antibody responses may be induced by VLPs that contain peptides inserted in their immunodominant regions. However, due to steric problems, the size of the peptides capable of being incorporated into VLPs while still permitting capsid assembly, is rather limited. While peptides genetically fused to either the N- or C-terminus of VLPs present fewer assembly problems, the immune responses obtained against such epitopes are often limited, most likely because the epitopes are not optimally exposed. In addition, such particles may be less stable in vivo. Here, we show that peptides and proteins engineered to contain a free cys can be chemically coupled to VLPs formed from the hepatitis B core antigen (HBcAg) containing a lys in the immuno-dominant region. By using this approach steric hindrance of capsid assembly is abrogated. Peptides or protein coupled to VLPs in an oriented fashion are shown to induce strong and protective B cell responses even against self-epitopes in the absence of adjuvants. This molecular assembly system may be used to induce strong B cell responses against most antigens.

Cross-presentation of virus-like particles by skin-derived CD8(-) dendritic cells: a dispensable role for TAP.

Virus-like particles (VLP) induce efficient CTL responses although they do not carry any genetic information. Here, we analyzed MHC class I associated presentation of VLP-derived CTL-epitopes in vivo. After intradermal injection of VLP containing the immunodominant epitope (p33) of lymphocytic choriomeningitis virus (p33-VLP), presentation of peptide p33 in draining lymph nodes was largely restricted to CD8(-) skin-derived dendritic cells (DC). Surprisingly, and in contrast to findings with tumor cells, TAP1-deficient DC and macrophages mediated efficient cross-presentation of VLP-derived p33 in vivo and in vitro. However, the ability of TAP1-deficient DC to cross-present p33-VLP was reduced compared to wild-type DC, indicating that in DC, both TAP-dependent and TAP-independent pathways were operative. In contrast, macrophages cross-presented p33-VLP normally in the absence of TAP. The TAP-dependent pathway of cross-presentation is therefore confined to DC while both macrophages and DC harbor the TAP-independent pathway. In summary, the results show that VLP-derived epitopes are cross-presented by CD8(-) DC in vivo in a partial TAP-independent fashion and highlight important differences in the processing machinery of DC versus macrophages.

Critical role for activation of antigen-presenting cells in priming of cytotoxic T cell responses after vaccination with virus-like particles.

Virus-like particles (VLPs) are known to induce strong Ab responses in the absence of adjuvants. In addition, VLPs are able to prime CTL responses in vivo. To study the efficiency of this latter process, we fused peptide p33 derived from lymphocytic choriomeningitis virus to the hepatitis B core Ag, which spontaneously assembles into VLPs (p33-VLPs). These p33-VLPs were efficiently processed in vitro and in vivo for MHC class I presentation. Nevertheless, p33-VLPs induced weak CTL responses that failed to mediate effective protection from viral challenge. However, if APCs were activated concomitantly in vivo using either anti-CD40 Abs or CpG oligonucleotides, the CTL responses induced were fully protective against infection with lymphocytic choriomeningitis virus or recombinant vaccinia virus. Moreover, these CTL responses were comparable to responses generally induced by live vaccines, because they could be measured in primary ex vivo (51)Cr release assays. Thus, while VLPs alone are inefficient at inducing CTL responses, they become very powerful vaccines if applied together with substances that activate APCs.

Regulation of IgG antibody responses by epitope density and CD21-mediated costimulation.

Epitope density and organization have been shown to be important factors for B cell activation in many animal model systems. However, it has been difficult to separate the role of antigen organization from the role of local antigen concentrations because highly organized antigens are usually particulate whereas non-organized antigens are more soluble. Hence, highly organized and non-organized antigens may interact with different cell types and in different locations within lymphoid organs. In order to assess the role of antigen organization in regulating B cell responses, we immunized mice with highly repetitive virus-like particles, which exhibit different epitope densities covalently attached to them. Therefore, the same particulate structure was used to present identical epitopes that differed in their degree of organization. Induction of epitope-specific IgM titers, reflecting early B cell activation, were unaffected by the degree of epitope density. Furthermore, the absence of Th cells or CD21/CD35 did not reduce the IgM response. In contrast, the degree of organization was a critical factor influencing the magnitude of the epitope-specific IgG response. Moreover, the threshold for IgG responses was shifted in the absence of CD21/CD35, resulting in the requirement for higher epitope densities to allow efficient IgG responses. Thus, IgG but not IgM responses are regulated by epitope density and B cell costimulatory thresholds.