Tazio Storni

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.

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.

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 Qβ 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.

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.

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.

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 51Cr 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.

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.

Direct intralymphatic injection of peptide vaccines enhances immunogenicity

Research to enhance the efficiency of vaccines focuses mainly on improving either the adjuvant or the type and form of the antigen. This study evaluates the influence of the administration route on the efficiency of a peptide‐based vaccine. Peptide vaccines are generally administered subcutaneously or intradermally, from where they must reach secondary lymphatic organs to induce an immune response. We analyzed the efficacy of peptide vaccines administered directly into a lymph node. Using a MHC class I‐binding peptide from lymphocytic choriomeningitis virus, we found that intralymphatic injection enhanced immunogenicity by as much as 106 times when compared to subcutaneous and intradermal vaccination. Intralymphatic administration induced CD8 T cell responses with strong cytotoxic activity and IFN‐γ production that conferred long‐term protection against viral infections and tumors. These results should have immediate implications for clinical immunotherapy of infectious disease and cancer.

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.

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.