MHC class II loading of high or low affinity peptides directed by Ii/peptide fusion constructs: implications for T cell activation

CD4(+) T cells recognize peptides presented on the cell surface of antigen presenting cells in the MHC class II context. The biosynthesis and transport of MHC class II molecules depend on the type II transmembrane invariant chain (Ii) and are tightly regulated processes. Ii is known to bind to the MHC class II peptide-binding groove via its class II-associated Ii peptide (CLIP) region early in the biosynthetic pathway to prevent premature peptide binding. In this study we have genetically exchanged CLIP with peptides of either high or low affinity for the class II peptide binding groove and utilized the properties of Ii to manipulate MHC class II loading. An inducible promoter controlled expression of the Ii/peptide fusion constructs, and presentation at different expression levels was studied. Both peptides were excised from Ii and presented on MHC class II molecules as shown by liquid chromatography-tandem mass spectrometry, but the high affinity peptide was presented more efficiently than the low affinity peptide. Both peptides were efficient in eliciting T cell responses at high Ii/peptide concentration independent of the duration of T cell stimulation. The peptides were also able to elicit an IL-2 response at low expression levels; however, the kinetic differed as the T cells required longer duration of T cell contact to reach a significant T cell response. This probably reflects the number of class II/peptide complexes at the cell surface and is discussed.     

DR αβ dimers released from complexes with invariant chain fail to stimulate alloreactive T cell clones

To demonstrate that DR αβ dimers still complexed to invariant chain (Ii) have not yet acquired peptides recognized by alloreactive T cells, complexes between DR molecules and Ii isolated from Epstein-Barr-virus (EBV)-transformed B cells were analyzed by affinity chromatography and gel filtration. First, it was shown that DR/Ii complexes inserted into artificial planar membranes (PM) failed to stimulate proliferative response of five alloreactive T cell clones and a polyclonal alloreactive Tcell line, while PM bearing mature DR αβ dimers from the same EBV-B cells were stimulatory for the Tcell clones and the Tcell line. These findings indicate that either Ii inhibits binding of peptides to DR molecules or Ii hinders T cells recognition of peptide/DR complexes. Second, to discriminate between these two possibilities, DR αβ dimers, which were artificially released from complexes between DR molecules and Ii, were inserted into PM. These DR αβ dimers were devoid of alloreactive stimulatory capacity while fully capable of binding and presenting a tetanus toxin synthetic peptide to a specific Tcell clone, indicating that DR molecules released from complexes with Ii are empty. This study, by showing that DR molecules bound to Ii do not bear peptides recognized by alloreactive T cells, supports the notion that association of Ii with class II major histocompatibility complex (MHC) molecules prevents premature peptide loading and hence favors encounter with peptides derived from proteins of the extracellular compartment. Since allogeneic class II MHC molecules released from complexes with Ii were not stimulatory for five out of five alloreactive Tcell clones and a polyclonal alloreactive Tcell line, these data also indicate that, in most cases, alloreactive T cells recognize ligands constituted by complexes between allogeneic class II MHC molecules and specific peptides which derive from the antigen-presenting cells themselves or serum proteins.     

Achieving stability through editing and chaperoning: regulation of MHC class II peptide binding and expression

Summary: In antigen‐presenting cells (APCs), loading of major histocompatibility complex class II (MHC II) molecules with peptides is regulated by invariant chain (Ii), which blocks MHC II antigen‐binding sites in pre‐endosomal compartments. Several molecules then act upon MHC II molecules in endosomes to facilitate peptide loading: Ii‐degrading proteases, the peptide exchange factor, human leukocyte antigen‐DM (HLA‐DM), and its modulator, HLA‐DO (DO). Here, we review our findings arguing that DM stabilizes a globally altered conformation of the antigen‐binding groove by binding to a lateral surface of the MHC II molecule. Our data imply changes in the interactions between specificity pockets and peptide side chains, complementing data from others that suggest DM affects hydrogen bonds. Selective weakening of peptide/MHC interactions allows DM to alter the peptide repertoire. We also review our studies in cells that highlight the ability of several factors to modulate surface expression of MHC II molecules via post‐Golgi mechanisms; these factors include MHC class II‐associated Ii peptides (CLIP), DM, and microbial products that modulate MHC II traffic from endosomes to the plasma membrane. In this context, we discuss possible mechanisms by which the association of some MHC II alleles with autoimmune diseases may be linked to their low CLIP affinity.     

Surface-expressed invariant chain (CD74) is required for internalization of human leucocyte antigen-DR molecules to early endosomal compartments

Transport of major histocompatibility complex (MHC) class II molecules to the endocytic route is directed by the associated invariant chain (Ii). In the endocytic pathway, Ii is proteolytically cleaved and, upon removal of residual Ii fragments, class II alpha beta dimers are charged with antigenic peptide and recognized by CD4+ T cells. Although distinct peptide-loading compartments such as MIIC (MHC class II loading compartment) and CIIV (MHC class II vesicles) have been characterized in different cells, there is growing evidence of a multitude of subcellular compartments in which antigenic peptide loading takes place. We employed a physiological cellular system in which surface Ii (CD74) and surface human leucocyte antigen (HLA)-DR were induced either alone or in combination. This was achieved by transient exposure of HT-29 cells to recombinant interferon-gamma (rIFN-gamma). Using distinct cellular variants, we showed that: (i) the majority of Ii molecules physically associate on the cell membrane with class II dimers to form DR alpha beta:Ii complexes; (ii) the presence of surface Ii is a prerequisite for the rapid uptake of HLA-DR-specific monoclonal antibodies into early endosomes because only the surface DR+/Ii+ phenotype, and not the DR+/Ii- variant, efficiently internalizes; and (iii) the HLA-DR:Ii complexes are targeted to early endosomes, as indicated by co-localization with the GTPase, Rab5, and endocytosed bovine serum albumin. Internalization of HLA-DR:Ii complexes, accommodation of peptides by DR alphabeta heterodimers in early endosomes and recycling to the cell surface may be a mechanism used to increase the peptide repertoire that antigen-presenting cells display to MHC class II-restricted T cells.     

Invriant chain peptides enhancing or inhibiting the presentation of antigenic peptides by major histocompatibility complex class II molecules

Two soluble invariant chain (Ii) peptides with overlapping sequences had contrasting effects on the presentation of antigenic peptides by murine A^d, A^k, E^d, and E^k major histocompatibility complex (MHC) class II molecules. Naturally produced class II-associated invariant chain peptides human (h)Ii81–104/murine (m)Ii80–103 inhibited antigen presentation on these MHC class II alleles in a manner consistent with competitive inhibition. The Ii-4 peptides hIi77–92/mIi76–91 enhanced presentation of antigenic peptides on I-E class II alleles by promoting the exchange of peptides at the cell surface. Treatment of antigenpresenting cells (APC) with Ii-4 before the addition of antigenic peptide greatly enhanced subsequent T cell responses, while treatment of APC with Ii–4 after antigenic peptide binding decreased subsequent T cell responses. The hIi81–104 and mIi80–103 peptides inhibited T cell responses in both types of assays. The binding of biotinylated antigenic peptide to MHC class II-transfected L cells, as measured by flow cytometry, was inhibited by mIi80-103 and enhanced by mIi-4. Segments of Ii fragments remaining associated with MHC class II, or released Ii peptides, appear to regulate the formation of stable antigenic peptide/MHC class II complexes either positively or negatively through interactions at or near the antigenic peptide binding site. These findings open a pathway for the design of novel therapeutics based on the structure and function of natural and rationally designed fragments of Ii.     

Structural principles of MHC class II antigen presentation

Normal immune surveillance depends on the ability of MHC class II molecules to bind peptide antigens and carry them to the cell surface for display to T cells. To do this efficiently, class II molecules must be able to bind peptides from a broad array of antigen sequences and retain them at the cell surface long enough for T-cell recognition to occur. Class II molecules accomplish this task through a combination of clever structural biochemistry and the help of at least two different molecular chaperones: the class II-associated invariant chain (Ii); and a non-peptide binding class II molecule termed H2-DM in mouse and HLA-DM in man (DM). Here, we compare the existing 3-dimensional structures of class II-peptide complexes in order to review the general principles of peptide binding and presentation. We extend this analysis to include the structures of proteins known to interact with MHC class II, focusing primarily on the Ii chain and DM.     

Cathepsin S required for normal MHC class II peptide loading and germinal center development

Major histocompatibility complex (MHC) class II molecules acquire antigenic peptides after degradation of the invariant chain (Ii), an MHC class II-associated protein that otherwise blocks peptide binding. Antigen-presenting cells of mice that lack the protease cathepsin S fail to process Ii beyond a 10 kDa fragment, resulting in delayed peptide loading and accumulation of cell surface MHC class II/10 kDa Ii complexes. Although cathepsin S-deficient mice have normal numbers of B and T cells and normal IgE responses, they show markedly impaired antibody class switching to IgG2a and IgG3. These results indicate cathepsin S is a major Ii-processing enzyme in splenocytes and dendritic cells. Its role in humoral immunity critically depends on how antigens access the immune system.     

A study of complexes of class II invariant chain peptide: Major histocompatibility complex class II molecules using a new complex-specific monoclonal antibody

Complexes of major histocompatibility complex (MHC) class II molecules containing invariant chain (Ii)-derived peptides, known as class II-associated invariant chain peptides (CLIP), are expressed at high levels in presentation-deficient mutant cells. Expression of these complexes in mutant and wild-type antigen-presenting cells suggests that they represent an essential intermediate in the MHC class II antigen-presenting pathway. We have generated a monoclonal antibody, 30-2, which is specific for these complexes. Using this antibody, we have found quantitative differences in CLIP: MHC class II surface expression in mutant and wild-type cells. Our experiments also show that CLIP: MHC class II complexes are preferentially expressed on the cell surface similar to total mature MHC class II molecules. These complexes are found to accumulate in the endosomal compartment in the process of endosomal Ii degradation. Analysis of the fine specificity of the antibody indicates that these complexes have Ii peptide bound to the peptide-binding groove.     

MHC II and the Endocytic Pathway: Regulation by Invariant Chain

The major histocompatibility complex (MHC) class I and II molecules perform vital functions in innate and adaptive immune responses towards invading pathogens. MHC class I molecules load peptides in the endoplasmatic reticulum (ER) and display them to the T cell receptors (TcR) on CD8⁺ T lymphocytes. MHC class II molecules (MHC II) acquire their peptides in endosomes and present these to the TcR on CD4+ T lymphocytes. They are vital for the generation of humoral immune responses. MHC II assembly in the ER and trafficking to endosomes is guided by a specialized MHC II chaperone termed the invariant chain (Ii). Ii self-associates into a trimer in the ER, this provides a scaffold for the assembly of three MHC II heterodimers and blocks their peptide binding grooves, thereby avoiding premature peptide binding. Ii then transports the nascent MHC II to more or less specialized compartment where they can load peptides derived from internalized pathogens.     

Low-avidity recognition by CD4+ T cells directed to self-antigens

Self-reactive T cells populate the peripheral immune system, and likely form the reservoir from which autoreactive cells are derived. We analyzed a panel of self and non-self peptides presented by HLA-DR4, a class II molecule associated with autoimmunity, by immunization of mice transgenic for HLA-DR4. Significant structural avidity for T cell recognition, as measured by MHC class II tetramer binding to CD4(+) T cells was only observed in mice immunized with the non-self antigens. T cell hybridomas were generated from mice immunized with the naturally processed self-peptide hGAD65 (552-572) and also from mice immunized with an influenza-derived non-self epitope (HA 306-318). T cells specific for the self peptide failed to bind tetramers and exhibited low functional avidity as measured by the peptide concentration required to reach half-maximum proliferation values. In contrast, T cells specific for the non-self HA (306-318) peptide exhibited high structural and functional avidity profiles. As recently described in studies of murine CD8(+) T cell function, the predominance of low avidity recognition of self-peptide epitopes may be a characteristic feature of CD4(+) T cells responding to autoantigens.     

MHC class II-associated invariant chain peptide replacement by T cell epitopes: engineered invariant chain as a vehicle for directed and enhanced MHC class II antigen processing and presentation

Proteolysis of the invariant chain (Ii) leads to the generation of abundant MHC class II-associated invariant chain peptides (CLIP), which bind in the MHC class II binding groove via supermotifs in a manner similar to that of antigenic peptides. We have engineered an Ii vector with the capacity to express any antigenic peptide of interest instead of CLIP, for T cell stimulation. When peripheral blood mononuclear cells (PBMC) were pulsed with Ii hybrids encoding T cell epitopes of tetanus toxin or acetylcholine receptor, stimulation of T cells was dramatically enhanced compared to stimulation after priming with either the native or recombinant proteins. Site-specific insertion of antigenic sequences into the CLIP region promoted enhanced antigenicity of Ii hybrids which were shown to be processed intracellularly in a chloroquine-sensitive compartment. Naturally processed T helper epitopes were visualized directly on the surface of PBMC and identified as analogs of CLIP associated with MHC class II molecules. This novel Ii vector provides a flexible and efficient system for the delivery of defined peptide epitopes to T cells which might be useful in the development of specific vaccines and in the study of intracellular processing.     

The impact of the non-classical MHC proteins HLA-DM and HLA-DO on loading of MHC class II molecules

Summary: Peptide binding to classical major histocompatibility complex (MHC) class II molecules is known to be determined by the properties of the class ii peptide binding groove but recently it turned out to be co-controlled by the activity of the non-classical MHC molecules HLA-DM and HLA-DO: HLA-DM functions as a mediator of peptide exchange. In addition, HLA-DM is a chaperone for MHC class II molecules in endosomal and lysosomal loading compartments because it stabilizes the empty MHC class Ii peptide binding groove and keeps it receptive for peptide loading until appropriate peptide ligands are captured. Since HLA-DM favors the generation of high-stability peptide-MHC class Ii complexes by releasing low-stability peptide ligands, DM activity affects the peptide repertoire presented on the ceil surface of antigen-presenting cells. HLA-DO is expressed mainly in B cells and binds tightly to HLA-DM thereby modulating its activity Together, HLA-DM and HLA-DO are critical factors in shaping the MHC class Il-associated self or foreign peptide repertoire of antigen presenting cells and, hence, govern initiation or prevention of an immune response.     

HLA-DM can partially replace the invariant chain for HLA-DR transport and surface expression in transfected endocrine epithelial cells

The function of HLA class II molecules as peptide presenters to CD4+ T cells depends on the expression of associated molecules such as the invariant chain (Ii) and DM responsible for the correct transport of and high-stability peptide binding to the class II dimers. In organs affected by autoimmune diseases, endocrine epithelial cells express class II molecules, which presumably are involved in the presentation of self-peptides to autoreactive T cells. We have transfected the rat insulinoma cell line RINm5F with different combinations of HLA-DR, Ii and HLA-DM cDNAs and have studied how Ii and DM affect the transport and stability of class II molecules expressed by the different transfectants. Immunofluorescence and biochemical analysis showed that cells transfected with DR and DM in the absence of Ii expressed mostly stable molecules in their surface, and showed a lower accumulation of DR molecules in the endoplasmic reticulum (ER) than cells expressing only DR. This suggests that, in the absence of invariant chain, DM molecules can not only exchange peptides other than class II-associated invariant chain peptide (CLIP) but may also be involved in the transport of class II molecules out of the ER towards the endosomal route. In addition, these data confirm that expression of DR alone or DR+Ii do not allow the formation of sodium dodecyl sulphate (SDS)-stable complexes, that cells expressing DR+Ii have most DR molecules occupied by CLIP and that Ii and DM molecules allow regular routing and peptide loading of class II molecules.     

Invariant chains with the class II binding site replaced by a sequence from influenza virus matrix protein constrain low-affinity sequences to MHC II presentation

Presentation of antigenic peptides by MHC II molecules is required to initiate CD4 T(h) cell responses. Some peptides, however, because of low affinity for MHC II, are not efficiently presented. A segment of the MHC II chaperon molecule, invariant chain (Ii), is known to bind early in biosynthesis with low affinity to the peptide binding groove. Here we have exploited the properties of Ii to manipulate the MHC II-loading pathway and to present low-affinity sequences. We used a deletion mutant of Ii where the promiscuous binding site to MHC II, which is adjacent to the groove binding segment, was deleted. A recombinant Ii (rIi) chimera, derived from this construct, was made in which the class II binding segment was exchanged for wild-type or single amino acid substitution variants of an HLA-DR1-restricted sequence from influenza matrix protein (MAT), which leads to MHC II allotype-specific binding. This rIi was expressed in antigen-presenting cells (APC) and introduced the MAT sequence into the MHC II-processing pathway. As expected, rIiMAT elicited antigen-specific, DR1-restricted T cell cytokine production and proliferation. Significantly, rIiMAT, that binds the HLA-DR4 allele with low affinity, elicited DR4-restricted IL-2 production but not proliferation. In contrast, exogenously provided MAT peptide failed to elicit any responses from DR4-restricted T cells. Compatible results were obtained with a single amino acid substitution variant (MAT(T)), which binds with high affinity to DR4 but low affinity to DR1. We conclude that loading of MHC II with antigenic peptides from endogenously synthesized rIi chimeras allows presentation of low-affinity sequences that cannot be presented if provided exogenously as peptides. Ii fusion proteins containing low-affinity antigenic sequences might be useful for vaccination with tumor antigens to overcome deficiencies in antigen presentation.     

Conformational alterations during biosynthesis of HLA-DR3 molecules controlled by invariant chain and HLA-DM

HLA-DM is known to catalyze the exchange of class II-associated invariant chain (Ii) peptide (CLIP) for cognate peptide during biosynthesis. In DM-negative cells HLA-DR3 molecules have been shown to predominantly present CLIP and to lack the DR3-specific mAb epitope 16.23, which has led to the assumption that CLIP prevents binding of mAb 16.23. In the present study we show that CLIP does not prohibit 16.23 epitope expression, but that the formation of this epitope is directly influenced by interactions of the DR molecule with Ii and DM. Detergent solubilized DR3 from wild-type as well as DM(-) cells bound CLIP in a 16.23(+) mode. On cells, however, neither CLIP nor antigenic peptide bound to DR3 in a 16.23(+) conformation, unless HLA-DM was expressed. Thus, HLA-DM appears to alter the conformation of DR3 in a peptide-independent fashion. Since in DM-deficient cells that also lack Ii, DR3 molecules assembled in a 16.23(+) conformation, we conclude that during biosynthesis Ii and DM exert opposing conformational constraints, characterized by suppressing or releasing 16.23 epitope expression. These results imply that DR3/peptide complexes, including DR3/ CLIP, can exist in two conformations depending on previous interaction with DM, but independent of the nature of the peptide bound. We show that these naturally occurring class II conformers can be selectively recognized by T cells.     

Human duodenal epithelial cells constitutively express molecular components of antigen presentation but not costimulatory molecules

Constitutive expression of major histocompatibility complex (MHC) class II molecules by duodenal epithelial cells (EC) suggests that they can present antigen to CD4(+) T cells. However, other molecular components including invariant chain (Ii), HLA-DM, and costimulatory molecules CD80, CD86 and CD40, are required for efficient T-cell activation. We have investigated whether normal human duodenal EC possess these molecules and whether they can mediate MHC class II antigen presentation. EC were isolated from duodenal biopsies from patients in whom pathology was excluded. Freshly-isolated duodenal EC did not stimulate autologous T-cell proliferation against purified protein derivative of tuberculin. Flow cytometry and immunoblot analysis revealed that duodenal EC constitutively express HLA-DR, Ii, and HLA-DM. Surface MHC class II associated invariant chain peptide (CLIP) was not detectable, suggesting that HLA-DM functions normally in CLIP removal. Duodenal EC expressed SDS-stable HLA-DR alphabeta heterodimers, indicating that peptide binding had occurred. Surface expression of CD80, CD86 or CD40 was not detected although mRNA for these costimulatory molecules was present in all samples. These results suggest that nondiseased human duodenal EC can process and present antigen by the MHC class II pathway, but that they may induce anergy, rather than activation, of local T cells.     

Recognition of core and flanking amino acids of MHC class II-bound peptides by the T cell receptor

CD4 T cells recognize peptides bound to major histocompatibility complex (MHC) class II molecules. Most MHC class II molecules have four binding pockets occupied by amino acids 1, 4, 6, and 9 of the minimal peptide epitope, while the residues at positions 2, 3, 5, 7, and 8 are available to interact with the T cell receptor (TCR). In addition MHC class II bound peptides have flanking residues situated outside of this peptide core. Here we demonstrate that the flanking residues of the conalbumin peptide bound to I-A(k) have no effect on recognition by the D10 TCR. To study the role of peptide flanks for recognition by a second TCR, we determined the MHC and TCR contacting amino acids of the I-A(b) bound Ealpha peptide. The Ealpha peptide is shown to bind I-A(b) using four alanines as anchor residues. TCR recognition of Ealpha peptides with altered flanking residues again suggested that, in general, no specific interactions occurred with the peptide flanks. However, using an HLA-DM-mediated technique to measure peptide binding to MHC class II molecules, we found that the peptide flanking residues contribute substantially to MHC binding.     

Poor loading of major histocompatibility complex class II molecules with endogenously synthesized short peptides in the absence of invariant chain

In normal antigen-presenting cells, newly synthesized major histocompatibility complex (MHC) class II molecules associate with the invariant chain (Ii) glycoprotein in the endoplasmic reticulum (ER). They are loaded with peptides only after proteolytic removal of the Ii in post-Golgi endocytic vesicles. Since the Ii inhibits peptide binding to MHC class II molecules, this association could protect MHC class II molecules from being loaded with endogenous peptides early after biosynthesis. If this were an important function of the Ii in vivo, MHC class II molecules synthesized in cells lacking the Ii should be loaded efficiently with short endogenous peptides in the ER; such peptides are known to be present there due to TAP-mediated import from the cytosol. To examine this possibility, we have studied peptide loading in HeLa transfectants expressing murine H-2A^k MHC class II molecules either alone or together with an excess of Ii. Endogenous peptides could readily be extracted from conformationally intact A^k αβ dimers of biosynthetically labeled Ii⁺ cells, whereas peptide loading was greatly (> 95%) diminished in the absence of Ii. Significant amounts of sodium dodecyl sulfate-(SDS) stable 55-kDa peptide: A^k complexes were only found in the Ii⁺ transfectants. In the absence of Ii, the MHC class II molecules instead formed stable complexes with long (20 and 50 kDa) polypeptides. Known A^k-binding peptides bound stably to A^k molecules on Ii^? cells, could be co-purified with them, and were resistant to release in SDS, suggesting that poor recovery of endogenous peptides was not due to decreased stability of A^k: peptide complexes in the absence of Ii. We conclude that protection of MHC class II molecules from endogenous short peptides does not appear to be a quantitatively important function of the Ii molecule, because peptide loading is inefficient in its absence.     

Forcing Tumor Cells to Present Their Own Tumor Antigens to the Immune System： a Necessary Design for an Efficient Tumor Immunotherapy

The general principle for tumor cells to escape from immune surveillance is to prevent tumor antigens from being recognized by the immune system. Many methods have been developed to increase the immunogenecity of the tumor cells. The most efficient methods are able to force tumor cells to present their own tumor antigens to the immune system. Stimulating Th cells by converting tumor cells into MHC class II /Ii- antigen presenting cells is one of the most efficient technologies. Using antisense methods, we suppress the expression of the Ii protein that normally co-expresses with MHC class II molecules and blocks the antigenic peptide binding site of MHC class II molecules during synthesis in the endoplasmic reticulum. In such tumor cells, the“unprotected“ MHC class II molecules pick up endogenous tumor antigenic peptides, which have been transported into the ER for binding to MHC class I molecules. Simultaneous presentation of tumor antigens by both MHC class I and II molecules generates a robust and long-lasting anti-tumor immune response. MHC class II /Ii- tumor cells are potent tumor cell vaccines and also cure a significant number of animals with renal and prostate tumors. We have developed analogous human gene vectors that are suitable for most patients and cancers.     

Suppression of major histocompatibility complex class II-associated invariant chain enhances the potency of an HIV gp120 DNA vaccine

Summary One function of the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) is to prevent MHC class II molecules from binding endogenously generated antigenic epitopes. Ii inhibition leads to MHC class II presentation of endogenous antigens by APC without interrupting MHC class I presentation. We present data that in vivo immunization of BALB/c mice with HIV gp120 cDNA plus an Ii suppressive construct significantly enhances the activation of both gp120-specific T helper (Th) cells and cytotoxic T lymphocytes (CTL). Our results support the concept that MHC class II-positive/Ii-negative (class II(+)/Ii(-)) antigen-presenting cells (APC) present endogenously synthesized vaccine antigens simultaneously by MHC class II and class I molecules, activating both CD4(+) and CD8(+) T cells. Activated CD4(+) T cells locally strengthen the response of CD8(+) CTL, thus enhancing the potency of a DNA vaccine.