Kinetics of protective antibodies are determined by the viral surface antigen.

Delayed and weak virus neutralizing antibody (nAb) responses represent a hallmark correlating not only with the establishment of persistent infection but also with unsuccessful vaccine development. Using a reverse genetic approach, we evaluated possible underlying mechanisms in 2 widely studied viral infection models. Swapping the glycoproteins (GPs) of lymphocytic choriomeningitis virus (LCMV, naturally persisting, noncytolytic, inefficient nAb inducer) and vesicular stomatitis virus (VSV, nonpersisting, cytolytic, potent nAb inducer) transferred the only target of nAb's from either virus to the other. We analyzed the nAb response to each of the 2 recombinant and parent viruses in infected mice and found that nAb kinetics were solely determined by the viral surface GP and not by the virus backbone. Moreover, the slowly and poorly nAb-triggering LCMV virion was a potent immunogenic matrix for the more antigenic VSV-GP. These findings indicate that the viral GP determines nAb kinetics largely independently of the specific viral infection context. They further suggest that structural features of viral GPs or coevolutionary adaptation of the virus's GP to the host's naive B cell repertoire, or both, may critically limit nAb kinetics and improvement of vaccine efficacy.

Water-borne, in situ crosslinked biomaterials from phase-segregated precursors.

A novel process for the preparation of water-borne biomaterials for hard tissue repair from injectable precursors is described, where the precursors form crosslinked materials in situ under physiological conditions. The precursors react by means of a Michael-type addition reaction that makes use of addition donors such as pentaerythritol tetrakis 3'-mercaptopropionate (QT) and addition acceptors such as poly(ethylene glycol) diacrylate 570 MW (PEGDA), pentaerythritol triacrylate (TA), and poly(propylene glycol) diacrylate 900 MW (PPODA). These crosslinked materials (at 75 wt% solid), prepared from water dispersions or reverse emulsions, showed ultimate strengths in compression of 1.8 +/- 0.2 and 6.7 +/- 0.5 MPa and ultimate deformations of 35 +/- 2+/- and 37 +/- 2%, respectively. Scanning electron microscopy (SEM) shows that the morphology of the precursors templated the morphology of the final materials. The current study indicates that it is possible to obtain injectable high-modulus materials that have appropriate mechanical properties and gelation kinetics for tissue augmentation and stabilization applications such as mechanical stabilization of the intervertebral disc annulus.

Spatial and temporal organization of adeno-associated virus DNA replication in live cells.

Upon cell entry, the genomes of herpes simplex virus type 1 (HSV-1) and adenovirus (Ad) associate with distinct nuclear structures termed ND10 or promyelocytic leukemia (PML) nuclear bodies (NBs). PML NB morphology is altered or disrupted by specific viral proteins as replication proceeds. We examined whether adeno-associated virus (AAV) replication compartments also associate with PML NBs, and whether modification or disruption of these by HSV-1 or Ad, both of which are helper viruses for AAV, is necessary at all. Furthermore, to add a fourth dimension to our present view of AAV replication, we established an assay that allows visualization of AAV replication in live cells. A recombinant AAV containing 40 lac repressor binding sites between the AAV inverted terminal repeats was constructed. AAV Rep protein and helper virus-mediated replication of this recombinant AAV genome was visualized by binding of enhanced yellow fluorescent protein-lac repressor fusion protein to double-stranded AAV replication intermediates. We demonstrate in live cells that AAV DNA replication occurs in compartments which colocalize with AAV Rep. Early after infection, the replication compartments were small and varied in numbers from 2 to more than 40 per cell nucleus. Within 4 to 8 h, individual small replication compartments expanded and fused to larger structures which filled out much of the cell nucleus. We also show that AAV replication compartments can associate with modified PML NBs in Ad-infected cells. In wild-type HSV-1-infected cells, AAV replication compartments and PML NBs did not coexist, presumably because PML was completely disrupted by the HSV-1 ICP0 protein. However, alteration or disruption of PML appears not to be a prerequisite for AAV replication, as the formation of replication compartments was normal when the ICP0 mutants HSV-1 dl1403 and HSV-1 FXE, which do not affect PML NBs, were used as the helper viruses; under these conditions, AAV replication compartments did not associate with PML NBs.

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.

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.