Heath M. Guay

Immunogenicity of Influenza Virus Vaccine Is Increased by Anti-Gal-Mediated Targeting to Antigen-Presenting Cells

ABSTRACT This study describes a method for increasing the immunogenicity of influenza virus vaccines by exploiting the natural anti-Gal antibody to effectively target vaccines to antigen-presenting cells (APC). This method is based on enzymatic engineering of carbohydrate chains on virus envelope hemagglutinin to carry the α-Gal epitope (Galα1-3Galβ1-4GlcNAc-R). This epitope interacts with anti-Gal, the most abundant antibody in humans (1% of immunoglobulins). Influenza virus vaccine expressing α-Gal epitopes is opsonized in situ by anti-Gal immunoglobulin G. The Fc portion of opsonizing anti-Gal interacts with Fcγ receptors on APC and induces effective uptake of the vaccine virus by APC. APC internalizes the opsonized virus to transport it to draining lymph nodes for stimulation of influenza virus-specific T cells, thereby eliciting a protective immune response. The efficacy of such an influenza vaccine was demonstrated in α1,3galactosyltransferase (α1,3GT) knockout mice, which produce anti-Gal, using the influenza virus strain A/Puerto Rico/8/34-H1N1 (PR8). Synthesis of α-Gal epitopes on carbohydrate chains of PR8 virus (PR8αgal) was catalyzed by recombinant α1,3GT, the glycosylation enzyme that synthesizes α-Gal epitopes in cells of nonprimate mammals. Mice immunized with PR8αgal displayed much higher numbers of PR8-specific CD8+ and CD4+ T cells (determined by intracellular cytokine staining and enzyme-linked immunospot assay) and produced anti-PR8 antibodies with much higher titers than mice immunized with PR8 lacking α-Gal epitopes. Mice immunized with PR8αgal also displayed a much higher level of protection than PR8 immunized mice after being challenged with lethal doses of live PR8 virus. We suggest that a similar method for increasing immunogenicity may be applicable to avian influenza vaccines.

MyD88 is required for the formation of long-term humoral immunity to virus infection.

Development of long-term humoral immunity is a major goal of vaccination, but the mechanisms involved in the formation of long-term Ab responses are still being determined. In this study, we identify a previously unknown requirement for MyD88, an adaptor molecule that mediates signals at most TLRs, for the generation of long-term humoral immunity during live virus infection. Polyoma virus-infected MyD88 knockout mice generated strong acute T cell-dependent antiviral IgM and IgG responses and developed germinal centers. Activation-induced cytidine deaminase, an enzyme required for isotype switching and somatic hypermutation, was also induced in germinal center B cells, similar to wild-type mice. However, MyD88 knockout mice failed to develop bone marrow plasma cells and did not maintain long-term serum antiviral Ab responses. The isotype distribution of antiviral IgG responses was also altered; serum IgG2a and IgG2b levels were diminished, whereas IgG1 responses were not affected. The requirement for MyD88 for the formation of long-term humoral immunity to polyoma virus was intrinsic to B cells and was independent of IL-1R and IL-18R, cytokine receptors that also signal through MyD88. Our findings show that MyD88-dependent signaling pathways in B cells are essential for effectively generating long-term Ab responses and implicate a role for TLR in the formation of long-term humoral immunity.

The Antiviral CD8+ T Cell Response Is Differentially Dependent on CD4+ T Cell Help Over the Course of Persistent Infection

Although many studies have investigated the requirement for CD4+ T cell help for CD8+ T cell responses to acute viral infections that are fully resolved, less is known about the role of CD4+ T cells in maintaining ongoing CD8+ T cell responses to persistently infecting viruses. Using mouse polyoma virus (PyV), we asked whether CD4+ T cell help is required to maintain antiviral CD8+ T cell and humoral responses during acute and persistent phases of infection. Though fully intact during acute infection, the PyV-specific CD8+ T cell response declined numerically during persistent infection in MHC class II-deficient mice, leaving a small antiviral CD8+ T cell population that was maintained long term. These unhelped PyV-specific CD8+ T cells were functionally unimpaired; they retained the potential for robust expansion and cytokine production in response to Ag rechallenge. In addition, although a strong antiviral IgG response was initially elicited by MHC class II-deficient mice, these Ab titers fell, and long-lived PyV-specific Ab-secreting cells were not detected in the bone marrow. Finally, using a minimally myeloablative mixed bone marrow chimerism approach, we demonstrate that recruitment and/or maintenance of new virus-specific CD8+ T cells during persistent infection is impaired in the absence of MHC class II-restricted T cells. In summary, these studies show that CD4+ T cells differentially affect CD8+ T cell responses over the course of a persistent virus infection.

Generation of Protective T Cell-Independent Antiviral Antibody Responses in SCID Mice Reconstituted with Follicular or Marginal Zone B Cells

B cells generated in the bone marrow of adult mice enter the periphery as transitional B cells and subsequently differentiate into one of two phenotypically and functionally distinct subsets, marginal zone (MZ) or follicular (Fo) B cells. Recent reports indicate, however, that in response to environmental cues, such as lymphopenia, mature Fo B cells can change to display phenotypic markers characteristic of MZ B cells. Previously, we found that splenic B cells transferred to SCID mice responded to polyoma virus (PyV) infection with T cell-independent (TI) IgM and IgG secretion, reducing the viral load and protecting mice from the lethal effect of the infection. The contribution of MZ and Fo B cell subsets to this antiviral TI-2 response, however, has not been addressed. In this study, we show that both sort-purified MZ and Fo B cells generate protective TI Ab responses to PyV infection when transferred into SCID mice. Moreover, the transferred Fo B cells in the spleens of the PyV-infected SCID mice change phenotype, with many of them displaying MZ B cell characteristics. These findings demonstrate the plasticity of the B cell subsets in virus-infected hosts and show for the first time that B cells derived exclusively from Fo B cells can effectively function in antiviral TI-2 responses.

Dynamics and magnitude of virus-induced polyclonal B cell activation mediated by BCR-independent presentation of viral antigen

Hypergammaglobulinemia and production of autoantibodies occur during many viral infections, and studies have suggested that viral antigen‐presenting B cells may become polyclonally activated by CD4 T cells in vivo in the absence of viral engagement of the BCR. However, we have reported that CD4 cells in lymphocytic choriomengitis virus (LCMV)‐infected mice kill adoptively transferred B cells coated with LCMV class II peptides. We report here that most of the surviving naïve B cells presenting class II MHC peptides undergo an extensive differentiation process involving both proliferation and secretion of antibodies. Both events require CD4 cells and CD40/CD40L interactions but not MyD88‐dependent signaling within the B cells. B cells taken from immunologically tolerant donor LCMV‐carrier mice with high LCMV antigen load became activated following adoptive transfer into LCMV‐infected hosts, suggesting that B cells present sufficient antigen for this process during a viral infection. No division or activation of B cells was detected at all in virus‐infected hosts in the absence of cognate CD4 T cells and class II antigen. This approach, therefore, formally demonstrates and quantifies a virus‐induced polyclonal proliferation and differentiation of B cells, which, due to their high proportion, would mostly have BCR not specific for the virus.