The design of a human immunodeficiency virus-1 (HIV-1) immunogen that can induce broadly reactive neutralizing antibodies is a major goal of HIV-1 vaccine development. Although rare human monoclonal antibodies (mAbs) exist that broadly neutralize HIV-1, HIV-1 envelope immunogens do not induce these antibody specificities. Here we demonstrate that the two most broadly reactive HIV-1 envelope gp41 human mAbs, 2F5 and 4E10, are polyspecific autoantibodies reactive with the phospholipid cardiolipin. Thus, current HIV-1 vaccines may not induce these types of antibodies because of autoantigen mimicry of the conserved membrane-proximal epitopes of the virus. These results may have important implications for generating effective neutralizing antibody responses by using HIV-1 vaccines.

SummaryAntigen-specific B cell responses to mucosally delivered proteins are dependent upon CD4-positive T helper (Th) cells, and the frequency of Thl and Th2 cell responses after oral immunization may determine the level and isotype of mucosal antibody responses. We have used a protein-based vaccine, tetanus toxoid (TT), together with the mucosal adjuvant cholera toxin (CT), for oral immunization of mice to study the nature of antigen-specific Th cell subsets induced in Peyer's patches (PP) of the gastrointestinal (GI) tract and in the spleen (SP) during peak antibody responses. Mice orally immunized with TT and CT responded with antigen-specific secretory immunoglobulin A (S-IgA) antibodies in the GI tract, and with both IgG and IgA antibody responses in serum. PP and SP CD4 + T cells from mice orally immunized with TT plus CT were cultured with antigen-coated latex microspheres for induction of proliferative responses and for enumeration of cytokine producing CD4 + T cells. Interestingly, both PP and SP CD4 + T cell cultures showed increased numbers of IL-4-and IL-5 (Th2-type)-producing, spot-forming cells (SFCs) after 21 d of immunization, while essentially no intefferon-3~ (IFN-'y) or IL-2 (Thl-type) SFCs were noted. Cytokine-spedfic Northern blots and RT-PCR also revealed that significant IL-4 and IL-5 mRNA levels, but not IFN-3, or IL-2 mRNA, were present in CD4 + T cells isolated from antigen-stimulated cultures. However, systemic immunization with TT and CT induced antigen-specific IgG and IgM but not IgA antibodies in serum. Further, both IL-2-and IFN-3,-producing Thl-type cells as well as IL-4-and IL-5-secreting Th2-type cells were generated in SP. Our results show that oral immunization with TT and the mucosal adjuvant CT selectively induced antigen-specific Th2-type responses which may represent the major helper cell phenotype involved in mucosal IgA responses in the GI tract.

Palpable swelling of regional lymph nodes is a common sequela of microbial infections but the mechanism responsible for the sequestration and subsequent coordination of lymphocyte responses within these dynamic structures remains poorly understood. Here we show that draining lymph nodes of mast cell-deficient mice did not demonstrate swelling after intradermal bacterial challenge. Testing of individual mast cell-derived products in this model indicated that tumor necrosis factor was the main mediator of nodal hypertrophy, whereas tryptase and histamine had no effect. After peripheral mast cell activation, both tumor necrosis factor concentrations and the recruitment of circulating T cells were increased within draining nodes. These results show a critical function for peripheral mast cell-derived tumor necrosis factor in regulating the hypertrophy of draining lymph nodes during infection.

Mast cells (MCs) have recently received recognition as prominent effectors in the regulation of immune cell migration to draining lymph nodes and lymphocyte activation. However, their role in the development of humoral immune responses is not clear. Here, we demonstrate that subcutaneous or nasal administration of small-molecule MC activators with vaccine antigens evokes large increases in antigen-specific serum immunoglobulin G (IgG) responses. These responses were MC dependent and correlated with increased dendritic cell and lymphocyte recruitment to draining lymph nodes. Nasal instillation of these formulations also evoked antigen-specific secretory IgA and provided protection against anthrax lethal toxin challenge in vitro and against vaccinia virus infection in vivo. Collectively, these results define the MC as an integral sensory arm of the adaptive immune system. Moreover, they highlight MC activators as a new class of vaccine adjuvants, capable of inducing protective antigen-specific immune responses through needle-free routes of administration.

During infection, signals from the periphery are known to reach draining lymph nodes (DLNs), but how these molecules, such as inflammatory cytokines, traverse the significant distances involved without dilution or degradation remains unclear. We show that peripheral mast cells, upon activation, release stable submicrometer heparin-based particles containing tumor necrosis factor and other proteins. These complexes enter lymphatic vessels and rapidly traffic to the DLNs. This physiological drug delivery system facilitates communication between peripheral sites of inflammation and remote secondary lymphoid tissues.

Vaccination represents the most effective control measure in the fight against infectious diseases. Local mucosal immune responses are critical for protection from, and resolution of, infection by numerous mucosal pathogens. Antigen processing across mucosal surfaces is the natural route by which mucosal immunity is generated, as peripheral antigen delivery typically fails to induce mucosal immune responses. However, we demonstrate in this article that mucosal immune responses are evident at multiple mucosal surfaces after parenteral delivery of Venezuelan equine encephalitis virus replicon particles (VRP). Moreover, coinoculation of null VRP (not expressing any transgene) with inactivated influenza virions, or ovalbumin, resulted in a significant increase in antigen-specific systemic IgG and fecal IgA antibodies, compared with antigen alone. Pretreatment of VRP with UV light largely abrogated this adjuvant effect. These results demonstrate that alphavirus replicon particles possess intrinsic systemic and mucosal adjuvant activity and suggest that VRP RNA replication is the trigger for this activity. We feel that these observations and the continued experimentation they stimulate will ultimately define the specific components of an alternative pathway for the induction of mucosal immunity, and if the activity is evident in humans, will enable new possibilities for safe and inexpensive subunit and inactivated vaccines.vaccine vector ͉ Venezuelan equine encephalitis virus ͉ viral immunology ͉ RNA virus

Mast cells (MCs) are best known for eliciting harmful reactions, mostly after primary immunity has been established. Here, we report that during E. coli infection, the primary humoral response in MC-deficient mice is significantly diminished, and was found to be less protective in a urinary tract infection (UTI) model compared to the response from MC-sufficient counterparts. MCs were found to recruit large numbers of dendritic cells (DCs) into the infected tissue site, which eventually migrated into draining lymph nodes (DLNs) over a prolonged time-course. This pattern of trafficking was facilitated by MC generated TNF, which increased the expression of E-selectin on local blood vessels. Antibody blockade of E-selectin inhibited DC recruitment into the site of infection and DLNs, and consequently impaired the primary humoral immune response. Thus, during infection, resident MCs contribute to the primary protective adaptive response through recruitment of DCs from the circulation into infected sites.

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