Human immunodeficiency virus type 1 gag-specific mucosal immunity after oral immunization with papillomavirus pseudoviruses encoding gag

Mucosal surfaces are the primary portals for human immunodeficiency virus (HIV) transmission. Because systemic immunization, in general, does not induce effective mucosal immune responses, a mucosal HIV vaccine is urgently needed. For this study, we developed papillomavirus pseudoviruses that express HIV-1 Gag. The pseudoviruses are synthetic, nonreplicating viruses, yet they can produce antigens for a long time in the immune system. Here we show that oral immunization of mice by the use of papillomavirus pseudoviruses encoding Gag generated mucosal and systemic Gag-specific cytotoxic T lymphocytes that effectively lysed Gag-expressing target cells. Furthermore, the pseudoviruses generated Gag-specific gamma interferon-producing T cells and serum immunoglobulin G (IgG) and mucosal IgA. In contrast, oral immunization with plasmid DNA encoding HIV-1 Gag did not induce specific immune responses. Importantly, oral immunization with the pseudoviruses induced Gag-specific memory cytotoxic T lymphocytes and protected mice against a rectal mucosal challenge with a recombinant vaccinia virus expressing HIV-1 Gag. Thus, papillomavirus pseudoviruses encoding Gag are a promising mucosal vaccine against AIDS.     

A neonatal Fc receptor-targeted mucosal vaccine strategy effectively induces HIV-1 antigen-specific immunity to genital infection

Strategies to prevent the sexual transmission of HIV include vaccines that elicit durable, protective mucosal immune responses. A key to effective mucosal immunity is the capacity for antigens administered locally to cross epithelial barriers. Given the role of neonatal Fc receptor (FcRn) in transferring IgG across polarized epithelial cells which line mucosal surfaces, FcRn might be useful for delivering HIV vaccine antigens across mucosal epithelial barriers to the underlying antigen-presenting cells. Chimeric proteins composed of HIV Gag (p24) fused to the Fc region of IgG (Gag-Fc) bind efficiently to airway mucosa and are transported across this epithelial surface. Mice immunized intranasally with Gag-Fc plus CpG adjuvant developed local and systemic immunity, including durable B and T cell memory. Gag-specific immunity was sufficiently potent to protect against an intravaginal challenge with recombinant vaccinia virus expressing the HIV Gag protein. Intranasal administration of a Gag-Fc/CpG vaccine protected at a distal mucosal site. Our data suggest that targeting of FcRn with chimeric immunogens may be an important strategy for mucosal immunization and should be considered a new approach for preventive HIV vaccines.     

Direct analysis of the dynamics of the intestinal mucosa CD8 T cell response to systemic virus infection

The CD8 T cell response to vesicular stomatitis virus infection was characterized in the spleen and intestinal mucosa using MHC tetramers. Surprisingly, the primary response persisted in the lamina propria long after the splenic response had declined. Furthermore, the response was characterized by a protracted effector phase in which cytolytic activity in the lamina propria, but not in the spleen, was maintained. The appearance of Ag-specific cells in the intestinal mucosa was largely, though not exclusively, a result of beta(7) integrin-mediated migration. Infection with Listeria monocytogenes or with vaccinia virus also led to sustained mucosal responses. After reinfection of vesicular stomatitis virus-primed mice with a serotypically distinct virus, a sustained recall response was detected in all tissues. In CD40(-/-) mice, the mucosal, but not the splenic, response was compromised, resulting in diminished mucosal memory. The recall response was CD40 independent and correlated with memory levels, indicating that the mucosal and systemic responses operated independently. These findings illustrated the integrated yet distinct nature of systemic vs mucosal immune responses.     

Generation of immune memory by haptenated derivatives of thymus-independent antigens in C57BL/6 mice. I. The differentiation of memory B lymphocytes into antibody-secreting cells depends on the nature of the thymus-independent carrier used for memory induction and/or revelation

It has been previously reported that trinitrophenylated lipopolysaccharide (TNP-LPS), a thymus-independent (TI)-1 antigen, elicits an anamnestic response to TNP in C57BL/6 mice. The ability of these mice to mount a secondary response to TI-2 antigens was analyzed. Priming with DNP-Ficoll or DNP-Dextran, both TI-2 antigens, resulted in an increased frequency of TNP-binding B lymphocytes. Evidence is presented that memory cell-induction by DNP-Ficoll does not require functional T cells. The differentiation into antibody-forming cells (AFC) of memory cells generated by DNP-Dextran or DNP-Ficoll cannot be obtained by a challenge with either antigen. There was no indication that the lack of a secondary response to TI-2 antigens was related to suppressive T cells interfering with memory expression. Memory cells induced by DNP-Dextran or DNP-Ficoll can nevertheless be activated by TNP-LPS. In contrast to the restricted sensitivity of TNP-memory cells generated by TI-2 antigens, TNP-LPS-induced memory cells are indifferently susceptible to TI-1 or TI-2 antigenic stimulation. These results are discussed in terms of memory B-cell subpopulations.     

Thy1+ NK [corrected] cells from vaccinia virus-primed mice confer protection against vaccinia virus challenge in the absence of adaptive lymphocytes

While immunological memory has long been considered the province of T- and B-lymphocytes, it has recently been reported that innate cell populations are capable of mediating memory responses. We now show that an innate memory immune response is generated in mice following infection with vaccinia virus, a poxvirus for which no cognate germline-encoded receptor has been identified. This immune response results in viral clearance in the absence of classical adaptive T and B lymphocyte populations, and is mediated by a Thy1(+) subset of natural killer (NK) cells. We demonstrate that immune protection against infection from a lethal dose of virus can be adoptively transferred with memory hepatic Thy1(+) NK cells that were primed with live virus. Our results also indicate that, like classical immunological memory, stronger innate memory responses form in response to priming with live virus than a highly attenuated vector. These results demonstrate that a defined innate memory cell population alone can provide host protection against a lethal systemic infection through viral clearance.     

Contributions of memory B cells to secondary immune response

The secondary immune response is one of the most important features of immune systems. During the secondary immune response, the immune system can eliminate the antigen, which has been encountered by the individual during the primary invasion, more rapidly and efficiently. Both T and B memory cells contribute to the secondary response. In this paper, we only concentrate on the functions of memory B cells. We explore a model describing the memory contributed by the specific long-lived clone which is maintained by continued stimulation with a small amount of antigens sequestered on the surfaces of the follicular dendritic cells (FDC). The behavior of the secondary response provided by the model can be compared with experimental observations. The model shows that memory B cells indeed play an important role in the secondary response. It is found that a single memory cell in a long-lived clone may not be long-lived. In the present note, the influences of relevant parameters on the secondary response are also explored.     

A Vibrio cholerae ghost-based subunit vaccine induces cross-protective chlamydial immunity that is enhanced by CTA2B, the nontoxic derivative of cholera toxin

The Vibrio cholerae ghost (rVCG) platform is an effective carrier and delivery system for designing efficacious Chlamydia vaccines. We investigated whether CTA2B, the nontoxic derivative of cholera toxin, can augment protective immunity conferred by an rVCG-based chlamydial vaccine and enhance cross-protection against heterologous chlamydial strains. An rVCG vaccine coexpressing chlamydial major outer membrane protein and CTA2B was genetically constructed and antigens were targeted to the inner membrane of V. cholerae before ghost production by gene E -mediated lysis. Effective immunomodulation by CTA2B was demonstrated by the ability of the vaccine construct to enhance the activation and maturation of dendritic cells in vitro . Also, C57BL/6 mice immunized via mucosal and systemic routes showed increased specific mucosal and systemic antibody and T-helper type-1 (Th1) responses, irrespective of the route. The enhanced production of IFN-γ, but not IL-4 by genital mucosal and splenic T cells, indicated a predominantly Th1 response. Clearance of the Chlamydia muridarum vaginal infection was significantly enhanced by codelivery of the vaccine with CTA2B, with the intravaginal route showing a moderate advantage. These results indicate that the rVCG-based vaccine is capable of inducing cross-protection against heterologous chlamydial serovars and that incorporation of mucosal adjuvants, such as CTA2B in the rVCG delivery platform, may enhance protective immunity.     

Perspectives on mucosal vaccines: is mucosal tolerance a barrier?

Mucosal administration of Ags induces specific Abs in external secretions and systemic unresponsiveness termed oral or mucosal tolerance. The dominant response depends on the species studied, the nature, dose, frequency, route of Ag application, and the use of adjuvants. The temporal sequence of Ag exposure determines the quality of the ensuing immune response; although initial mucosal Ag exposure results in systemic T cell hyporesponsiveness, pre-existing systemic responses are refractory to the tolerizing effects of mucosal Ag encounter. Mucosal and systemic humoral responses may be induced concomitantly with diminished systemic T cell responses, thereby permitting Ab-mediated containment of mucosal Ags without stimulation of the systemic immune compartment. B cell Ig isotype switching and differentiation toward IgA production share common regulatory mechanisms with the suppression of T cells. Optimization of mucosal vaccination strategies has the potential for enhancing protective immune responses and suppressing systemic responses to autoantigens desirable for the treatment of autoimmune diseases.     

Induction and visualization of mucosal memory CD8 T cells following systemic virus infection.

Whether CD8 T cell memory exists outside secondary lymphoid organs is unclear. Using an adoptive transfer system that enables tracking of OVA-specific CD8 T cells, we explored the antigenic requirements for inducing CD8 T cell memory and identified intestinal mucosa memory cells. Although systemic immunization with soluble OVA induced clonal expansion, memory CD8 cells were not produced. In contrast, infection with virus-encoding OVA induced memory CD8 cells in the periphery and the lamina propria and intraepithelial compartments of the intestinal mucosa. Mucosal memory cells expressed a distinct array of adhesion molecules as compared with secondary lymphoid memory cells, suggesting that there may be separate mucosal and systemic memory pools. Mucosal CD8 memory cells rapidly produced IFN-gamma after Ag stimulation. Reactivation of memory cells by Ag feeding resulted in increased cell size and up-regulation of CD28 and CD11c. CD8 mucosal memory cells exhibited ex vivo lytic activity that was up-regulated dramatically following Ag reencounter in vivo. Interestingly, reactivation of memory cells did not require CD28-mediated costimulation. The ability of the intestinal mucosa to maintain CD8 memory cells provides a potential mechanism for effective mucosal vaccination.     

Enteric immunization reveals a T cell network for IgA responses and suggests that humans possess a common mucosal immune system

The local IgA response is a result of two related events, the induction of sensitized T and B cells in gut- or b ronchial-associated lymphoreticular tissues (GALT or BALT) and the final differentiation of IgA plasma cells in mucosal tissues where IgA is produced and transported to become secretory IgA (S-IgA) antibodies into external secretions. Oral administration of various types of antigens/vaccines may result in two types of response, i.e., S-IgA antibodies at mucosa and systemic unresponsiveness to antigen, a state termed oral tolerance. Regulatory T cells in GALT help account for both S-IgA responses and oral tolerance and thus serve to fine tune responses to orally encountered antigens. Studies in animal models and humans have shown that oral administration of antigens sensitize lymphoid cells in GALT which subsequently home to mucosa and result in S-IgA responses in several external secretions. The significant promise of oral vaccines for prevention of microbial diseases including neisserial diseases is discussed.     

Inappropriate expression of IgD from a transgene inhibits the function of antigen-specific memory B cells

IgD expression has been shown to be downmodulated upon mitogenic or antigenic activation of B cells. To investigate whether this decrease is of functional significance we studied a mouse strain that expresses transgenic IgD on all B cells. The rearranged gene encoding the heavy chain of this IgD requires endogenous gene rearrangement before it can be expressed; therefore, normal B cell development is not affected. As a result, both transgenic IgD and endogenous IgM and IgD are expressed on all peripheral B cells. We show that the presence of extraneous IgD does not affect normal B cell activation by polyclonal stimulators, nor does it affect the primary IgM or IgG responses to TI or TD antigens. However, the secondary memory response is significantly diminished. The decrease is not attributable to a defective generation of memory B cells; instead the activation of memory cells appears to be compromised. Since the depressed response can be overcome by prior aggregation of the transgenic IgD with allotype-specific anti-IgD antibodies, it appears that persistence of the transgenic IgD on memory cells may influence their ability to be activated. Thus, the decrease in IgD expression on normal B cells after activation may be necessary for optimal activation of memory cells.     

Thy1+ Nk Cells from Vaccinia Virus-Primed Mice Confer Protection against Vaccinia Virus Challenge in the Absence of Adaptive Lymphocytes

While immunological memory has long been considered the province of T- and B- lymphocytes, it has recently been reported that innate cell populations are capable of mediating memory responses. We now show that an innate memory immune response is generated in mice following infection with vaccinia virus, a poxvirus for which no cognate germline-encoded receptor has been identified. This immune response results in viral clearance in the absence of classical adaptive T and B lymphocyte populations, and is mediated by a Thy1⁺ subset of natural killer (NK) cells. We demonstrate that immune protection against infection from a lethal dose of virus can be adoptively transferred with memory hepatic Thy1⁺ NK cells that were primed with live virus. Our results also indicate that, like classical immunological memory, stronger innate memory responses form in response to priming with live virus than a highly attenuated vector. These results demonstrate that a defined innate memory cell population alone can provide host protection against a lethal systemic infection through viral clearance.     

Immunological importance of the second gut segment of carp. I. Uptake and processing of antigens by epithelial cells and macrophages

Uptake and transport of soluble (ferritin) and particulate ( Vibrio anguillarum ) antigen from intestinal lumen to mucosal macrophages was studied using immunocytochemical and electron-microscopical techniques. Both antigens were taken up by epithelial cells of the second gut segment, reached the supranuclear vacuoles and were finally transported to large intraepithelial macrophages. In contrast with particulate antigen, antigenic determinants of ferritin were demonstrated in the basal part of the epithelium and appeared to be released at the mucosal site.
After anal intubation many small macrophages penetrated the intestinal epithelium, took up antigen and after 24 h disappeared again from the intestinal mucosa. This feature was antigen independent and also occurred after anal PBS-intubation. The larger, less mobile macrophages stayed in the intestinal epithelium and finally exposed antigenic determinants (of both antigens) at their outer surface, suggesting an antigen-presenting function. Whether these large macrophages can induce a local or mucosal immune response and whether the smaller mobile macrophages are involved in a systemic response is discussed.     

Systemic vaccination prevents the total destruction of mucosal CD4 T cells during acute SIV challenge

BACKGROUND: Acute human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infections are accompanied by a systemic loss of memory CD4 T cells, with mucosal sites serving as a major site for viral replication, dissemination and CD4 T cell depletion. Protecting the mucosal CD4 T cell compartment thus is critical to contain HIV, and preserve the integrity of the mucosal immune system. The primary objective of this study was to determine if systemic vaccination with DNA/rAd-5 encoding SIV-mac239-env, gag and pol could prevent the destruction of CD4 T cells in mucosal tissues. METHODS: Rhesus macaques were immunized with DNA/r-Ad-5 encoding SIV genes and compared with those immunized with sham vectors following high dose intravenous challenge with SIVmac251. SIV specific CD4 and CD8 T cell responses, cell associated viral loads and mucosal CD4 T cell dynamics were evaluated. RESULTS: Strong SIV specific immune responses were induced in mucosal tissues of vaccinated animals as compared with sham controls. These responses expanded rapidly following challenge suggesting a strong anamnestic response. Immune responses were associated with a decrease in cell associated viral loads, and a loss of fewer mucosal CD4 T cells. Approximately 25% of mucosal CD4 T cells were preserved in vaccinated animals as compared with <5% in sham controls. These results demonstrate that systemic immunization strategies can induce immune responses in mucosal tissues that can protect mucosal CD4 T cells from complete destruction following challenge. CONCLUSIONS: Preservation of mucosal CD4 T cells can contribute to maintaining immune competence in mucosal tissues and provide a substantial immune benefit to the vaccinees.     

Development of a nasal vaccine for chronic hepatitis B infection that uses the ability of hepatitis B core antigen to stimulate a strong Th1 response against hepatitis B surface antigen

There are estimated to be 350 million chronic carriers of hepatitis B infection worldwide. Patients with chronic hepatitis B are at risk of liver cirrhosis with associated mortality because of hepatocellular carcinoma and other complications. An important goal, therefore, is the development of an effective therapeutic vaccine against chronic hepatitis B virus (HBV). A major barrier to the development of such a vaccine is the impaired immune response to HBV antigens observed in the T cells of affected patients. One strategy to overcome these barriers is to activate mucosal T cells through the use of nasal vaccination because this may overcome the systemic immune downregulation that results from HBV infection. In addition, it may be beneficial to present additional HBV epitopes beyond those contained in the traditional hepatitis B surface antigen (HbsAg) vaccine, for example, by using the hepatitis B core antigen (HBcAg). This is advantageous because HBcAg has a unique ability to act as a potent Th1 adjuvant to HbsAg, while also serving as an immunogenic target. In this study we describe the effect of coadministration of HBsAg and HBcAg as part of a strategy to develop a more potent and effective HBV therapeutic vaccine.     

The role of T cells in IgG production; thymus-dependent antigens induce B cell memory in the absence of T cells.

B cell memory was shown to develop in congenitally athymic (nu/nu) mice after injection with small amounts of thymus-dependent antigens, in particular heterologous serum proteins, such as fown gamma-globulin (FGG) or DNP-bovine-serum albumin (DNP-BSA). Large doses of proteins (10 mg) tended to produce a specific B cell unresponsiveness, although there was still some evidence of B cell priming. The antigen did not have to be in a multivalent form to interact with B cell so as to induce immunologic memory or tolerance. In contrast to the induction of B cell memory, the production of IgG antibody in this system was found to be strongly T cell dependent. Thymus-independent antigens like LPS or POL with pronounced adjuvant effects on IgG production in normal or surgically thymectomized mice, could not replace T cells in allowing an IgG response against thymus-dependent antigens in congenitally athymic mice. However, the action of T cells once activated is likely to be non-antigen-specific, since it was shown that supernatants of antigen-activated-syngeneic T cells stimulated IgG production in cultures of primed B cell populations non-antigen-specifically.     

Increased levels of (class switched) memory B cells in peripheral blood of current smokers

There is increasing evidence that a specific immune response contributes to the pathogenesis of COPD. B-cell follicles are present in lung tissue and increased anti-elastin titers have been found in plasma of COPD patients. Additionally, regulatory T cells (Tregs) have been implicated in its pathogenesis as they control immunological reactions. We hypothesize that the specific immune response in COPD is smoke induced, either by a direct effect of smoking or as a result of smoke-induced lung tissue destruction (i.e. formation of neo-epitopes or auto antigens). Furthermore, we propose that Tregs are involved in the suppression of this smoke-induced specific immune response.The presence of B cells, memory B cells and Tregs was assessed by flow cytometry in peripheral blood of 20 COPD patients and 29 healthy individuals and related to their current smoking status.COPD patients had lower (memory) B-cell percentages and higher Treg percentages in peripheral blood than healthy individuals, with a significant negative correlation between these cells. Interestingly, current smokers had higher percentages of (class-switched) memory B cells than ex-smokers and never smokers, irrespective of COPD.This increase in (class-switched) memory B cells in current smokers is intriguing and suggests that smoke-induced neo-antigens may be constantly induced in the lung. The negative correlation between B cells and Tregs in blood is in line with previously published observations that Tregs can suppress B cells. Future studies focusing on the presence of these (class switched) memory B cells in the lung, their antigen specificity and their interaction with Tregs are necessary to further elucidate the specific B-cell response in COPD.     

Functional heterogeneity of virgin and memory B murine lymphocytes revealed by the utilization of cyclosporin A: an overview

The effects of cyclosporin A on the generation and revelation of B memory cells by thymus-independent (TI) antigens was investigated. A class 1 (TNP-LPS) and a class 2 (TNP-Ficoll) TI antigens were used for priming an elicitation. Evidence is presented that cyclosporin A does not interfere with the generation of hapten-specific (TNP) B memory cells by TNP-LPS or DNP-Ficoll. Cyclosporin A does not affect the revelation of B memory cells by TNP-LPS, but inhibits their revelation by TNP-Ficoll. These findings are discussed in terms of two distinct B cell lineages leading to antibody-forming cells and memory cells precursors, and in terms of heterogeneity of B memory cells.     

Successful priming and tolerization of T cells to orally administered antigens in B-cell-deficient mice

B cells have been shown to function as APCs capable of inducing both T cell priming and tolerization. Recently, B cells were also revealed to be essential in the organogenesis of Payer's patches (PPs), which have been supposed to play an important role in the initiation of mucosal immune responses. In this study, we examined the roles of B cells in T cell response to orally administrated antigen using B-cell-deficient mice. It was revealed that (1) both a single high dose and repeated low doses of orally administered OVA successfully induced tolerance of T cells in B-cell-deficient mice and (2) oral administration of OVA with cholera toxin successfully primed T cells in B-cell-deficient mice. Thus, it was revealed that B cells are not required for both priming and tolerization of T cells to orally administered antigens. These results also contradict the supposed roles of PPs in mucosal immune responses.     

Axon growth and guidance genes identify T-dependent germinal centre B cells

Selection of B cells subjected to hypermutation in germinal centres (GC) during T cell-dependent (TD) antibody responses yields memory cells and long-lived plasma cells that produce high affinity antibodies biased to foreign antigens rather than self-antigens. GC also form in T-independent (TI) responses to polysaccharide antigens but failed selection results in GC involution and memory cells are not generated. To date there are no markers that allow phenotypic distinction of T-dependent and TI germinal centre B cells. We compared the global gene expression of GC B cells purified from mice immunized with either TD or TI antigens and identified eighty genes that are differentially expressed in TD GC. Significantly, the largest cluster comprises genes involved in growth and guidance of neuron axons such as Plexin B2, Basp1, Nelf, Shh, Sc4mol and Sult4alpha. This is consistent with formation of long neurite (axon and dendrite)-like structures by mouse and human GC B cells, which may facilitate T:B cell interactions within GC, affinity maturation and B cell memory formation. Expression of BASP1 and PLEXIN B2 protein is very low or undetectable in resting and TI GC B cells, but markedly upregulated in GC B cells induced in the presence of T cell help. Finally we show some of the axon growth genes upregulated in TD-GC B cells including Basp1, Shh, Sult4alpha, Sc4mol are also preferentially expressed in post-GC B cell neoplasms.