John L. VanCott

CD4 T Cells Are the Only Lymphocytes Needed To Protect Mice against Rotavirus Shedding after Intranasal Immunization with a Chimeric VP6 Protein and the Adjuvant LT(R192G)

ABSTRACT Intranasal immunization of mice with a chimeric VP6 protein and the mucosal adjuvant Escherichia coli heat labile toxin LT(R192G) induces nearly complete protection against murine rotavirus (strain EDIM [epizootic diarrhea of infant mice virus]) shedding for at least 1 year. The aim of this study was to identify the protective lymphocytes elicited by this new vaccine candidate. Immunization of mouse strains lacking one or more lymphocyte populations revealed that protection was dependent on αβ T cells but mice lacking γδ T cells and B cells remained fully protected. Furthermore, depletion of CD8 T cells in immunized B-cell-deficient mice before challenge resulted in no loss of protection, while depletion of CD4 T cells caused complete loss of protection. Therefore, αβ CD4 T cells appeared to be the only lymphocytes required for protection. As confirmation, purified splenic T cells from immunized mice were intraperitoneally injected into Rag-2 mice chronically infected with EDIM. Transfer of 2 × 106 CD8 T cells had no effect on shedding, while transfer of 2 × 105 CD4 T cells fully resolved shedding in 7 days. Interestingly, transfer of naive splenic CD4 T cells also resolved shedding but more time and cells were required. Together, these results establish CD4 T cells as effectors of protection against rotavirus after intranasal immunization of mice with VP6 and LT(R192G).

The Role of Th1 and Th2 Cells for Mucosal IgA Responsesa

We have used cytokine-knockout mice to help determine the precise requirements for CD4+ Th cell regulation of IgA responses. In these studies, we have used two different oral delivery systems to induce mucosal and systemic antibody responses to the vaccine TT. In normal mice, oral administration of TT with CT as adjuvant induces Th2 cells and cytokines, which give rise to mucosal IgA and serum IgG1, IgA, and IgE responses. On the other hand, oral immunization with rSalmonella expressing Tox C results in Th1-type responses as well as Th2 cell-derived IL-10 and macrophage-derived IL-6, which correlate with mucosal IgA and serum IgG2a antibody responses. Two major conclusions can be drawn from our studies with these two regimens in normal, IFN-gamma-/-, and IL-4-/- mice. First, oral administration of rSalmonella, which elicits classical Th1-type responses also induces significant mucosal IgA responses when given to mice with defective Th1- (IFN-gamma-/-) or Th2- (IL-4-/-) cytokine pathways. Interestingly, we detect Th2-type cells producing IL-10 and macrophage-secreting IL-6 in both normal and cytokine-deficient mice, and we postulate that these two cytokines are of most importance for murine IgA responses. Second, oral administration of TT plus CT as adjuvant induces classical Th2-type responses in both normal and IFN-gamma-/- mice. Further, lack of IL-4 results in failure to induce mucosal IgA responses. Thus, the IL-4 pathway is necessary for the CT adjuvant effect for mucosal IgA responses after oral immunization with a protein vaccine.

Functional Mapping of Protective Domains and Epitopes in the Rotavirus VP6 Protein

ABSTRACT The purpose of this study was to determine which regions of the VP6 protein of the murine rotavirus strain EDIM are able to elicit protection against rotavirus shedding in the adult mouse model following intranasal (i.n.) immunization with fragments of VP6 and a subsequent oral EDIM challenge. In the initial experiment, the first (fragment AB), middle (BC), or last (CD) part of VP6 that was genetically fused to maltose-binding protein (MBP) and expressed inEscherichia coli was examined. Mice (BALB/c) immunized with two 9-μg doses of each of the chimeras and 10 μg of the mucosal adjuvant LT(R192G) were found to be protected against EDIM shedding (80, 92, and nearly 100% reduction, respectively; P≤ 0.01) following challenge. Because CD produced almost complete protection, we prepared four E. coli-expressed, MBP-fused chimeras containing overlapping fragments of the CD region (i.e., CD1, CD2, CD3, and CD4) whose lengths ranged from 61 to 67 amino acid residues. Following i.n. immunization, CD1, CD2, and CD4 induced significant (P ≤ 0.004) protection (88, 84, and 92% reduction, respectively). In addition, 11 peptides (18 to 30 residues) of the CD region with between 0 and 13 overlapping amino acids were synthesized. Two 50-μg doses of each peptide with LT(R192G) were administered i.n. to BALB/c mice. Five peptides were found to elicit significant (P ≤ 0.02) protection. Moreover, a 14-amino-acid region within peptide 6 containing a putative CD4+ T-cell epitope was found to confer nearly complete protection, suggesting a protective role for CD4+ T cells. Mice that were protected by fragments BC and CD1 and four of the five protective synthetic peptides did not develop measurable rotavirus antibodies in serum or stool, implying that protection induced by these domains was not dependent on antibody. Together, these observations suggest that multiple regions of VP6 can stimulate protection, a region of VP6 as small as 14 amino acids containing a CD4+ T-cell epitope can stimulate nearly complete protection, and protection mediated by a subset of epitopes in the VP6 protein does not require antibodies in BALB/c mice.

The Role of Interferons in Rotavirus Infections and Protection

Type I and type II interferons (IFNs) play a critical role in control of a number of viral infections. To study whether altered and reduced functional capacities of type I and type II IFNs would affect rotavirus-induced diarrhea and viral replication, we obtained signal transducers and activators of transcription 1 (Stat1) knock-out mice (Stat1(-/-)) that lack many IFN-induced responses. We found that suckling Stat1(-/-) and immunocompetent mice orally infected with rotavirus experienced diarrhea and shed rotavirus with similar intensity. However, adult Stat1(-/-) mice shed up to 100-fold more homologous murine rotavirus and heterologous rhesus rotavirus antigen in their stools than did immunocompetent mice 2-6 days after infection. Clearance of rotavirus in stools from adult Stat1(-/-) mice occurred at the same time as in wild-type (WT) control mice. Clearance in Stat1(-/-) mice correlated with a potent antibody response and a mixed Th1 and Th2 response, whereas in WT control mice, clearance correlated with a weaker antibody response and a polarized Th1 response. Stat1(-/-) mice were fully protected against subsequent challenge. Moreover, vaccination of adult Stat1(-/-) mice with a rotavirus VP6 protein and the mucosal adjuvant Escherichia coli heat-labile toxin LT (R192G) elicited 94% protection, as measured by the total reduction in viral shedding for the group in comparison to unimmunized controls. Thus, modulating IFN function through the loss of Stat1 caused a defective innate immune response in adult mice but had no effect on rotavirus-induced diarrhea and replication in suckling mice. Furthermore, adult Stat1(-/-), IFN-gamma, and IFN-alpha/beta receptor(-/-) (IFNAR-2(-/-)) mice infected with rotavirus or vaccinated with VP6 vaccine and adjuvant were fully protected against rotavirus shedding following a subsequent challenge with rotavirus.

Mice Develop Effective but Delayed Protective Immune Responses When Immunized as Neonates either Intranasally with Nonliving VP6/LT(R192G) or Orally with Live Rhesus Rotavirus Vaccine Candidates

ABSTRACT Rotavirus vaccines are delivered early in life, when the immune system is immature. To determine the effects of immaturity on responses to candidate vaccines, neonatal (7 days old) and adult mice were immunized with single doses of either Escherichia coli-expressed rotavirus VP6 protein and the adjuvant LT(R192G) or live rhesus rotavirus (RRV), and protection against fecal rotavirus shedding following challenge with the murine rotavirus strain EDIM was determined. Neonatal mice immunized intranasally with VP6/LT(R192G) were unprotected at 10 days postimmunization (dpi) and had no detectable rotavirus B-cell (antibody) or CD4+ CD8+ T-cell (rotavirus-inducible, Th1 [gamma interferon and interleukin-2 {IL-2}]-, Th2 [IL-5 and IL-4]-, or ThIL-17 [IL-17]-producing spleen cells) responses. However, by 28 and 42 dpi, these mice were significantly (Pu2009≥ 0.003) protected and contained memory rotavirus-specific T cells but produced no rotavirus antibody. In contrast, adult mice were nearly fully protected by 10 dpi and contained both rotavirus immunoglobulin G and memory T cells. Neonates immunized orally with RRV were also less protected (P = 0.01) than adult mice by 10 dpi and produced correspondingly less rotavirus antibody. Both groups contained few rotavirus-specific memory T cells. Protection levels by 28 dpi for neonates or adults were equal, as were rotavirus antibody levels. This report introduces a neonatal mouse model for active protection studies with rotavirus vaccines. It indicates that, with time, neonatal mice develop full protection after intranasal immunization with VP6/LT(R192G) or oral immunization with a live heterologous rotavirus and supports reports that protection depends on CD4+ T cells or antibody, respectively.

Protective Immunity to Rotavirus Shedding in the Absence of Interleukin-6: Th1 Cells and Immunoglobulin A Develop Normally

ABSTRACT We investigated whether interleukin-6 (IL-6) was required for the development of immunoglobulin A (IgA)- and T-helper 1 (Th1)-associated protective immune responses to rotavirus by using adult IL-6-deficient mice [BALB/c and (C57BL/6 × O1a)F2 backgrounds]. Naive IL-6− mice had normal frequencies of IgA plasma cells in the gastrointestinal tract. Consistent with this, total levels of IgA in fecal extracts, saliva, and sera were unaltered. In specific response to oral infection with rhesus rotavirus, IL-6−and IL-6+ mice exhibited efficient Th1-type gamma interferon responses in Peyers patches with high levels of serum IgG2a and intestinal IgA. Although there was an increase in Th2-type IL-4 in CD4+ T cells from IL-6− mice following restimulation with rotavirus antigen in the presence of irradiated antigen-presenting cells, unfractionated Peyers patch cells failed to produce a significant increase in IL-4. Moreover, virus-specific IgG1 in serum was not significantly increased in IL-6− mice in comparison with IL-6+ mice. Following oral inoculation with murine rotavirus, IL-6− and IL-6+ mice mediated clearance of rotavirus and mounted a strong IgA response. When IL-6− and IL-6+ mice [(C57BL/6 × O1a)F2 background] were orally inoculated with rhesus rotavirus and later challenged with murine rotavirus, all of the mice maintained high levels of IgA in feces and were protected against reinfection. Thus, IL-6 failed to provide unique functions in the development of IgA-secreting B cells and in the establishment of Th1-associated protective immunity against rotavirus infection in adult mice.

Induction of pneumococcal polysaccharide-specific mucosal immune responses by oral immunization

Liposome and cholera toxin (CT) are considered to be effective antigen delivery vehicles and adjuvants for mucosal vaccines. The effect of these antigen delivery systems on adjuvant responses to mucosally administered pneumococcal polysaccharide (Pnup) was investigated in this study. Both mucosal (e.g. oral) and systemic (i.p.) immunization of mice with purified preparations of Pnup type 23F induced antigen-specific IgM responses in sera. Interestingly, oral immunization of as little as 10 micrograms of Pnup type 23F was sufficient to induce systemic IgM responses. Pnup-specific IgM antibodies peaked by day 7 and no booster responses were evident after a second dose on day 14. In order to examine whether IgG and IgA Pnup-specific immune responses are induced by mucosal immunization, the mucosal adjuvant CT was mixed with Pnup type 23 as an oral vaccine. Co-oral administration of CT and Pnup type 23F resulted in the induction of Pnup-specific faecal IgA antibodies. These results were confirmed by detecting antigen-specific IgA-spot-forming cells in mononuclear cell suspensions prepared from the intestine of immunized mice. These findings suggest that oral immunization with Pnup in the presence of mucosal adjuvants, such as CT, could induce Pnup-specific IgA responses whereas Pnup alone did not. In an attempt to further enhance antigen-specific antibody responses, Pnup type 23F was encapsulated in liposomes and used as mucosal vaccine. However, immunogenicity of Pnup was not improved.

A T cell/B cell/epithelial cell internet for mucosal inflammation and immunity

ConclusionsMucosal immune responses are strongly regulated by CD4+ T cells and their derived cytokines. In this regard, IFN-γ−/− mice (i.e., which lack Th1 and have elevated Th2 cells) showed strong mucosal Th2-type responses together with S-IgA production, while IL-4−/− (e.g., dominant Th1 and lack of Th2 cells) mice had impaired mucosal Th2 and IgA responses following oral delivery of TT and CT. However, when rSalmonella or radenovirus were used for antigen delivery, significant levels of mucosal IgA responses were induced in both IFN-γ−/− and IL-4−/− mice. The choice of the antigen delivery system which leads to optimal Th and B cell interactions are important for the induction of effective IgA responses, even in situations where the immune system is compromised. It is clear that Th2-type cytokines are important in mucosal IgA responses; however, other cytokine combinations can compensate for mucosal immunity in situations in which Th2 cell responses are absent. Mucosally induced tolerance may be one approach to prevent several systemic immune disorders; however, the mechanism of this phenomenon still needs to be elucidated. Our recent findings have suggested that IFN-γ may play an important role in induction of systemic unresponsiveness since oral tolerance was not induced in IFN-γ−/− mice.Our studies as well as those of others indicated that at least two phases of a triad of cell interactions are important for the mucosal immune system. First, it has been shown that epithelial cell-produced IL-7 and SCF andαβ T cell-derived IL-2 are essential activation and growth signals for intestinalγδ T cells. Second, our studies with TCRδ knockout mice have suggested that mucosalγδ T cells also play a critical role in the regulation of mucosal IgA responses. Thus, a mucosal internet amongγδ T cells,αβ T cells, and IgA B cells appear critical for mucosal homeostasis and for regulation of specific mucosal immune responses.

Mucosal immunity: Regulation by helper T cells and a novel method for detection

The mechanisms which regulate mucosal IgA responses to orally administered protein vaccines are not yet fully elucidated. We have used two delivery systems, soluble tetanus toxoid (TT) with the mucosal adjuvant cholera toxin (CT) and recombinant Salmonella expressing Tox C, a fragment of TT, to assess the nature of CD4+ T helper (Th) cells and derived cytokines which support mucosal IgA responses in both normal and cytokine knockout (interferon gamma knockout; IFN-gamma-/- and IL-4-/-) mice. Our results provide important new information regarding Th cell and cytokine regulation of mucosal IgA responses. Whereas TT coadministered with CT induces predominant TT-specific Th2-type responses, rSalmonella delivery of Tox C induced dominant Th1-type responses along with synthesis of the Th2-cytokine IL-10. Both vaccine regimen elicited high levels of mucosal S-IgA and IL-6 production by macrophages. Further oral immunization of IFN-gamma-/- and IL-4-/- mice with rSalmonella Tox C also induced macrophage-derived IL-6 and Th2-derived IL-10 as well as S-IgA responses, suggesting that IFN-gamma from Th1-type cells as well as traditional Th2 cells producing IL-4 and IL-5 are not essential for mucosal IgA responses. Rather, induction of second level Th2 cells producing IL-10 together with high levels of IL-6 from other cell sources may be sufficient for mucosal IgA responses in the absence of traditional Th2 cells. These studies were facilitated by the development of a sensitive new luminometry assay which allowed detection of cytokines and cell surface molecules which are below the levels of detection by current solid phase assays.

Measurement of HIV-1 Specific and Total Antibody Secreting Cells by ELISPOT

B-cells play an important role in protection against pathogens, and they secrete specific antibodies in serum and mucosal secretions upon antigenic stimulation contributing to immune exclusion and clearance of pathogens. The frequency of antibody forming cells (AFC) in specific organs is often a reflection of the route of antigen exposure, i.e., systemic, oral, or intranasal, as well as of antigenic load. Enumeration of AFC was originally performed by plaque-forming cell assay measuring lysis of sheep red blood cells. The nature of this assay, that requires coupling of the antigen to sheep red blood cells (SRBC), made detection of various antigen-specific AFC rather cumbersome. However, the development of the enzyme linked immunodetection of AFCs (ELISPOT) combined with the development of standardized lymphocyte isolation techniques enables detection of AFC secreting antibodies specific for many different antigens and derived from various immunologic effector sites (1-6).