Mark R. McDermott

Bronchus- and nasal-associated lymphoid tissues

Summary:  The bronchus‐associated lymphoid tissue (BALT) and the nasal‐associated lymphoid tissue (NALT) constitute organized lymphoid aggregates that are capable of T‐ and B‐cell responses to inhaled antigens. BALT, located mostly at bifurcations of the bronchus in animals and humans, is present in the fetus and develops rapidly following birth, especially in the presence of antigens. Humoral immune responses elicited by BALT are primarily immunoglobulin A secretion both locally and by BALT‐derived B cells that have trafficked to distant mucosal sites. Similarly located T‐cell responses have been noted. On the basis of these findings, the BALT can be thought of as functionally analogous to mucosal lymphoid aggregates in the intestine and is deemed a member of the common mucosal immunologic system. NALT has been described principally in the rodent nasal passage as two separate lymphoid aggregates. It develops after birth, likely in response to antigen, and B‐ and T‐cell responses parallel those that occur in BALT. It is not known whether NALT cells traffic to distant mucosal sites, although mucosal responses have been detected after nasal immunization. NALT appears from many studies to be a functionally distinct lymphoid aggregate when compared with BALT and Peyers patches. It may exist, however, in humans as a diffuse collection of isolated lymphoid follicles.

Monitoring foreign gene expression by a human adenovirus-based vector using the firefly luciferase gene as a reporter

We have constructed a helper-independent adenovirus type 5-luciferase recombinant (Ad5-Luc 3) containing the firefly luciferase gene flanked by simian virus 40 (SV40) regulatory sequences inserted in the early region 3 (E3) of the Ad5 genome. Expression of luciferase in cells infected with Ad5-Luc3 was relatively efficient. In HeLa cells approximately 20 micrograms luciferase per 10(6) cells was made by 36 h post-infection and a 62 kilo-Dalton (kDa) luciferase band was clearly visible in a [35S]methionine-labeled Ad5-Luc 3-infected cell extract analyzed directly by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results of experiments in which cultured cells were infected with Ad5-Luc 3 in the presence or absence of 1-beta-D-arabinofuranosyl cytosine (AraC) showed that the majority of luciferase expression was dependent on viral DNA replication. This suggested that the enzyme was probably translated primarily from mRNA derived from transcripts expressed from the major late promoter of Ad5. An anti-luciferase antibody was raised in a rabbit and used to further characterize the luciferase expressed in HeLa cells infected with Ad5-Luc 3 by immunoprecipitations and Western blot analyses. The half-life of luciferase expressed in HeLa cells infected with Ad5-Luc 3 was calculated to be approximately 6-8 h by pulse chase analysis. Luciferase is likely to be a useful marker for monitoring virus dissemination and gene expression in experimental animals because assays for enzymatic activity are extremely sensitive and backgrounds are low in all tissues. In mice inoculated intraperitoneally (i.p.) with Ad5-Luc 3, luciferase activity was detected in the liver, spleen, kidney, and lung. A single i.p. inoculation of mice with Ad5-Luc 3 was sufficient to raise anti-luciferase antibody and Ad5 neutralizing antibody which persisted for at least 8 weeks. Even in the presence of circulating anti-luciferase and Ad5 neutralizing antibodies, luciferase activity could be detected in the livers, spleens, and kidneys of mice inoculated i.p. a second time with Ad5-Luc 3.

Impairment of host-versus-graft reaction in pregnant mice: II. Selective suppression of cytotoxic T-cell generation correlates with soluble suppressor activity and with successful allogeneic pregnancy☆☆☆

Abstract The fetus resulting from an allogeneic (interstrain) mating represents a type of graft that is not rejected by the mother. We have studied the lymph nodes draining the uterus (DLN) of C3H mice which had been mated to DBA/2 males. In order to determine the mechanism by which allogeneic pregnancy inhibits the generation of cytotoxic T cells (CTL) reactive with paternal H-2 antigens, we show that the frequency of precursor cells that develop into CTL in the DLN is not reduced, and thus pregnancy suppresses the development of CTL from their precursors. This suppression, which has been associated with mitomycin C-resistant suppressor cells in the DLN, tends to be anatomically localized to the DLN, acts without specificity for paternal H-2 antigens, and is associated with a soluble suppressive activity which does not pass through an ultrafiltration membrane (25,000-dalton cutoff). Although CTL generation was suppressed in the DLN, the antibody response to sheep erythrocyte antigens was not impaired. Allogeneically mated CBA strain mice which spontaneously resorbed their fetuses appeared to lack the suppression of CTL generation found in successful pregnancies. The presence of cell-associated suppressor activity in the DLN appears to correlate with the occurrence of successful allogeneic pregnancy in mice.

A Common Mucosal Immunologic System Involving the Bronchus, Breast and Bowel

The immune response of the upper respiratory tract is characterized by a predominance of secretory IgA anitbody in local secretions and IgA containing cells in the lamina propria (1). The amount of IgA which is present at various levels of the respiratory tract differs since much more IgA is found high, than low, in the respiratory tract (2). The amount of IgA relative to IgG diminishes in secretions derived from lower in the tract, so that washings derived from the alveolar spaces show a ratio of the two immunoglobulins which approximates that of serum (2).

Mucosal and systemic antiviral antibodies in mice inoculated intravaginally with herpes simplex virus type 2

Herpes simplex virus type 2 (HSV-2) causes lethal illness after intravaginal (IVAG) inoculation into BALB/cJ mice. In the present studies, we demonstrated in mice that primary IVAG vaccination with an attenuated strain of HSV-2 induced humoral immunity in sera and in vaginal secretions. Secondary genital exposure to HSV-2 enhanced this response. However, intraperitoneal exposure to attenuated HSV-2 elicited an antiviral antibody response in sera but not in vaginal secretions. In both sera and vaginal secretions, antiviral IgG antibodies were the major isotype. Systemic exposure to HSV-2 elicited antibodies only in sera that were specific for the major viral antigens whereas IVAG inoculation with HSV-2 stimulated both serum and vaginal antibody responses. Intravenous transfer of antiviral monoclonal antibodies protected against systemic HSV-2 infection but were ineffective against vaginal infection due to a lack of transudation into vaginal secretions. These results suggested that local humoral immunity in the genital tract is important in resistance to HSV-2.

Novel polymer-grafted starch microparticles for mucosal delivery of vaccines.

Recent studies have demonstrated that systemic and mucosal administration of soluble antigens in biodegradable microparticles can potentiate antigen‐specific humoral and cellular immune responses. However, current microparticle formulations are not adequate for all vaccine antigens, necessitating the further development of microparticle carrier systems. In this study, we developed a novel microparticle fabrication technique in which human serum albumin (HSA) was entrapped in starch microparticles grafted with 3‐(triethoxysilyl)‐propyl‐terminated polydimethylsiloxane (TS‐PDMS), a biocompatible silicone polymer. The immunogenicity of HSA was preserved during the microparticle fabrication process. Following intraperitoneal immunization of mice, TS‐PDMS‐grafted microparticles (MP) dramatically enhanced serum IgG responses compared with ungrafted MP and soluble HSA alone (P < 0.001). When delivered orally, both TS‐PDMS‐grafted and ungrafted microparticles elicited HSA‐specific IgA responses in gut secretions, in contrast to orally administered soluble antigen. Indeed, TS‐PDMS‐grafted microparticles stimulated significantly stronger serum IgG (P < 0.005) and IgA (P < 0.001) responses compared with those elicited by ungrafted microparticles. These findings indicate that TS‐PDMS‐grafted starch microparticles have potential as systemic and mucosal vaccine delivery vehicles.

Intranasal immunization with polymer‐grafted microparticles activates the nasal‐associated lymphoid tissue and draining lymph nodes

Waldeyer’s ring is located at the juncture of the respiratory and alimentary tracts, where it is bombarded by inhaled and ingested antigens. However, knowledge of its exact function or consequences of its removal is incomplete. Recently, the murine nasal‐associated lymphoid tissue (NALT) has been reported to have functional similarities to Waldeyer’s ring and, thus, might be a suitable model to examine the function of oronasopharyngeal lymphoid tissues. To explore the capability of NALT to incite local mucosal and systemic immunity, we immunized mice intranasally (i.n.) with 3‐(triethoxysilyl)‐propyl‐terminated polydimethylsiloxane (TS‐PDMS)‐grafted microparticles (MP), an inoculant previously shown to induce robust systemic and mucosal humoral immunity following intragastric (i.g.) administration. We demonstrated that i.n. immunization with low doses of microentrapped, but not soluble, human serum albumin (HSA) evoked robust circulating IgG responses (P<0·05). Additionally, NALT cells isolated from MP‐treated mice proliferated in vitro when restimulated with HSA (P<0·05), suggesting that i.n. immunization with HSA‐containing MP incited specific immunity via NALT cell activation. Coinciding with these observations, after i.n. MP administration HSA‐specific spot‐forming cells (SFC) were observed in NALT, and later posterior cervical lymph nodes (pCLN) and spleen (SPL), suggesting that the observed MP‐induced specific systemic antibody responses emanated from the NALT. We also showed that i.n. immunization with HSA‐containing TS‐PDMS‐grafted MP stimulated interleukin‐4 (IL‐4)‐secreting lymphocytes in the NALT. This cytokine microenvironment was probably responsible for driving the IgG1 sera response observed after i.n. MP administration, via the migration of NALT‐derived IgG1‐committed B cells. Interestingly, unlike i.g. MP administration, i.n. immunization with HSA‐containing MP did not evoke detectable specific IgA in any lymphoid tissue examined, or in nasal secretions, probably reflecting differences between NALT and other mucosae‐associated lymphoid tissues (MALT).

Immunogenicity in mice of tandem repeats of an epitope from herpes simplex gD protein when expressed by recombinant adenovirus vectors

The antigenic and immunogenic potential was examined of human adenovirus type 5 (Ad) recombinants carrying and expressing from one to four tandem repeats of a linear neutralizing epitope from the gD protein of herpes simplex virus type 1 (HSV-1) as a fusion with the beta-galactosidase protein. The fusion proteins produced by these Ad vectors in infected cell culture reacted with a herpes simplex virus (HSV) epitope-specific monoclonal antibody to a degree dependent on the number of epitope repeats in the protein. Mice immunized by intraperitoneal injection of the Ad vectors developed an anti-HSV immune response as measured by ELISA and by HSV-1 neutralization assays. The mean antibody titre induced by a single injection of the Ad vector increased with the number of epitope repeats expressed by the recombinant. Any animal that had developed a serum-neutralizing titre of at least 1:80 survived challenge with a normally lethal dose of HSV-2 administered by the intraperitoneal route. Recombinant vectors expressing four repeats of the HSV epitope were as effective in antibody induction and protection as an adenovirus vector carrying and expressing the entire HSV gD protein. These results suggest that the expression of tandem repeats of appropriate epitopic sequences by adenovirus vectors may provide a safe and effective method of immunizing against HSV infection.

Polymer-grafted starch microparticles for oral and nasal immunization

Microparticle delivery systems for oral vaccine administration are receiving considerable attention. A novel silicone polymer‐grafted starch microparticle system was developed that is efficacious both orally and intranasally. Unlike most other microparticle systems, this novel system does not appear to retard the release of antigen or to protect antigen from degradation. The results indicate that a unique physiochemical relationship occurs between protein antigen and silicone in a starch matrix that facilitates the mucosal immunogenicity of antigen. This leads to predominance of Th2 antibody response. Taken together, these findings indicate that this novel microparticle system may be advantageous for the delivery of small quantities of antigen, especially intranasally, and may be useful for the induction of oral tolerance.

Vaccination by cholera toxin conjugated to a herpes simplex virus type 2 glycoprotein D peptide

Immunization of BALB/cJ mice with a peptide corresponding to residues 1 to 23 of glycoprotein D [gD(1-23)] from herpes simplex virus type 2 (HSV-2) elicits antibody responses which correlate with protection against lethal HSV-2 infection. In the present study, we examined the ability of cholera toxin (CTX) to act as an immunogenic carrier for gD(1-23). The number of gD(1-23) residues conjugated to CTX affected its binding to GM1 ganglioside and physiological toxicity, both of which are factors affecting oral immunogenicity. The antibody response elicited after intraperitoneal (i.p.) immunization with the CTX-gD(1-23) conjugate was protective against a lethal i.p. challenge with HSV-2. In other experiments, mice were immunized i.p. on day 0 and subsequent immunizations conducted on days 14 and 28 were administered either intragastrically or intravaginally (i.vag.). Intraperitoneal priming followed by either i.p or intragastric boosting resulted in anti-HSV-2 antibodies in vaginal washings and in protection against a lethal i.vag. challenge with HSV-2. Intraperitoneal priming followed by i.vag. boosting did not elicit anti-HSV-2 antibodies in vaginal washings and did not protect mice against a lethal i.vag. challenge with HSV-2. These results suggest that CTX can act as a systemic and an oral delivery molecule for the covalently linked gD(1-23) peptide and that such conjugates can elicit protective immune responses against systemic and genital HSV-2 infection.