Jerry R. McGhee

A Clinical Inflammatory Syndrome Attributable to Aerosolized Lipid- DNA Administration in Cystic Fibrosis

Immunologic reactivity to lipid-DNA conjugates has traditionally been viewed as less of an issue than with viral vectors. We performed a dose escalation safety trial of aerosolized cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the lower airways of eight adult cystic fibrosis patients, and monitored expression by RT-PCR. The cDNA was complexed to a cationic lipid amphiphile (GL-67) consisting of a cholesterol anchor linked to a spermine head group. CFTR transgene was detected in three patients at 2-7 days after gene administration. Four of the eight patients developed a pronounced clinical syndrome of fever (maximum of 103.3EF), myalgias, and arthralgia beginning within 6 hr of gene administration. Serum IL-6 but not levels of IL-8, IL-1, TNF-alpha, or IFN-gamma became elevated within 1-3 hr of gene administration. No antibodies to the cationic liposome or plasmid DNA were detected. We found that plasmid DNA by itself elicited minimal proliferation of peripheral blood mononuclear cells taken from study patients, but led to brisk immune cell proliferation when complexed to a cationic lipid. Lipid and DNA were synergistic in causing this response. Cellular proliferation was also seen with eukaryotic DNA, suggesting that at least part of the immunologic response to lipid-DNA conjugates is independent of unmethylated (E. coli-derived) CpG sequences that have previously been associated with innate inflammatory changes in the lung.

Alternate Mucosal Immune System: Organized Peyer’s Patches Are Not Required for IgA Responses in the Gastrointestinal Tract

The progeny of mice treated with lymphotoxin (LT)-β receptor (LTβR) and Ig (LTβR-Ig) lack Peyer’s patches but not mesenteric lymph nodes (MLN). In this study, we used this approach to determine the importance of Peyer’s patches for induction of mucosal IgA Ab responses in the murine gastrointestinal tract. Immunohistochemical analysis revealed that LTβR-Ig-treated, Peyer’s patch null (PP null) mice possessed significant numbers of IgA-positive (IgA+) plasma cells in the intestinal lamina propria. Further, oral immunization of PP null mice with OVA plus cholera toxin as mucosal adjuvant resulted in Ag-specific mucosal IgA and serum IgG Ab responses. OVA-specific CD4+ T cells of the Th2 type were induced in MLN and spleen of PP null mice. In contrast, when TNF and LT-α double knockout (TNF/LT-α−/−) mice, which lack both Peyer’s patches and MLN, were orally immunized with OVA plus cholera toxin, neither mucosal IgA nor serum IgG anti-OVA Abs were induced. On the other hand, LTβR-Ig- and TNF receptor 55-Ig-treated normal adult mice elicited OVA- and cholera toxin B subunit-specific mucosal IgA responses, indicating that both LT-αβ and TNF/LT-α pathways do not contribute for class switching for IgA Ab responses. These results show that the MLN plays a more important role than had been appreciated until now for the induction of both mucosal and systemic Ab responses after oral immunization. Further, organized Peyer’s patches are not a strict requirement for induction of mucosal IgA Ab responses in the gastrointestinal tract.

Oral QS-21 Requires Early IL-4 Help for Induction of Mucosal and Systemic Immunity

The highly purified saponin derivative, QS-21, from the Quillaja saponaria Molina tree has been proved to be safe for parenteral administration and represents a potential alternative to bacterial enterotoxin derivatives as a mucosal adjuvant. Here we report that p.o. administration of QS-21 with the vaccine protein tetanus toxoid elicited strong serum IgM and IgG Ab responses, which were only slightly enhanced by further oral immunization. The IgG Ab subclass responses were predominantly IgG1 followed by IgG2b for the 50-μg p.o. dose of QS-21, whereas the 250-μg p.o. dose also induced IgG2a and IgG3 Abs. Low oral QS-21 doses induced transient IgE Ab responses 7 days after the primary immunization, whereas no IgE Ab responses were seen in mice given the higher QS-21 dose. Further, low but not high p.o. QS-21 doses triggered Ag-specific secretory IgA (S-IgA) Ab responses. Th cell responses showed higher IFN-γ (Th1-type) and lower IL-5, IL-6, and IL-10 (Th2-type) secretion after the high QS-21 p.o. dose than after low doses. Interestingly, the mucosal adjuvant activity of low oral QS-21 doses was diminished in IL-4−/− mice, suggesting a role for this cytokine in the initiation of mucosal immunity by oral QS-21. In summary, our results show that oral QS-21 enhances immunity to coadministered Ag and that different doses of QS-21 lead to distinct patterns of cytokine and serum Ab responses. We also show that an early IL-4 response is required for the induction of mucosal immunity by oral QS-21 as adjuvant.

A double-blind, placebo-controlled study of Mycobacterium-specific human immune responses induced by intradermal bacille Calmette-Guérin vaccination.

Recent studies have indicated that type 1 T cell responses (potent interferon-gamma and cytolytic responses, with absence of interleukin-4 production) are important for protective immunity against mycobacteria. These observations suggest that assays of type 1 T cell responses may be useful as surrogate markers of protective immunity in the evaluation of new tuberculosis vaccines. To be useful as surrogate markers, immunologic assays must distinguish between vaccine recipients and control subjects in clinical trials. Previous studies have shown that bacille Calmette-Guérin (BCG) vaccination can induce human type 1 T cell responses, but randomized trials have not been done to determine whether measurement of these responses can distinguish between BCG recipients and control subjects. We have conducted a double-blind, placebo-controlled trial of intradermal vaccination with two different BCG strains. We compared the mean lymphoproliferative, cytotoxic, Th1 and Th2 cytokine, and antibody responses detected in BCG and placebo recipients. These studies demonstrated that significant increases in Mycobacterium-specific T cell proliferative responses and type 1 cytokine responses were induced by BCG when compared with results with a placebo. In addition, BCG induced significant increases in Mycobacterium-specific antibody responses with an isotype profile characteristic of a type 1 cytokine bias. T cell and antibody assays involving the use of mycobacterial whole cell lysates or live BCG were able to discriminate between BCG and placebo recipients better than were assays using mycobacterial culture filtrates. These studies provide important information for the development of immunologic assays that might be useful as surrogate markers of protective immunity in future trials of new tuberculosis vaccines.

Therapeutic manipulation of the immune system: enhancement of innate and adaptive mucosal immunity.

The mucosal immune system has evolved alongside, but separate, from the general systemic immune system. As a major consequence of this dichotomy, only immune responses initiated in mucosal inductive sites can result in effective immunity in mucosal tissues themselves. Oral tolerance, as usually assessed as orally-induced systemic unresponsiveness, contributes to mucosal homoeostasis by preventing unwanted immune reactions to food or environmental antigens. It is now established that tolerance can also be induced by the nasal route and mucosally-induced tolerance is being actively investigated for immune therapy against a number of diseases. Nontoxic derivatives of cholera toxin and the heat labile toxin of Escherichia coli as well as chimeric enterotoxins have been developed. These molecules retain the mucosal adjuvant activity of native enterotoxins and are effective at inducing targeted Th1 or Th2- type immune responses. Mucosal delivery of cytokines as adjuvants represents a safer alternative to parenteral cytokine injection. Nasally administered cytokines such as IL-1 and IL-12 or chemokines including RANTES, lymphotactin, MIP-1 beta, all act as mucosal adjuvants for co-administered antigens. Each of these cytokines promote specific pattern of CD4(+) T helper cell cytokine responses that could be exploited for targeted immune therapy. Although GALT and NALT are both parts of the Common Mucosal Immune System, there are major differences between orally and nasally induced immune responses. Nasal vaccines more effectively promote protective immunity in the genitourinary tract than do oral vaccines. In addition, aging affects mucosal tolerance or immunity in GALT more than is seen in NALT. Therapeutic manipulation of mucosal immunity involves regulation of CD4(+) T cell cytokine responses and thus, should require a careful examination of the host status, including the occurrence of inflammatory bowel diseases.

New Strategies for Oral Immunization

The potential of biocompatible and biodegradable microspheres as a controlled release oral vaccine delivery system has been examined. Orally-administered 1-10 11m microspheres composed of poly (DL-Iactide-co-glycolide) were specifically taken up into the Peyers patch lymphoid tissue of the gut, where those ~ 5 !lID remained for up to 35 days. Microspheres < 5 11m disseminated within macrophages to the mesenteric lymph nodes and spleen. In contrast to soluble staphylococcal enterotoxin B toxoid, oral immunization with enterotoxoid in microspheres induced circulating toxin-specific antibodies and a concurrent secretory IgA anti-toxin response in saliva and gut fluid.

A study of the single radial hemolysis in gel system—I factors affecting the model

Abstract Mouse hemolytic 19S antibodies can be quantitatively measured if they are allowed to radially diffuse in an agar plate containing the erythrocyte antigen. After diffusion and subsequent lysis with complement a linear relationship can be seen between the area of lysis and the square root of the antibody dilution. It is found that the temperature does not affect the area of lysis obtained; however, the erythrocyte concentration, agar concentration and time do affect the slope of the line for any antibody preparation. When care is taken, only 4·37 per cent error was observed.

A study of the single radial hemolysis in gel system—II application of the model to mouse 19S hemolytic antibodies

Abstract A quantitative method for the determination of mouse anti-SRBC has been described in the previous paper (Hiramoto et al., 1971). The mathematical formulation used to derive the slope was least square; theoretical and practical uses of these comparisons are given below. In addition, reconstruction experiments were performed to further test this system. In the first experiment, an antiserum was partially diluted and then compared with a different antiserum standard. In the second experiment, the same antiserum was diluted by one-third and compared with the original.

A simple method for quantitation of hemolytic antibodies

Abstract A simple and reproducible method for the quantitation of hemolytic SRBC serum is presented. The method involves the use of agar-SRBC plates, and antibody is allowed to diffuse radially. After a sufficient amount of time, the plates can be developed by the direct addition of complement. After zones of lysis were complete, the area of lysis was found to be directly proportional to the square root of the serum dilution.

Methodology a study of the single radial hemolysis in gel system-III. Quantitation of complement☆

Abstract Application of the RHG method for quantification of complement (C′) is described. The measurements of potency obtained by the radial hemolysis in gel plates for different lots of C′ agree with the values obtained by conventional tube titration. With properly standardized plates, the RHG method can determine the CH 50 units of different lots of C′ using an experimentally determined area of lysis ( y ) value obtained for CH 50 /ml. This also allows the estimation of residual CH 50 /ml after complement fixation by antigen-antibody.