Development of an adjuvant-active nonionic block copolymer for use in oil-free subunit vaccines formulations.

Nonionic block copolymers, synthesized from repeating units of oxypropylene and oxyethylene, can be designed so that individual copolymers have unique physical properties with differential levels of adjuvant activity. We have designed high molecular weight block copolymers that spontaneously assemble into 500 nm-3 mum particles when formulated with protein antigens in aqueous solutions at physiological pH. The adjuvant activity of one of these copolymers, termed CRL1005, was compared to selected research adjuvants using ovalbumin (OVA) as the prototype vaccine antigen. Suboptimal doses of OVA were formulated with complete and incomplete. Freund's adjuvant (CFA/IFA), alum Quil-A saponins Ribi Adjuvant System (RAS) or the CRL1005 copolymer and these formulations were used to immunize C57BL/6 mice. The CRL1005 copolymer appeared to be more potent than either Quil-A or alum and comparable to the RAS formulation, based on the numbers of responding mice and the OVA-specific antibody titers. Alum. RAS and Quil-A all augmented the production of IgG1 and IgG2l, similarly whereas only the CFA/IFA boosted IgG2a levels significantly. The effect of adjuvants on relative antibody affinity was more variable with the CRL1005 and CFA/IFA inducing antibodies with the highest affinity scores. This high molecular weight nonionic copolymer is nontoxic in aqueous formulations and should therefore be compatible with a wide variety of protein or polysaccharide vaccine antigens.

Cytokines enhance neutrophils from human immunodeficiency virus-negative donors and AIDS patients to inhibit the growth of Mycobacterium avium in vitro.

Mycobacterium avium is one of the most prevalent opportunistic infections in AIDS patients, and neither prophylaxis nor treatment against M. avium is effective. To evaluate host defense mechanisms against mycobacterial infections, studies investigated whether neutrophils from AIDS patients could inhibit the growth of M. avium in vitro and what cytokines enhance neutrophil function against M. avium. Peripheral blood neutrophils from human immunodeficiency virus-negative and AIDS patients were incubated with media, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-8, or macrophage-inhibitory proteins and infected with M. avium, and the inhibition of bacterial growth was determined. G-CSF (1000 U/mL) and GM-CSF (2000 U/mL) stimulated neutrophils from AIDS patients to significantly inhibit M. avium growth. These results demonstrate that neutrophils from AIDS patients can respond to exogenously supplied G-CSF or GM-CSF by inhibiting the growth of M. avium.

Interleukin-12 enhances antigen-specific proliferation of peripheral blood mononuclear cells from HIV-positive and negative donors in response to Mycobacterium avium.

OBJECTIVE: To determine if the addition of recombinant (r) human interleukin (IL)-12 enhances in vitro proliferative responses to Mycobacterium avium of peripheral blood mononuclear cells (PBMC) from HIV-positive donors with CD4 cell counts < 100 x 10(6)/l. DESIGN AND METHODS: PBMC proliferative responses to virulent and avirulent serovars of M. avium in the presence and absence of exogenously added IL-12 were determined in 24 HIV-positive and 11 HIV-negative donors by 3H-thymidine uptake assay. Changes in CD4 and CD8 cell populations after IL-12 treatment and M. avium stimulation were analyzed by FACS. RESULTS: IL-12 significantly enhanced proliferation of PBMC to both virulent and avirulent M. avium from all 24 HIV-positive donors (P = 0.0001) although the magnitude varied for each donor. In contrast, addition of IL-12 to PBMC from HIV-negative donors only increased the proliferative responses to the virulent M. avium serovar 4 (P = 0.0044). PBMC from HIV-positive donors in the presence of IL-12 responded better to the avirulent serovar of M. avium than the virulent serovar 4. Proliferative responses of HIV-positive donors to M. avium alone, however, were significantly less (P = 0.0013) than that of HIV-negative donors. Increased proliferative responses of HIV-positive donors were independent of CD4 counts. No significant changes in the ratio of CD4+ to CD8+ T cells occurred in either HIV-positive or negative donors under any culture conditions. CONCLUSION: In vitro proliferative responses of PBMC from HIV-positive donors to M. avium were significantly enhanced by the addition of human rIL-12, which was not dependent on their CD4 cell counts. The use of IL-12 as an enhancer of cell-mediated immunity in AIDS patients against M. avium infections deserves further study.

Opposing regulatory effects of thioredoxin and eosinophil cytotoxicity-enhancing factor on the development of human immunodeficiency virus 1.

Exogenous recombinant human thioredoxin (rTRX, > or = 500 nM), a dithiol reductase enzyme, inhibited the expression of human immunodeficiency virus (HIV) 1BaL in human macrophages (M phi) by 71% (range, 26-100%), as evaluated by p24 antigen production and the integration of provirus at 14 d after infection. The stoichiometric reducing agent N-acetylcysteine (NAC) also inhibited HIV production, but to a lesser degree, and only at 30,000-fold higher concentrations. Exogenous rTRX is cleaved by M phi to generate the inflammatory cytokine, eosinophil cytotoxicity-enhancing factor (ECEF). In contrast to rTRX, rECEF (concentrations from 50 pM to 2 microM) enhanced the production of HIV by 67% (range, 33-92%). Thus, whereas TRX is a potent inhibitor of the expression of HIV in human M phi, cleavage of TRX to ECEF creates a mediator with the opposite effect. TRX also inhibited the expression of integrated provirus in the chronically infected OM 10.1 cell line, showing that it can act at a step subsequent to viral infection and integration.