In the present study we evaluated the role of B cells in acquired immunity to Salmonella infection by using gene-targeted B-cell-deficient innately susceptible mice on a C57BL/6 background (Igh-6 −/−).Igh-6 −/− mice immunized with a live, attenuated aroA Salmonella enterica serovar Typhimurium vaccine strain showed impaired long-term acquired resistance against the virulent serovar Typhimurium strain C5.Igh-6 −/− mice were able to control a primary infection and to clear the inoculum from the reticuloendothelial system. However, Igh-6 −/− mice, unlikeIgh-6 +/+ C57BL/6 controls, did not survive an oral challenge with strain C5 at 4 months after vaccination. Transfer of immune serum did not restore resistance inIgh-6 −/− mice. Total splenocytes and purified CD4+ T cells obtained fromIgh-6 −/− mice 4 months after vaccination showed reduced ability to release Th1-type cytokines (interleukin 2 and gamma interferon) upon in vitro restimulation with serovar Typhimurium soluble cell extracts compared to cells obtained fromIgh-6 +/+ C57BL/6 control mice. Therefore, the impaired resistance to oral challenge with virulent serovar Typhimurium observed in B-cell-deficient mice, which cannot be restored by passive transfer of Salmonella-immune serum, may be in part due to a reduced serovar Typhimurium-specific T-cell response following primary immunization.

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Ookinete formation from mature Plasmodium berghei gametocytes in vitro was partially inhibited by 0.05-0.1 microM atovaquone and almost totally blocked at a concentration of 0.25 microM. Microgametocyte exflagellation was not affected by atovaquone at concentrations up to 300 microM. Ookinete formation was also inhibited in culture when addition of 0.20 microM atovaquone was delayed by 4 hr, by which time DNA replication was likely to have been completed. Inhibition of ookinete formation by atovaquone was not reversed by orotic acid. Plasmodium berghei-infected Anopheles stephensi mosquitoes were fed a second blood meal 4, 7, 14, and 20 days postinfection (p.i.) from mice that had been treated with atovaquone or control diluent 8 hr previously. Atovaquone blood feeds on day 4 reduced oocyst numbers on days 6-12, although sporozoite numbers in the thorax and abdomen on day 20 were not significantly reduced. Blood feeds on day 7 slowed oocyst growth, blood feeds on day 14 did not significantly reduce sporozoite numbers, and feeds to mosquitoes on day 20 p.i. had no effect on transmission to naive mice. Sporozoite invasion of human hepatoma cells was unaffected by the presence of atovaquone.

The activity of atovaquone against Plasmodium berghei ANKA during sporogonic development has been examined. Anopheles stephensi mosquitoes were fed on gametocyte infected mice which had been treated 8 h previously with atovaquone or diluent alone. Mosquito midguts were examined for oocysts, and salivary gland infections were estimated using an ELISA for the circumsporozoite protein (CSP). The number of oocysts per midgut fell by at least 97% when mosquitoes were fed on mice dosed with 0.1-10 mg atovaquone/kg body weight. This was paralleled by a decrease in the prevalence of oocyst-infected mosquitoes from 70-90% in controls to 40% or 10% respectively. No oocysts were observed at a dose of 100 mg/kg. CSP ELISA results indicated that mosquitoes fed on atovaquone failed to produce sporozoites. Mosquitoes which fed on gametocytaemic, atovaquone-treated mice (0.1-100 mg/kg) did not transmit malaria to naive mice. These results demonstrate that atovaquone has a highly potent inhibitory activity against the mosquito stages of P. berghei.

The ability of enterotoxin-based mucosal adjuvants to induce CD8+ MHC class I-restricted CTL responses to a codelivered bystander Ag was examined. Escherichia coli heat-labile toxin (LT), or derivatives of LT carrying mutations in the A subunit (LTR72, LTK63), were tested in parallel with cholera toxin (CT) or a fusion protein consisting of the A1 subunit of CT fused to the Ig binding domain of Staphylococcus aureus protein A (called CTA1-DD). Intranasal (i.n.) immunization of C57BL/6 mice with CT, CTA1-DD, LT, LTR72, LTK63, but not rLT-B, elicited MHC class I-restricted CD8+ T cell responses to coadministered OVA or the OVA CTL peptide SIINFEKL (OVA257–264). CT, LT, and LTR72 also induced CTL responses to OVA after s.c. or oral coimmunization whereas LTK63 only activated responses after s.c. coimmunization. rLT-B was unable to adjuvant CTL responses to OVA or OVA257–264 administered by any route. Mice treated with an anti-CD4 mAb to deplete CD4+ T cells mounted significant OVA-specific CTL responses after i.n. coadministration of LT with OVA or OVA257–264. Both 51Cr release assays and IFN-γ enzyme-linked immunospot assays indicated that IFN-γ−/− and IL-12 p40−/− gene knockout mice developed CTL responses equivalent to those detected in normal C57BL/6 mice. The results highlight the versatility of toxin-based adjuvants and suggest that LT potentiates CTL responses independently of IL-12 and IFN-γ and probably by a mechanism unrelated to cross-priming.