Certain lambda-P22 hybrids, providing that they express the P22 C1 protein, fail to grow in Escherichia coli with the sipB391 mutation. We show that sipB391, previously located to the 57-min region of the E. coli chromosome, is a large deletion that extends into the 3' end of ssrA, a gene encoding the small stable 10Sa RNA. This deletion, apparently created by the excision of a cryptic prophage, CP4-57 (identified by Kirby et al. [J. E. Kirby, J. E. Trempy, and S. Gottesman, J. Bacteriol. 176:2068-2081]), leaves most of ssrA intact but removes the sequence encoding the 3' end of the precursor form of 10Sa RNA. The lack of functional 10Sa RNA, resulting from either the excision of CP4-57 or insertional inactivation of ssrA, appears to be responsible for the inhibition of lambda-P22 growth in E. coli with the sipB391 mutation. We propose that 10Sa RNA acts either directly or indirectly to facilitate removal of C1 protein from its DNA target site.

Recent studies have shown that immune responses against the cell-traversal protein for Plasmodium ookinetes and sporozoites (CelTOS) can inhibit parasite infection. While these studies provide important evidence toward the development of vaccines targeting this protein, it remains unknown whether these responses could engage the Plasmodium falciparum CelTOS in vivo. Using a newly developed rodent malaria chimeric parasite expressing the P. falciparum CelTOS (PfCelTOS), we evaluated the protective effect of in vivo immune responses elicited by vaccination and assessed the neutralizing capacity of monoclonal antibodies specific against PfCelTOS. Mice immunized with recombinant P. falciparum CelTOS in combination with the glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) or glucopyranosyl lipid adjuvant-liposome-QS21 (GLA-LSQ) adjuvant system significantly inhibited sporozoite hepatocyte infection. Notably, monoclonal antibodies against PfCelTOS strongly inhibited oocyst development of P. falciparum and Plasmodium berghei expressing PfCelTOS in Anopheles gambiae mosquitoes. Taken together, our results demonstrate that anti-CelTOS responses elicited by vaccination or passive immunization can inhibit sporozoite and ookinete infection and impair vector transmission.

The 10Sa RNA, encoded by the E. coli ssrA gene, appears to modulate action of some DNA-binding proteins. When ssrA is inactivated, lacZ expression from the lac operon, as well as galK from a gal operon fused to a phage lambda promoter, is reduced from that observed in bacteria wild-type for ssrA. These differences are not observed if the relevant repressor is inactive, suggesting that in the absence of 10Sa RNA binding of LacI and lambda cI repressors is enhanced. Gel mobility shifts show that 10Sa RNA binds these repressors and that an excess of 10Sa RNA competes for binding of lambda cI with a DNA fragment containing the OR2 repressor-binding sequence. Similar observations were made in studies of the E. coli LexA repressor and phage P22 C1 transcription activator proteins. These results suggest that direct interaction with 10Sa RNA may explain this modulation of protein-DNA interactions.

The circumsporozoite protein (CSP) of Plasmodium falciparum is a major surface protein, which forms a dense coat on the sporozoite's surface. Preclinical research on CSP and clinical evaluation of a CSP fragment-based RTS, S/AS01 vaccine have demonstrated a modest degree of protection against P. falciparum, mediated in part by humoral immunity and in part by cell-mediated immunity. Given the partial protective efficacy of the RTS, S/AS01 vaccine in a recent Phase 3 trial, further improvement of CSP-based vaccines is crucial. In this report, we describe the preclinical development of a full-length, recombinant CSP (rCSP)-based vaccine candidate against P. falciparum malaria suitable for current Good Manufacturing Practice (cGMP) production. Utilizing a novel high-throughput Pseudomonas fluorescens expression platform, we demonstrated greater efficacy of full-length rCSP as compared to N-terminally truncated versions, rapidly down-selected a promising lead vaccine candidate, and developed a high-yield purification process to express immunologically active, intact antigen for clinical trial material production. The rCSP, when formulated with various adjuvants, induced antigen-specific antibody responses as measured by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA), as well as CD4+ T-cell responses as determined by ELISpot. The adjuvanted rCSP vaccine conferred protection in mice when challenged with transgenic P. berghei sporozoites containing the P. falciparum repeat region of CSP. Furthermore, heterologous prime/boost regimens with adjuvanted rCSP and an adenovirus type 35-vectored CSP (Ad35CS) showed modest improvements in eliciting CSP-specific T-cell responses and anti-malarial protection, depending on the order of vaccine delivery. Collectively, these data support the importance of further clinical development of adjuvanted rCSP, either as a stand-alone product or as one of the components in a heterologous prime/boost strategy, ultimately acting as an effective vaccine candidate for the mitigation of P. falciparum-induced malaria.

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