In Vivo -Expressed Proteins of Virulent Leptospira interrogans Serovar Autumnalis N2 Elicit Strong IgM Responses of Value in Conclusive Diagnosis

Abstract

bLeptospirosis is a serious zoonosis that is underdiagnosed because of limited access to laboratory facilities in Southeast Asia, Central and South America, and Oceania. Timely diagnosis of locally distributed serovars of high virulence is crucial for successful care and outbreak management. Using pooled patient sera, an expression gene library of a virulent Leptospira interrogans serovar Autumnalis strain N2 isolated in South India was screened. The identified genes were characterized, and the purified recombinant proteins were used as antigens in IgM enzyme-linked immunosorbent assay (ELISA) either singly or in combination. Sera (n ‫؍‬ 118) from cases of acute leptospirosis along with sera (n ‫؍‬ 58) from healthy subjects were tested for reactivity with the identified proteins in an ELISA designed to detect specific IgM responses. We have identified nine immunoreactive proteins, ArgC, RecA, GlpF, FliD, TrmD, RplS, RnhB, Lp28.6, and Lrr44.9, which were found to be highly conserved among pathogenic leptospires. Apparently, the proteins ArgC, RecA, GlpF, FliD, TrmD, and Lrr44.9 are expressed during natural infection of the host and undetectable in in vitro cultures. Among all the recombinant proteins used as antigens in IgM ELISA, ArgC had the highest sensitivity and specificity, 89.8% and 95.5%, respectively, for the conclusive diagnosis of leptospirosis. The use of ArgC and RecA in combination for IgM ELISA increased the sensitivity and specificity to 95.7% and 94.9%, respectively. ArgC and RecA thus elicited specific IgM responses and were therefore effective in laboratory confirmation of Leptospira infection.

Protective immunity of recombinant LipL21 and I-LipL21 against Leptospira interrogans serovar Autumnalis N2 infection

“…interrogans serovar Autumnalis strain N2 was maintained by regular sub-culturing in EllinghausenMcCullough-Johnson-Harris (EMJH) bovine serum albumin-Tween 80 medium (Difco Laboratories, USA) at the Medical Microbiology Laboratory, Bharathidasan University, Tiruchirappalli, India. All procedures with L. interrogans were conducted using MACS (Mouse Adapted Challenge Strain) as previously described [15]. The Escherichia coli strains NovaBlue and BL21 (DE3) (Novagen, Madison, USA) were used for cloning and expression of recombinant proteins.…”

Heterologous DNA prime-protein boost immunization with RecA and FliD offers cross-clade protection against leptospiral infection

“…All procedures with L . interrogans were conducted using leptospiral MACS (Mouse Adapted Challenge Strain) as per our earlier report 15 . Escherichia coli strains like DH5α, BL21 (DE3), and BL21 (DE3) pLysS (Novagen, Madison, Wis.) were routinely grown in Luria-Bertani (LB) medium (Sigma Aldrich, St Louis, MO).…”

“…weilli and L . biflexa as described earlier 15 . The coding sequences were aligned using CLC Main workbench (version 7.9.1), based on the ClustalW algorithm.…”

“…Apart from Lig proteins, none of the other leptospiral candidates proved its efficacy. Recently, we discovered proteins that are expressed during leptospiral infection ( in vivo expressed proteins) in the host and these proteins were shown to elicit strong IgM responses for conclusive diagnosis of leptospirosis 15 . Considering the immune response during natural infection among the human cases, evidencing their durable immunogenicity we developed an efficient DNA prime-protein boost strategy with our newly identified proteins RecA and FliD.…”

Peptide specific monoclonal antibodies of Leptospiral LigA for acute diagnosis of leptospirosis

“…The MACS strains were passaged twice in vitro and used for preparation of heat extracted antigens 13 .…”