BackgroundProduction of human monoclonal antibodies that exhibit broadly neutralizing activity is needed for preventing HIV-1 infection, however only a few such antibodies have been generated till date. Isolation of antibodies by the hybridoma technology is a cumbersome process with fewer yields. Further, the loss of unstable or slowly growing clones which may have unique binding specificities often occurs during cloning and propagation and the strongly positive clones are often lost. This has been avoided by the process described in this paper, wherein, by combining the strategy of EBV transformation and recombinant DNA technology, we constructed human single chain variable fragments (scFvs) against the third variable region (V3) of the clade C HIV-1 envelope.ResultsAn antigen specific phage library of 7000 clones was constructed from the enriched V3- positive antibody secreting EBV transformed cells. By ligation of the digested scFv DNA into phagemid vector and bio panning against the HIV-1 consensus C and B V3 peptides followed by random selection of 40 clones, we identified 15 clones that showed V3 reactivity in phage ELISA. DNA fingerprinting analysis and sequencing showed that 13 out of the 15 clones were distinct. Expression of the positive clones was tested by SDS-PAGE and Western blot. All the 13 anti-V3 scFvs showed cross-reactivity against both the clade C and B V3 peptides and did not show any reactivity against other unrelated peptides in ELISA. Preliminary neutralization assays indicated varying degrees of neutralization of clade C and B viruses. EBV transformation, followed by antigen selection of lines to identify specific binders, enabled the selection of phage from un-cloned lines for scFv generation, thus avoiding the problems of hybridoma technology. Moreover, as the clones were pretested for antigen binding, a comparatively small library sufficed for the selection of a considerable number of unique antigen binding phage. After selection, the phage clones were propagated in a clonal manner.ConclusionsThis strategy can be efficiently used and is cost effective for the generation of diverse recombinant antibodies. This is the first study to generate anti-V3 scFvs against HIV-1 Clade C.
Neutralizing monoclonal antibodies are being found to be increasingly useful in viral infections. In hepatitis B infection, antibodies are proven to be useful for passive prophylaxis. The preS1 region (21–47a.a.) of HBV contains the viral hepatocyte-binding domain crucial for its attachment and infection of hepatocytes. Antibodies against this region are neutralizing and are best suited for immune-based neutralization of HBV, especially in view of their not recognizing decoy particles. Anti-preS1 (21–47a.a.) antibodies are present in serum of spontaneously recovered individuals. We generated a phage-displayed scFv library using circulating lymphocytes from these individuals and selected four preS1-peptide specific scFvs with markedly distinct sequences from this library. All the antibodies recognized the blood-derived and recombinant preS1 containing antigens. Each scFv showed a discrete binding signature, interacting with different amino acids within the preS1-peptide region. Ability to prevent binding of the preS1 protein (N-terminus 60a.a.) to HepG2 cells stably expressing hNTCP (HepG2-hNTCP-C4 cells), the HBV receptor on human hepatocytes was taken as a surrogate marker for neutralizing capacity. These antibodies inhibited preS1-hepatocyte interaction individually and even better in combination. Such a combination of potentially neutralizing recombinant antibodies with defined specificities could be used for preventing/managing HBV infections, including those by possible escape mutants.
Typhoid fever caused by human restricted Salmonella typhi presents a considerable health burden on developing South-Asian nations like India. The suboptimal sensitivity and specificity associated with culture-based isolation of etiological agent and the extensively used surface antigen-based serological assays often lead to misdiagnosis and inappropriate antimicrobial treatment. The increasing reports of the emergence of resistant strains and undefined disease burden signify the critical need for an inexpensive, reliable, easy-to-use, and highly sensitive diagnostic test for typhoid fever. Utilizing S. typhi-specific and immunogenic antigens in sero-diagnostic assays could lead to precise diagnosis of acute typhoid and prompt treatment. In this study, we report cloning, expression, and purification of recombinant Cytolethal distending toxin subunit B (CdtB) of S. typhi, which is reported to be highly specific, immunogenic, and expressed only upon S. typhi infection. We further evaluated the purified recombinant CdtB for its diagnostic potential in an IgM-based indirect ELISA format using 33 human samples. Twenty-one serum samples from blood culture confirmed cases (n = 21) of typhoid and 12 samples from healthy controls (n = 12) were tested. The assay showed sensitivity of 100% and specificity of 83.3% respectively with positive and negative predictive values of 91.3 and 100% respectively. Efficient detection of specific IgM antibodies indicates that CdtB could be highly valuable in sero-diagnosis of acute typhoid and rapid screening of clinical samples.
Recently conducted human phase- I trials showed protective effect of anti-HIV-1 broadly neutralizing antibodies (bnAbs). The V3 region of the HIV-1 envelope is highly conserved as it is the co-receptor binding site, and is highly immunogenic. Recombinant single-chain antibody fragments (scFvs) can serve as potential tools for construction of chimeric/bispecific antibodies that can target different epitopes on the HIV-1 envelope. Previously, we have constructed a V3 specific human scFv phage recombinant library by a combinational approach of Epstein-Barr virus (EBV) transformation and antigen (V3) preselection, using peripheral blood mononuclear cells (PBMCs), from a subtype C HIV-1 infected antiretroviral naive donor. In the present study, by biopanning this recombinant scFv phage library with V3B (subtype B) and V3C (subtype C) peptides, we identified unique cross reactive anti-V3 scFv monoclonals. These scFvs demonstrated cross-neutralizing activity when tested against subtype A, subtype B, and subtype C viruses. Further, molecular modeling of the anti-V3 scFvs with V3C and V3B peptides predicted their sites of interaction with the scFvs, providing insights for future immunogen design studies. A large collection of such monoclonal antibody fragments with diverse epitope specificities can be useful immunotherapeutic reagents along with antiretroviral drugs to prevent HIV-1 infection and disease progression.
Typhoid fever is a significant global health problem with highest burden on the developing world. The severity of typhoid is often underestimated, and currently available serological diagnostic assays are inadequate due to lack in requisite sensitivity and specificity. This underlines an absolute need to develop a reliable and accurate diagnostics that would benefit long-term disease control and treatment and to understand the real disease burden. Here, we have utilized flagellin protein of S. typhi that is surface accessible, abundantly expressed, and highly immunogenic, for developing immunodiagnostic tests. Flagellin monomers are composed of conserved amino-terminal and carboxy-terminal, and serovar-specific middle region. We have generated a panel of murine monoclonal antibodies (mAbs) against the middle region of flagellin, purified from large culture of S. typhi to ensure its native conformation. These mAbs showed unique specificity and very high affinity toward S. typhi flagellin without showing any cross-reactivity with other serovars. Genetic analysis of mAbs also revealed high frequency of somatic mutation due to antigenic selection process across variable region to achieve high binding affinity. These antibodies also displayed stable binding in stringent reaction conditions for antigen-antibody interactions, like DMSO, urea, KSCN, guanidinium HCl, and extremes of pH. One of the mAbs potentially reversed the TLR5-mediated immune response, in vitro by inhibiting TLR5-flagellin interaction. In our study, binding of these mAbs to flagellin, with high affinity, present on bacterial surface, as well as in soluble form, validates their potential use in developing improved diagnostics with significantly higher sensitivity and specificity.
Liver is a key organ involved in the regulation of both systemic as well as local inflammatory responses. Hepatic inflammation is the hallmark of viral hepatitis caused by non-cytopathic Hepatitis B and C viruses (HBV and HCV). Both HBV and HCV induce several inflammatory responses, causing persistent liver injury, which manifests into progressive diseased state. This ultimately leads to fibrosis, cirrhosis and eventually hepatocellular carcinoma. The disease progression is mediated by a complex interplay of molecular pathways involving both viral and hepatic factors. The complex cellular crosstalk, involving pro-inflammatory cytokines, during the liver injury also causes extra hepatic disorders such as artherosclerosis, glomerulonephritis, arthritis, cardio vascular and brain disease. In addition, these viral infections have been reported to contribute to non-Hodgkin lymphoma, cholangio carcinoma and pancreatic cancer. Host genetics also play an important role in the HBV/HCV mediated viral hepatitis and the accumulation of mutations in the host genes responsible for mounting antiviral effects (cytokines and interferon receptors) has been shown to produce differential immune responses among individuals and increase susceptibility to chronic infections. Viral proteins are known to modulate the host immune response by a variety of mechanisms such as by exerting direct or indirect effects on the cytokine pathways, oxidative stress, miRNAs and other cellular processes. However, the complete network of cytokines involved in the disease pathogenesis is yet not fully understood. This review analyzes the interplay of inflammatory molecules and viral proteins, the impact of local and systemic inflammation during HBV and HCV infection and the contribution of host genetics to such responses.
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