Biology subjects

Malladi, S. K.

Publications and source records attributed to Malladi, S. K..

6 recordsLinked to original sources

bioRxiv2024

Monoclonal antibodies targeting the FimH adhesin protect against uropathogenic E. coli UTI

As antimicrobial resistance increases, urinary tract infections (UTIs) are expected to pose an increased burden in morbidity and expense on the healthcare system, increasing the need for alternative antibiotic-sparing treatments. Most UTIs are caused by uropathogenic Escherichia coli (UPEC), while Klebsiella pneumoniae causes a significant portion of non-UPEC UTIs. Both bacteria express type 1 pili tipped with the mannose-binding FimH adhesin critical for UTI pathogenesis. We generated and biochemically characterized 33 murine monoclonal antibodies (mAbs) to FimH. Two mAbs protected mice from E. coli UTI. Mechanistically, we show that this protection is Fc-independent and mediated by the ability of these mAbs to sterically block FimH function. Our data reveals that FimH mAbs hold promise as an antibiotic-sparing treatment strategy.

microbiology↗

bioRxiv2024

Defining a highly conserved B cell epitope in the receptor binding motif of SARS-CoV-2 spike glycoprotein

SARS-CoV-2 mRNA vaccines induce robust and persistent germinal centre (GC) B cell responses in humans. It remains unclear how the continuous evolution of the virus impacts the breadth of the induced GC B cell response. Using ultrasound-guided fine needle aspiration, we examined draining lymph nodes of nine healthy adults following bivalent booster immunization. We show that 77.8% of the B cell clones in the GC expressed as representative monoclonal antibodies recognized the spike protein, with a third (37.8%) of these targeting the receptor binding domain (RBD). Strikingly, only one RBD-targeting mAb, mAb-52, neutralized all tested SARS- CoV-2 strains, including the recent KP.2 variant. mAb-52 utilizes the IGHV3-66 public clonotype, protects hamsters challenged against the EG.5.1 variant and targets the class I/II RBD epitope, closely mimicking the binding footprint of ACE2. Finally, we show that the remarkable breadth of mAb-52 is due to the somatic hypermutations accumulated within vaccine-induced GC reaction. One Sentence SummaryBooster SARS-CoV-2 mRNA vaccine recruits and broadens GC B cell responses targeting a highly conserved site on receptor binding domain of spike glycoprotein.

immunology↗

bioRxiv2022

SARS-CoV-2 Omicron boosting induces de novo B cell response in humans

The primary two-dose SARS-CoV-2 mRNA vaccine series are strongly immunogenic in humans, but the emergence of highly infectious variants necessitated additional doses of these vaccines and the development of new variant-derived ones1-4. SARS-CoV-2 booster immunizations in humans primarily recruit pre-existing memory B cells (MBCs)5-9. It remains unclear, however, whether the additional doses induce germinal centre (GC) reactions where reengaged B cells can further mature and whether variant-derived vaccines can elicit responses to novel epitopes specific to such variants. Here, we show that boosting with the original SARS- CoV-2 spike vaccine (mRNA-1273) or a B.1.351/B.1.617.2 (Beta/Delta) bivalent vaccine (mRNA-1273.213) induces robust spike-specific GC B cell responses in humans. The GC response persisted for at least eight weeks, leading to significantly more mutated antigen-specific MBC and bone marrow plasma cell compartments. Interrogation of MBC-derived spike-binding monoclonal antibodies (mAbs) isolated from individuals boosted with either mRNA-1273, mRNA-1273.213, or a monovalent Omicron BA.1-based vaccine (mRNA-1273.529) revealed a striking imprinting effect by the primary vaccination series, with all mAbs (n=769) recognizing the original SARS-CoV-2 spike protein. Nonetheless, using a more targeted approach, we isolated mAbs that recognized the spike protein of the SARS-CoV-2 Omicron (BA.1) but not the original SARS-CoV-2 spike from the mRNA-1273.529 boosted individuals. The latter mAbs were less mutated and recognized novel epitopes within the spike protein, suggesting a naive B cell origin. Thus, SARS-CoV-2 boosting in humans induce robust GC B cell responses, and immunization with an antigenically distant spike can overcome the antigenic imprinting by the primary vaccination series.

immunology↗

bioRxiv2022

Highly Thermotolerant SARS-CoV-2 Vaccine Elicits Neutralising Antibodies Against Delta and Omicron in Mice

As existing vaccines fail to completely prevent COVID-19 infections or community transmission, there is an unmet need for vaccines that can better combat SARS-CoV-2 variants of concern (VOC). We have previously developed highly thermo-tolerant monomeric and trimeric receptor binding domain derivatives that can withstand 100{degrees}C for 90 minutes and 37{degrees}C for four weeks, and help eliminate cold chain requirements. We show that mice immunised with these vaccine formulations elicit high titres of antibodies that neutralise SARS-CoV-2 variants VIC31 (with Spike: D614G mutation), Delta and Omicron (BA.1.1) VOC. Compared to VIC31, there was an average 14.4-fold reduction in neutralisation against BA.1.1 for the three monomeric antigen-adjuvant combinations, and 16.5-fold reduction for the three trimeric antigen-adjuvant combinations; the corresponding values against Delta were 2.5 and 3.0. Our findings suggest that monomeric formulations are suitable for the upcoming Phase I human clinical trials, and that there is potential for increasing efficacy with vaccine matching to improve responses against emerging variants. These findings are consistent with in silico modelling and AlphaFold predictions which show that while oligomeric presentation can be generally beneficial, it can make important epitopes inaccessible, and also carries the risk of eliciting unwanted antibodies against the oligomerisation domain.

immunology↗

bioRxiv2021

A highly thermotolerant, trimeric SARS-CoV-2 receptor binding domain derivative elicits high titers of neutralizing antibodies

The Receptor Binding Domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We designed a trimeric, highly thermotolerant glycan engineered RBD by fusion to a heterologous, poorly immunogenic disulfide linked trimerization domain derived from cartilage matrix protein. The protein expressed at a yield of [~]80-100 mg/liter in transiently transfected Expi293 cells, as well as CHO and HEK293 stable cell lines and formed homogeneous disulfide-linked trimers. When lyophilized, these possessed remarkable functional stability to transient thermal stress of upto 100 {degrees}C and were stable to long term storage of over 4 weeks at 37 {degrees}C unlike an alternative RBD-trimer with a different trimerization domain. Two intramuscular immunizations with a human-compatible SWE adjuvanted formulation, elicited antibodies with pseudoviral neutralizing titers in guinea pigs and mice that were 25-250 fold higher than corresponding values in human convalescent sera. Against the beta (B.1.351) variant of concern (VOC), pseudoviral neutralization titers for RBD trimer were [~] three-fold lower than against wildtype B.1 virus. RBD was also displayed on a designed ferritin-like Msdps2 nanoparticle. This showed decreased yield and immunogenicity relative to trimeric RBD. Replicative virus neutralization assays using mouse sera demonstrated that antibodies induced by the trimers neutralized all four VOC to date, namely B.1.1.7, B.1.351, P.1 and B.1.617.2 without significant differences. Trimeric RBD immunized hamsters were protected from viral challenge. The excellent immunogenicity, thermotolerance, and high yield of these immunogens suggest that they are a promising modality to combat COVID-19, including all SARS-CoV-2 VOC to date.

microbiology↗

bioRxiv2020

Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment

Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Several use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these novel, nucleic acid based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the Receptor Binding Domain (RBD) of the viral spike protein. We describe a monomeric, glycan engineered RBD protein fragment that is expressed at a purified yield of 214mg/L in unoptimized, mammalian cell culture and in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100{degrees}C when lyophilized, upto 70{degrees}C in solution and stable for over four weeks at 37{degrees}C. In prime:boost guinea pig immunizations, when formulated with the MF59 like adjuvant AddaVax, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ~415 against replicative virus, comparing favourably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.

biophysics↗