key: cord-0767128-jh39yygg authors: Sangesland, Maya; Yousif, Ashraf S.; Ronsard, Larance; Kazer, Samuel W.; Zhu, Alex Lee; Gatter, G. James; Hayward, Matthew R.; Barnes, Ralston M.; Quirindongo-Crespo, Maricel; Rohrer, Daniel; Lonberg, Nils; Kwon, Douglas; Shalek, Alex K.; Lingwood, Daniel title: A Single Human V(H)-gene Allows for a Broad-Spectrum Antibody Response Targeting Bacterial Lipopolysaccharides in the Blood date: 2020-08-25 journal: Cell Rep DOI: 10.1016/j.celrep.2020.108065 sha: aa09d6f193af15840f616cc0c908b9106b64b542 doc_id: 767128 cord_uid: jh39yygg B cell receptors (BCRs) display a combination of variable (V)-gene-encoded complementarity determining regions (CDRs) and adaptive/hypervariable CDR3 loops to engage antigens. It has long been proposed that the former tune for recognition of pathogens or groups of pathogens. To experimentally evaluate this within the human antibody repertoire, we perform immune challenges in transgenic mice that bear diverse human CDR3 and light chains but are constrained to different human V(H)(-)genes. We find that, of six commonly deployed V(H) sequences, only those CDRs encoded by IGHV1-2(∗)02 enable polyclonal antibody responses against bacterial lipopolysaccharide (LPS) when introduced to the bloodstream. The LPS is from diverse strains of gram-negative bacteria, and the V(H)-gene-dependent responses are directed against the non-variable and universal saccrolipid substructure of this antigen. This reveals a broad-spectrum anti-LPS response in which germline-encoded CDRs naturally hardwire the human antibody repertoire for recognition of a conserved microbial target. The ligand binding surface of antibodies is generated by diverse gene-encoded and hypervariable loops. Sangesland et al. demonstrate that certain geneencoded loops are needed for human antibodies to accommodate the shapes of some bacterial antigens and naturally tune for recognition of conserved microbial features therein. The adaptive immune system generates diverse sets of antibodies through the recombination of B cell receptor (BCR) gene segments. For each germline BCR, affinity to incoming antigen is provided by complementarity determining regions (CDRs) that are either directly encoded by antibody variable (V) genes (CDR1 and CDR2) or assembled de novo through stochastic N-junctional diversification of antibody D and J segments (CDR3). Most BCR diversity is concentrated within the antibody heavy-chain CDR3 (CDRH3), which occupies a central position within the germline antigen binding site (Glanville et al., 2009; Schroeder and Cavacini, 2010) . CDRH3 loops vary highly in length, sequence, and shape, and because of this diversity, they are the principal determinants of antigen specificity (Kuroda et al., 2008; Morea et al., 1998; North et al., 2011; Oliva et al., 1998; Saada et al., 2007; Schroeder and Cavacini, 2010; Shirai et al., 1999; Wedemayer et al., 1997; Xu and Davis, 2000) . The remaining CDRs are genetically encoded and structurally restricted to a few canonical loop conformations that have generally been considered functionally redundant (Al-Lazikani et al., 1997; Martin and Thornton, 1996; North et al., 2011) , although some bacterial and viral proteins bear natural affinities for V-gene-specific features outside the antigen binding surface, triggering non-productive/superantigen B cell responses (Silverman and Goodyear, 2006; Villar et al., 2016) . In the current study, we harnessed mouse genetics to mechanistically interrogate natural antigen specificities endowed by human antibody genes in vivo. Our results show that despite their limited diversity, human heavy chain variable (V H ) genes are not functionally redundant and that specific germline-encoded input is both needed and tuned to accommodate conserved bacterial structures when present in the bloodstream. It has long been hypothesized that the encoded CDR repertoire was shaped to endow antigen-specific recognition of conserved microbial features (Chothia and Lesk, 1987; Chothia et al., 1989; Cohn and Langman, 1990) . Although this hypothesis has been refined and applied to account for human antibody specificities (Henry Dunand and Wilson, 2015; Lerner, 2011; Lonberg, 2005) , experimental evaluation of germline-encoded recognition of recurrent microbial patterns has largely been explored within mice in which genetic manipulation has enabled mechanistic studies in vivo (Chen et al., 2009; Mi et al., 2000) . Early reports noting V H -gene bias in hybridomas reactive to phosphorylcholine (PC), a lipid head group displayed by bacterial and eukaryotic cell membranes (Crews et al., 1981; Gearhart et al., 1981) , revealed a germline-encoded target specificity that is now recognized as a core feature of natural antibodies: circulating polyreactive immunoglobulins that are produced constitutively by murine B1 cells and provide important bacterial defense activities (Chen et al., 2009; Grö nwall et al., 2012; Holodick et al., 2017; Mi et al., 2000; Savage and Baumgarth, 2015) . Natural antibodies with V H -gene-biased polyreactive substrate specificities have also been identified in humans that, like their murine counterparts, are produced from specialized B cell subsets (Grö nwall et al., 2012; Nguyen and Baumgarth, 2016) . However, in these cases, the microbial targets do not serve as the natural B cell priming agents, because natural antibodies are produced constitutively, possibly through BCR ligation by polyreactive self-antigens (Grö nwall et al., 2012; Holodick et al., 2017; Savage and Baumgarth, 2015) . In this study, we sought to mechanistically evaluate whether conventional B cell responses from the human antibody repertoire (triggered by BCR ligation with exogenous antigen) bear natural specificities for conserved microbial substrate. Individual germline antibody CDRs have already shown clear evidence of differing utilities, because CDR sequences are differentially deployed following immune exposure to certain antigens (Brorson et al., 1999; Hardy et al., 1989; Henry Dunand and Wilson, 2015; Hwang et al., 2014; Kirik et al., 2017; Lerner, 2011; Lonberg, 2005; Pape et al., 2018; Schickel et al., 2017; Wang et al., 2014; Willis et al., 2013) , most recently observed for antibody responses to SARS-CoV-2 within convalescent individuals (Cao et al., 2020; Robbiani et al., 2020; Wec et al., 2020b) . In humans, such germline-endowed affinity for antigen can also seed genetically reproducible or public broadly neutralizing antibody (bnAb) responses against numerous pathogens, including influenza virus, HIV, hepatitis C virus, hepatitis B virus, and yellow fever virus (Avnir et al., 2014; Flyak et al., 2018; Golsaz-Shirazi et al., 2015; Hehle et al., 2020; Joyce et al., 2016; Lerner, 2011; Lingwood et al., 2012; Lucas et al., 1991; Pappas et al., 2014; Scheid et al., 2011; Tzarum et al., 2019; Wec et al., 2020a; Wheatley et al., 2015; Wu et al., 2011; Zhou et al., 2013 Zhou et al., , 2015 . We have experimentally demonstrated that such public epitope targeting by human BCRs enables vaccine amplification of pathway-reproducible antibody responses that broadly neutralize influenza virus . However, much of the work defining natural antigen substrate for human antibodies has relied on retrospective analyses of immune responses, in which patterns in antibody gene usage are first identified and then subsequently defined through biochemical activities in vitro. To mechanistically evaluate human antibody V H -gene contribution to the polyclonal response within the living organism, we engineered transgenic animals that harbored human CDRH3 and light-chain (LC) diversities but were simultaneously constrained to different user-defined V H -genes (encoding CDRH1 + CDRH2). Using this approach, we defined the in vivo V H -gene contribution to extrafollicular responses against bacterial antigens in the blood, simple antiseptic B cell reactions that enable rapid humoral targeting of bacterial lipids and carbohydrates and in which response titers reflect antigen recognition by germline BCRs (Cerutti et al., 2013; Garraud et al., 2012; Limet et al., 1987 Limet et al., , 1989 MacLennan et al., 2003; Shishido et al., 2012) . We observed that specific V H-gene input was needed to engage and respond to lipopolysaccharide (LPS), a defining antigen of gram-negative bacteria. Notably, we found that the human V Hgene IGHV1-2*02, known for genetically endowing public bnAb responses against HIV (Gristick et al., 2016; Scheid et al., 2011; Wu et al., 2011; Zhou et al., 2013 Zhou et al., , 2015 , was required to accommodate this key glycolipid antigen when prepared from diverse bacterial sources. We then mapped this V H -endowed polyclonal target specificity to the conserved substructure of LPS. Animals using non-IGHV1-2 V H -genes failed to accommodate LPS, despite bearing similar CDRH3 and LC diversities, B cell frequencies, and capacity to respond to other thymus-independent (TI) and thymus-dependent (TD) antigen geometries when present in the blood. Hence, we conclude that despite their limited diversity, individual human antibody V H sequences are not functionally redundant and that specific germline-encoded CDRs naturally endow the human antibody repertoire with a broadspectrum response targeting a conserved bacterial feature. We have previously shown that mice homozygous for the HC2 locus are fully constrained to user-defined usage of single human V H -genes while containing a humanized CDRH3 length distribution and amino acid composition that is similar to that of the human repertoire . By systematically altering the V H sequence, this system provides an opportunity to mechanistically evaluate human V H -gene contribution to the polyclonal antibody response within the living organism. The number and frequency of B and T lymphocytes, antibody response titers, and somatic hypermutation within these mice are comparable to wild-type (WT) C57BL/6 . We previously used the HC2 locus to fully constrain the antibody response to single human V H-genes: IGHV1-2*02 or IGHV1-69*01, in which antibody LC diversity was provided by the murine repertoire . To more closely recapitulate the human repertoire in the present study, we replaced the murine LC repertoire with KCo5, an engineered human kappa chain locus, as previously established for the HC2 platform Lonberg, 2005; Xu and Davis, 2000) ( Figure 1A ). As expected, deep sequencing of the immunoglobulin M (IgM) BCR repertoires of these transgenic mice, termed IGHV1-2 + hIgK and IGHV1-69 + hIgK, revealed CDRH3 length and amino acid distributions that were similar to each other (>13.0 3 10 6 aligned BCR reads per genotype) and to the human CDRH3 profile, as mined from a recent human IgM repertoire analysis (DeWitt et al., 2016) ( Figures 1B and 1C) . Hence, these animals provided a system to experimentally vary human antibody V H -gene usage to mechanistically evaluate V H contribution to antigen engagement in the context of an otherwise unconstrained polyclonal human-like BCR repertoire. We then applied this approach to evaluate B cell antigen recognition within the rapid blood-based extrafollicular antibody responses, normally triggered by bacterial lipids and carbohydrates (Cerutti et al., 2013; Garraud et al., 2012; Limet et al., 1987 Limet et al., , 1989 Ma-cLennan et al., 2003; Shishido et al., 2012) . Antiseptic responses to bloodborne bacterial lipids and carbohydrates are primarily TI IgM arising from B cells residing in the marginal zone (MZ) of the spleen (Cerutti et al., 2013; Garraud et al., 2012; MacLennan et al., 2003; Mond et al., 1995; Vos et al., 2000) . We measured the frequencies and total numbers of B cells (CD3 À /CD19 + ), T cells (CD3 + /CD19 À ), and MZ B cells (CD3 À / CD19 + /B220 + /MHCII + /CD23 high /CD23 low ) (Garraud et al., 2012; Kanayama et al., 2005) within our transgenic animals ( Figures 2A and 2B ). We found that the animal genotypes contained similar numbers and frequencies of these immune cell subsets. Furthermore, to confirm that the intravenously (i.v.) introduced bacterial antigens trafficked to and perfused the extrafollicular space, we i.v. injected fluorescent bacterial LPS and then imaged the CD169 macrophage surface boundary layer encapsulating the spleen follicles. We found that the fluorescent antigen perfused the MZ and was captured within this space in both transgenic mouse genotypes ( Figure 2C ). Hence, B cells from our mice bearing human antibody repertoires with different V Hgenes appeared similarly poised to initiate humoral responses against extrafollicular antigenic stimuli, particularly bacterial LPS. (Cerutti et al., 2013; Garraud et al., 2012; MacLennan et al., 2003; Mond et al., 1995; Shishido et al., 2012; Vos et al., 2000) and can be can be tracked 3, 7, and 14 days after i.v. immune challenge (Cerutti et al., 2013; Chappell et al., 2012; MacLennan et al., 2003; Rubtsov et al., 2005) . Moreover, sequential immunization with TI antigen will not boost antibody response titers, because there is no T cell help to coordinate B cell germinal center formation and development of B cell memory (Mond et al., 1995; Tittle and Rittenberg, 1980; Vos et al., 2000) . We found that both IGHV1-2 and IGHV1-69 mice produced robust extrafollicular IgM responses against TNP-ficoll, a standard TI-2 antigen that elicits antibodies through BCR crosslinking only, in the absence of innate immune receptor stimulation (Mond et al., 1995; Ö ner et al., 2016; Tittle and Rittenberg, 1980; Vos et al., 2000) ( Figure 3A) . We observed the same result following immunization of HCo12 mice, which use the same human CDRH3 and LC diversity but deploy five common human V H -genes (IGHV5-51 + IGHV3-23 + IGHV3-30 + IGHV1-69 +IGHV4-34) (Figures 3A and 3B ). All mouse genotypes also produced robust IgM responses against ovalbumin when this control TD antigen was administered i.v. (Figure 3A ). Surprisingly, however, only the IGHV1-2 mice generated bloodborne A B C (A) Representative gating strategy on lymphocytes harvested from the spleens of IGHV1-69 and IGHV1-2 mouse genotypes. CD3-and CD19-positive cells were gated negatively against a blue live/dead marker. Singlets were defined by forward scatter height (FSCH) versus forward scatter area (FSCA). MZ B cells were defined as CD3 -/CD19 + /B220 + /MHCII + /CD23 low /CD21 + , as shown by the gating scheme in red. (B) Frequency and number of CD3 T cells, CD19 B cells, and CD3 -/CD19 + /B220 + /MHCII + /CD23 low /CD21 + (MZ) B cells were quantified in IGHV1-69 versus IGHV1-2 mice (p > 0.05, two-sided Student's t test, n = 3 mice per genotype). (C) To ensure that LPS perfused the MZ in both animal genotypes, fluorescent LPS (red) was injected into the bloodstream, and its collection in the spleen MZ was monitored 1 h later by immunofluorescence. Green indicates the CD169 metallophilic macrophage layer demarking the separation of spleen follicles (F) and extrafollicular MZ (marked by the dotted line). Scale bar, 50 mm. IgM responses against LPS, which were prepared from diverse bacterial strains spanning two phylogenic orders: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Salmonella enterica (Figures 3B and 3C ). This demonstrated that despite housing comparable levels of CDRH3 and LC diversities and B cells receptive to other TI and TD antigen geometries, polyclonal accommodation of diverse LPS structures was functionally reliant on CDRs encoded by IGHV1-2. Given that V H -gene-dependent immunoreceptor activity was observed for diverse sources of LPS, we hypothesized that IGHV1-2-encoded CDRs may endow target solutions that recognize conserved features of this antigen, perhaps the conserved saccrolipid substructure of bacterial LPS ( Figure 4A ). To test this, we immunized our animals with a uniform Kdo2-lipid (D) IgM responses against LPS, the principle TI antigen from gram-negative bacteria. LPS was prepared from bacteria spanning the orders Pseudomonadales and Enterobacterales (mean ± SD, n = 3 per genotype). Pre-immune readings were subtracted from these curves. Cell Reports 32, 108065, August 25, 2020 5 Report ll A preparation with chemically homogeneous acyl chain structures, and consequently the most potent LPS-based TLR4 agonist yet described, reported as 10-fold higher than conventional LPS preparations (Saito et al., 2010) . As with the heterogeneous LPS inocula, we observed V H -gene-dependent accommodation of Kdo2-lipid A in vivo, as evidenced by robust IgM responses in IGHV1-2 animals and dampened immunogenicity in the IGHV1-69 and HCo12 genotypes ( Figure 4B ). We therefore surmised that the natural polyclonal B cell target specificity endowed by the IGHV1-2 sequence mapped to the conserved, universal building block of bacterial LPS. Genetically hardwired specificities for conserved microbial patterns are hallmarks of the innate immune system and have long been proposed for B cell antigen receptors (Chothia and Lesk, 1987; Chothia et al., 1989; Cohn and Langman, 1990; Henry Dunand and Wilson, 2015; Hwang et al., 2014; Lerner, 2011; Lonberg, 2005) . However, such germline-endowed affinities for specific features common to pathogens or groups of pathogens have been difficult to mechanistically prove in vivo. This is because the ligand binding surface of the BCR is composed of a chimeric display of encoded and non-encoded CDRs unique to each lymphocyte clone (Glanville et al., 2009; Kuroda et al., 2008; Morea et al., 1998; Oliva et al., 1998; Saada et al., 2007; Schroeder and Cavacini, 2010; Shirai et al., 1999; Wedemayer et al., 1997; Xu and Davis, 2000) . How then can mechanistic causality be assigned to any one component in vivo? Much of the work defining natural human antibody substrate has relied on retrospective analyses of human immune responses in which patterns in antibody gene usage are first identified and then biochemically evaluated in vitro. In this study, we harnessed mouse genetics to mechanistically interrogate BCR antigen specificities (B) Extrafollicular IgM responses against Kdo2-lipid A when introduced into the bloodstream of IGHV1-2, IGHV1-69, and HCo12 animals. IgM responses were monitored 3, 7, and 14 days post-immune challenge (mean ± SD, n = 3 per genotype). Pre-immune readings were subtracted from these curves. endowed by individual human antibody genes. We engineered a system in which all antibody CDRs were humanized, allowing unconstrained diversity with the exception of those encoded by the V H sequence, which were 100% restricted and systematically varied. Through this approach, we could experimentally define human V H -gene contribution to the polyclonal antigen response as a whole and as a function of immune challenge within the living organism. We evaluated this parameter within extrafollicular B cell responses to bloodborne LPS, simple and rapid antiseptic reactions in which antibody response titers reflect germline BCR engagement capacity (Cerutti et al., 2013; Garraud et al., 2012; MacLennan et al., 2003; Shishido et al., 2012) . Our results indicated that of six common human V H sequences, only IGHV1-2 could support robust polyclonal accommodation of LPS, which was prepared from diverse sources spanning two phylogenic orders of gram-negative bacteria. Importantly, this was achieved by genetically endowing for recognition of the non-variable saccrolipid core of this antigen, revealing a broad-spectrum antiseptic response against a conserved microbial feature, akin to the pattern recognition receptor activities of the innate immune system. In a pioneering study, Xu and Davis (2000) reported that a single V H -gene mouse generated antibodies against any protein antigen through CDRH3 diversity alone, functionally establishing this motif as the principle determinant of B cell antigen recognition in vivo. The centrally positioned CDRH3 supplies the human BCR repertoire with >10 6 times the diversity of antibody V-geneencoded CDRs and is thought to enable target solutions against any antigenic surface (Glanville et al., 2009; Kuroda et al., 2008; Morea et al., 1998; Oliva et al., 1998; Saada et al., 2007; Schroeder and Cavacini, 2010; Shirai et al., 1999; Wedemayer et al., 1997; Xu and Davis, 2000) . Diverse CDRH3-and LC-associated targeting solutions have been observed for antibody responses against LPS and other bacterial antigens (Haji-Ghassemi et al., 2015; Nguyen et al., 2003) and would also be expected for the polyclonal anti-LPS responses we observe. However, we demonstrate a deterministic feature that functionally gates the polyclonal response as a whole, namely, that specific human V H -gene input is obligate for engaging bloodborne LPS, despite the presence of equivalent CDRH3 and LC diversities across our transgenic mouse genotypes. The LPS response deficits occurred in animals that were otherwise able to respond to control TI and TD antigens and were not explained by the capacity to perfuse the spleen MZ with LPS or the number of responding B cells in residence. LPS is also a potent agonist for Toll-like receptor 4 (TLR4) and a powerful B cell adjuvant (Pone et al., 2012; Schweighoffer et al., 2017) , and it is particularly potent in the Kdo2-lipid A format (Saito et al., 2010) . The failure to accommodate these antigen geometries, even in the presence of such TLR stimulation, underscores our conclusions that despite their limited diversity, human V H -genes are not functionally redundant and that some human antiseptic responses are germline encoded. The amino acid sequence XXGG(S/T/G) within the CDRH2 loops of mouse immunoglobulins has previously been reported as a lipid A binding motif (Haji-Ghassemi et al., 2016) . This motif is present in the CDRH2 from IGHV1-2*02; however, it is also present within IGHV3-23*01, contained within HCo12 mice, which did not respond to bloodborne LPS. Consequently, LPS binding through this motif may not be broadly transplantable to the human V H -genes of our study, although differential expression of V H sequences within the HCo12 mouse also cannot be ruled out. In conclusion, we demonstrate that conventional B cell responses can be tuned by human-gene-endowed affinities for a conserved microbial pattern, akin to the receptors of the innate immune system. This discovery was enabled by manipulating mouse genetics to experimentally define V H -gene contribution to B cell antigen recognition within an otherwise unrestricted human-like antibody repertoire. Beyond its potential clinical importance in combating sepsis, a germline-encoded anti-LPS response may reflect a primordial/evolutionarily retained capacity that, in the case of IGHV1-2*02, could have provided a foundation for later unanticipated germline utilities, such as the nonrandom use of this V H sequence in human bnAbs against HIV (Gristick et al., 2016; Scheid et al., 2011; Wu et al., 2011; Zhou et al., 2013 Zhou et al., , 2015 . 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Science Structural insights into the evolution of an antibody combining site H5N1 Vaccine-Elicited Memory B Cells Are Genetically Constrained by the IGHV Locus in the Recognition of a Neutralizing Epitope in the Hemagglutinin Stem Human germline antibody gene segments encode polyspecific antibodies Focused evolution of HIV-1 neutralizing antibodies revealed by structures and deep sequencing Diversity in the CDR3 region of V(H) is sufficient for most antibody specificities Hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection Multidonor analysis reveals structural elements, genetic determinants, and maturation pathway for HIV-1 neutralization by VRC01-class antibodies Structural Repertoire of HIV-1-Neutralizing Antibodies Targeting the CD4 Supersite in 14 Donors RESOURCE AVAILABILITY Lead Contact Further information and requests for reagents should be directed to and will be fulfilled by Daniel Lingwood Materials Availability There are restrictions to the availability of the IGHV1-69, IGHV1-2 and HCo12 mice due to a MTA with Bristol-Myers Squibb. Data and Code Availability Antibody sequences from this study were The animals were maintained within Ragon Institute's HPPF barrier facility and all experiments were conducted with institutional IACUC approval (MGH protocol 2014N000252). In this study, both male and female animals, aged 6-10 weeks, were used. METHOD DETAILS BCR sequencing CDRH3 diversity within the transgenic mice was measured by deep sequencing the BCRs from antigen naive IgM B cells in bulk according to our previously deployed method we reconfigured the sequenced naive IgM BCRs and the mined human data to generate frequency plots of CDRH3 length and assigned Kabat in R10 (RPMI with 10% FBS) in the presence of 1mg/ml collagenase D (Roche collagenase D) and then filtered through a 70mm cell strainer. The splenocytes were resuspended within ACK lysis buffer to remove red blood cells (Lonza) and then washed with PBS. The cells were first stained Each antibody was used at a final dilution of 1:100 within the master mix. The samples were then run on a 5 laser LSR Fortessa (BD Biosciences) and data were analyzed using FlowJo software version 9 Imaging antigen capture in the spleen To confirm that our transgenic mice could capture bacterial LPS within the spleen marginal zone, we intravenously injected 15mg fluorescent LPS (E.coli Serotype 055:B5, Fluor568 conjugate, Thermofisher) and then removed the spleens at 1 after the injection. We prepared the spleens for immunostaining by submerging $5mm thick pieces within These samples were air-dried overnight and then fixed in ice-cold acetone for 10 min. Following rehydration in PBS, we blocked the sections using ''Background sniper'' (Biocare) within a within a dark humid chamber and then incubated the chamber with anti-CD169-FITC diluted 1:500 in PBS containing 0.1% Tween20) Biosearch Technologies); or 15mg of kdo2-Lipid A (Di[3-deoxy-D-manno-octulosonyl]-lipid A, Avanti Polar Lipids) Excess binding sites were then blocked by addition of 10mg/ml BSA in HS buffer (50mM HEPES, 0.15mM NaCl, pH 7.4) at 200ml/well. After blocking, the sera (pre-bleed and immune sera) were added through serial dilution in HS buffer containing 1mg/ml BSA, starting at 1:20 (v/v). This was allowed to bind for 3 h at 37 C. The plate was then washed (3x) with HS buffer containing 1mg/ml BSA and then incubated with anti-IgM-HRP at 1:5000 dilution in HS buffer + 1mg/ml BSA. The wells were then washed (3x) as above and developed by addition of TMB substrate (Sigma) followed by quenching with 1N sulphuric acid The plates were blocked with 2% BSA in PBS for 1 h and then washed (3x) with PBS. After blocking, the pre and post immune sera was serially diluted into PBS [starting at 1:20 (v/v)] and then added to the wells and incubated for 1 h. The plates were washed (3x) in PBS containing 0.5% Tween 20 (PBST) and incubated with the anti-IgM-HRP diluted at 1:5000. The wells were then washed in PBST and developed using TMB substrate followed by quenching by sulphuric acid