Jody D. Berry

DNA Vaccination with the Major Outer-Membrane Protein Gene Induces Acquired Immunity to Chlamydia trachomatis (Mouse Pneumonitis) Infection

The efficacy of DNA vaccination for prevention of Chlamydia trachomatis infection was studied using the murine model of pneumonia induced by the mouse pneumonitis (MoPn) isolate of C. trachomatis. Intramuscular DNA immunization with two chlamydial genes, one that encodes the major outer-membrane protein (MOMP) and one that encodes a cytoplasmic enzyme (cytosine triphosphate [CTP] synthetase) were tested. The MOMP DNA vaccine but not the CTP synthetase DNA vaccine generated significant delayed-type hypersensitivity and serum antibodies to MoPn elementary bodies and reduced the peak growth of MoPn by >100-fold following lung challenge infection. MOMP DNA immunization suggests a new approach to vaccine development for prevention of human chlamydial infection.

Development and characterisation of neutralising monoclonal antibody to the SARS-coronavirus

Abstract There is a global need to elucidate protective antigens expressed by the SARS-coronavirus (SARS-CoV). Monoclonal antibody reagents that recognise specific antigens on SARS-CoV are needed urgently. In this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mAbs) against the SARS-CoV is presented, based upon their specificity, binding requirements, and biological activity. Initial screening by ELISA, using highly purified virus as the coating antigen, resulted in the selection of 103mAbs to the SARS virus. Subsequent screening steps reduced this panel to seventeen IgG mAbs. A single mAb, F26G15, is specific for the nucleoprotein as seen in Western immunoblot while five other mAbs react with the Spike protein. Two of these Spike-specific mAbs demonstrate the ability to neutralise SARS-CoV in vitro while another four Western immunoblot-negative mAbs also neutralise the virus. The utility of these mAbs for diagnostic development is demonstrated. Antibody from convalescent SARS patients, but not normal human serum, is also shown to specifically compete off binding of mAbs to whole SARS-CoV. These studies highlight the importance of using standardised assays and reagents. These mAbs will be useful for the development of diagnostic tests, studies of SARS-CoV pathogenesis and vaccine development.

Potent and selective inhibition of SARS coronavirus replication by aurintricarboxylic acid

Abstract The severe acute respiratory syndrome virus (SARS) is a coronavirus that instigated regional epidemics in Canada and several Asian countries in 2003. The newly identified SARS coronavirus (SARS-CoV) can be transmitted among humans and cause severe or even fatal illnesses. As preventive vaccine development takes years to complete and adverse reactions have been reported to some veterinary coronaviral vaccines, anti-viral compounds must be relentlessly pursued. In this study, we analyzed the effect of aurintricarboxylic acid (ATA) on SARS-CoV replication in cell culture, and found that ATA could drastically inhibit SARS-CoV replication, with viral production being 1000-fold less than that in the untreated control. Importantly, when compared with IFNs α and β, viral production was inhibited by more than 1000-fold as compared with the untreated control. In addition, when compared with IFNs α and β, ATA was approximately 10 times more potent than IFN α and 100 times more than interferon β at their highest concentrations reported in the literature previously. Our data indicated that ATA should be considered as a candidate anti-SARS compound for future clinical evaluation.

Analysis of the Original Antigenic Sin Antibody Response to the Major Outer Membrane Protein of Chlamydia trachomatis

The anamnestic antibody response to the Chlamydia trachomatis major outer membrane protein (MOMP) was evaluated in mice after priming with serovar C and boosting either with the homologous serovar or with heterologous serovars (A, H, K, and B). Microimmunofluorescence antibody responses demonstrated that boosting with heterologous serovars strongly recalled antibody to serovar C, typical of an original antigenic sin (OAS) response. Boosting with serovars antigenically related to serovar C (A, H, and K) recalled antibody to the variable domain 1 (VD1) peptide of the MOMP of serovar C as determined by a pin-peptide ELISA. Complete amino acid substitution analysis of the VD1 peptide epitope of the MOMP showed that the original antigenic sin response to each boosting serovar contained antibodies with unique patterns of VD1 peptide recognition. The data suggest that antigenically related C. trachomatis serovars differentially recruit B cell lineages from a heterogeneous memory B cell pool that had been induced by priming with the original serovar and thus account for the OAS antibody response.

Chlamydia trachomatis CTP synthetase: molecular characterization and developmental regulation of expression

Chlamydia trachomatis is a nucleotide parasite, being entirely dependent on its host eukaryotic cell for a supply of ATP, GTP, and UTP. Chlamydiae are not, however, auxotrophic for CTP, as they are able both to transport CTP from the host and synthesize CTP de novo via a chlamydial CTP synthetase. This study addresses the developmental regulation of CTP synthetase over the course of the C. trachomatis life cycle. Given the distinct life stages of C. trachomatis, analysis of temporal changes in gene expression and regulation of protein activity is the key to unravelling the mechanism of pathogenesis of this bacterium. The results of immunodetection analysis indicate that CTP synthetase is present in C. trachomatis elementary bodies and reticulate bodies and that it is widespread in other chlamydial strains. Reverse transcriptase–polymerase chain reaction (RT–PCR) and metabolic labelling experiments show that CTP synthetase is transcribed and translated primarily during the mid‐ and late stages of the chlamydial growth cycle. In addition, C. trachomatis CTP synthetase was transcribed with the CTP utilizing enzyme CMP‐2‐keto‐3‐deoxyoctanoic acid synthetase (CMP‐KDO synthetase) as part of a polycistronic mRNA. The co‐expression of these two enzymes suggests a role for CTP synthetase in lipopolysaccharide biosynthesis, potentially channelling CTP directly to CMP‐KDO synthetase. The ability of the intact operon to complement CTP synthetase and CMP‐KDO deficiencies in mutant Escherichia coli strains indicates that both enzymes are efficiently translated from a single messenger RNA. Kinetic analysis revealed that the C. trachomatis CTP synthetase possessed co‐operativity patterns typical of both prokaryotic and eukaryotic CTP synthetases. However, the Km of the enzyme for UTP was lower than that of E. coli CTP synthetase, presumably in response to the low intracellular concentration of this nucleotide in C. trachomatis.

Rapid monoclonal antibody generation via dendritic cell targeting in vivo.

Dendritic cells (DC) are the professional antigen-presenting cells of the immune system. Previous studies have demonstrated that targeting foreign antigens to DC leads to enhanced antigen (Ag)-specific responses in vivo. However, the utility of this strategy for the generation of MAbs has not been investigated. To address this question we immunized mice with IgG-peptide conjugates prepared with the hamster anti-murine CD11c MAb N418. Synthetic peptides corresponding to two different exposed regions of DC-specific ICAM-3 grabbing nonintegrin (DC-SIGN), a human C-type lectin, were conjugated to N418 using thiol-based chemistry. The N418 MAb served as the targeting molecule and synthetic peptides as the Ag (MAb-Ag). A rapid and peptide specific serum IgG response was produced by Day 7 when the synthetic peptides were linked to the N418 MAb, compared to peptide co-delivered with the N418 without linkage. Spleen cells from N418-peptide immunized mice were fused on Day 10, and three IgG1/k monoclonal antibodies (MAbs) were selected to one of the peptide epitopes (MID-peptide). One of the MAbs, Novik 2, bound to two forms of recombinant DC-SIGN protein in enzyme-linked immunosorbent assay (ELISA), and was specifically inhibited by the MID-peptide in solution. Two of these MAbs show specific binding to DC-SIGN expressed by cultured human primary DC. We conclude that in vivo DC targeting enhances the immunogenicity of synthetic peptides and is an effective method for the rapid generation of MAbs to predetermined epitopes.

Molecular characterization of a panel of murine monoclonal antibodies specific for the SARS-coronavirus

Abstract The availability of monoclonal antibodies (mAbs) specific for the SARS-coronavirus (SARS-CoV) is important for the development of both diagnostic tools and treatment of infection. A molecular characterization of nine monoclonal antibodies raised in immune mice, using highly purified, inactivated SARS-CoV as the inoculating antigen, is presented in this report. These antibodies are specific for numerous viral protein targets, and six of them are able to effectively neutralize SARS-CoV in vitro, including one with a neutralizing titre of 0.075nM. A phylogenetic analysis of the heavy and light chain sequences reveals that the mAbs share considerable homology. The majority of the heavy chains belong to a single Ig germline V-gene family, while considerably more sequence variation is evident in the light chain sequences. These analyses demonstrate that neutralization ability can be correlated with specific murine VH-gene alleles. For instance, one evident trend is high sequence conservation in the VH chains of the neutralizing mAbs, particularly in CDR-1 and CDR-2. The results suggest that optimization of murine mAbs for neutralization of SARS-CoV infection will likely be possible, and will aid in the development of diagnostic tools and passive treatments for SARS-CoV infection.

Molecular analysis of monoclonal antibodies to group variant capsular polysaccharide of Neisseria meningitidis: recurrent heavy chains and alternative light chain partners.

Abstract We determined the molecular sequence of monoclonal antibodies (mAbs) to serogroups B and C capsular polysaccharides (PS) of Neisseria meningitidis. N. meningitidis infections are a leading cause of bacterial septicemia and meningitis in humans. Antibodies to PS are fundamental to host defense and diagnostics. The polysaccharide capsule of group B N. meningitidis is poorly immunogenic and thus is an important model for studying pathogen-host co-evolution through understanding the molecular basis of the host immune response. We used a modified reverse-transcriptase PCR to amplify and sequence the V-genes of murine hybridomas produced against types B and C capsular PS. Databank analysis of the sequences encoding the V-genes of type C capsular PS mAb, 4-2-C, reveal that heavy chain alleles are recurrently used to encode this specificity in mice. Interestingly, a V-gene from the same germline family also encodes the V-domain of mAbs 2-2-B, which targets the antigenically distinct serogroup B capsular PS. Somatic mutation, junctional diversity and alternative light chains collectively impart the specificity for these serologically distinct epitopes. Knowledge of the specific immunoglobulin genes used to target common bacterial virulence factors may lead to insights on pathogen-host co-evolution, and the potential use of this information in pre-symptomatic diagnosis is discussed.

Development of functional human monoclonal single-chain variable fragment antibody against HIV-1 from human cervical B cells.

A panel of novel recombinant single-chain variable fragment (scFv) antibody against human immunodeficiency virus type-1 (HIV-1) was isolated and characterized. We generated human scFvs using RNA harvested from cervical B lymphocytes of Kenyan prostitutes who are highly exposed to HIV-1, but remain persistently seronegative. The variable regions of the heavy (VH) and light (VL) chain antibody genes were selected as hybrids using guided-selection with the VL and VH, respectively, of a derivative of IgGb(12) using the phagemid vector pComb3X. IgGb(12) is a previously well-characterized HIV-1 neutralizing human monoclonal antibody (MAb). One of the hybrid scFv, IgA6/4L, neutralizes HIV-1 infectivity in in vitro cell culture assay. The cervical VH and VL chain antibody genes were connected by a DNA linker and subcloned in pComb3X. The cervical scFv clones were functional in recognizing HIV-1 gp120 by enzyme-linked immunosorbant assay (ELISA) and on cells in flow cytometry. Whole IgGb(12) does not inhibit binding of clones IgA6/5k nor IgA6/30lambda to gp120, which suggests that they bind different epitopes. Nucleotide sequence analysis of the cervical scFv show the clones are unique and reveal interesting characteristics of human cervical V gene pools. This work demonstrates, for the first time, cloning of a functional scFv MAb to a sexually transmitted disease pathogen from local cervical B-cell pools in exposed humans.

Neutralizing epitopes of the SARS-CoV S-protein cluster independent of repertoire, antigen structure or mAb technology.

Neutralizing antibody responses to the surface glycoproteins of enveloped viruses play an important role in immunity. Many of these glycoproteins, including the severe acute respiratory syndrome-coronavirus (SARS-CoV) spike (S) protein form trimeric units in the membrane of the native virion. There is substantial experimental and pre-clinical evidence showing that the S protein is a promising lead for vaccines and therapeutics. Previously we generated a panel of monoclonal antibodies (mAbs) to whole inactivated SARS-CoV which neutralize the virus in vitro. 1,2 Here, we define their specificity and affinity, map several of their epitopes and lastly characterise chimeric versions of them. Our data show that the neutralizing mAbs bind to the angiotensin-converting enzyme 2 (ACE2) receptor-binding domain (RBD) of the SARS S protein. Three of the chimeric mAbs retain their binding specificity while one conformational mAb, F26G19, lost its ability to bind the S protein despite high level expression. The affinity for recombinant S is maintained in all of the functional chimeric versions of the parental mAbs. Both parental mAb F26G18 and the chimeric version neutralize the TOR2 strain of SARS-CoV with essentially identical titres (2.07 and 2.47 nM, respectively). Lastly, a comparison with other neutralizing mAbs to SARS-CoV clearly shows that the dominance of a 33 amino acid residue loop of the SARS-CoV RBD is independent of repertoire, species, quaternary structure, and importantly, the technology used to derive the mAbs. In cases like this, the dominance of a compact RBD antigenic domain and the central role of the S protein in pathogenesis may inherently create immunoselection pressure on viruses to evolve more complex evasion strategies or die out of a host species. The apparent simplicity of the mechanism of SARS-CoV neutralization is in stark contrast to the complexity shown by other enveloped viruses.