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Bacillus anthracis (Ames strain) chromosome-derived open reading frames (ORFs), predicted to code for surface exposed or virulence related proteins, were selected as B. anthracis -specific vaccine candidates by a multistep computational screen of the entire draft chromosome sequence (February 2001 version, 460 contigs, The Institute for Genomic Research, Rockville, Md.). The selection procedure combined preliminary annotation (sequence similarity searches and domain assignments), prediction of cellular localization, taxonomical and functional screen and additional filtering criteria (size, number of paralogs). The reductive strategy, combined with manual curation, resulted in selection of 240 candidate ORFs encoding proteins with putative known function, as well as 280 proteins of unknown function. Proteomic analysis of two-dimensional gels of a B. anthracis membrane fraction, verified the expression of some gene products. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analyses allowed identification of 38 spots cross-reacting with sera from B. anthracis immunized animals. These spots were found to represent eight in vivo immunogens, comprising of EA1, Sap, and 6 proteins whose expression and immunogenicity was not reported before. Five of these 8 immunogens were preselected by the bioinformatic analysis (EA1, Sap, 2 novel SLH proteins and peroxiredoxin/AhpC), as vaccine candidates. This study demonstrates that a combination of the bioinformatic and proteomic strategies may be useful in promoting the development of next generation anthrax vaccine.

The Fv is the smallest antigen binding fragment of the antibody and is made of the variable domains of the light and heavy chains, V L and V H , respectively. The 26-kDa Fv is amenable for structure determination in solution using multi-dimensional hetero-nuclear NMR spectroscopy. The human monoclonal antibody 447-52D neutralizes a broad spectrum of HIV-1 isolates. This anti-HIV-1 antibody elicited in an infected patient is directed against the third variable loop (V3) of the envelope glycoprotein (gp120) of the virus. The V3 loop is an immunodominant neutralizing epitope of HIV-1. To obtain the 447-52D Fv for NMR studies, an Escherichia coli bicistronic expression vector for the heterodimeric 447-52D Fv and vectors for single chain Fv and individually expressed V H and V L were constructed. A pelB signal peptide was linked to the antibody genes to enable secretion of the expressed polypeptides into the periplasm. For easy cloning of any antibody gene without potential modification of the antibody sequence, restriction sites were introduced in the pelB sequence and following the termination codon. A set of oligonucleotides that prime the leader peptide genes of all potential antibody human antibodies were designed as backward primers. The forward primers for the V L and V H were based on constant region sequences. The 447-52D Fv could not be expressed either by a bicistronic vector or as single chain Fv, probably due to its toxicity to Escherichia coli . High level of expression was obtained by individual expression of the V H and the V L chains, which were then purified and recombined to generate a soluble and active 447-52D Fv fragment. The V L of mAb 447-52D was uniformly labeled with 13 C and 15 N nuclei (U– 13 C/ 15 N). Preliminary NMR spectra demonstrate that structure determination of the recombinant 447-52D Fv and its complex with V3 peptides is feasible.

A genomic analysis of the Bacillus anthracis virulence plasmid pXO1, aimed at identifying potential vaccine candidates and virulence-related genes, was carried out. The 143 previously defined open reading frames (ORFs) (R. T. Okinaka, K. Cloud, O. Hampton, A. R. Hoffmaster, K. K. Hill, P. Keim, T. M. Koehler, G. Lamke, S. Kumano, J. Mahillon, D. Manter, Y. Martinez, D. Ricke, R. Svensson, and P. J. Jackson, J. Bacteriol. 181: 6509-6515, 1999) were subjected to extensive sequence similarity searches (with the nonredundant and unfinished microbial genome databases), as well as motif, cellular location, and domain analyses. A comparative genomics analysis was conducted with the related genomes of Bacillus subtilis , Bacillus halodurans , and Bacillus cereus and the pBtoxis plasmid of Bacillus thuringiensis var. israeliensis. As a result, the percentage of ORFs with clues about their functions increased from ∼30% (as previously reported) to more than 60%. The bioinformatics analysis permitted identification of novel genes with putative relevance for pathogenesis and virulence. Based on our analyses, 11 putative proteins were chosen as targets for functional genomics studies. A rapid and efficient functional screening method was developed, in which PCR-amplified full-length linear DNA products of the selected ORFs were transcribed and directly translated in vitro and their immunogenicities were assessed on the basis of their reactivities with hyperimmune anti- B. anthracis antisera. Of the 11 ORFs selected for analysis, 9 were successfully expressed as full-length polypeptides, and 3 of these were found to be antigenic and to have immunogenic potential. The latter ORFs are currently being evaluated to determine their vaccine potential.

We report the draft whole-genome sequence of the nonproteolytic Bacillus anthracis V770-NP1-R strain. Compared to those of other B. anthracis strains, the genome exhibits unique mutations in multiple targets potentially affecting proteolytic functions. One of these mutations is a deletion that disrupts the NprR quorum-sensing regulator of the NprA protease.

The 0.5β monoclonal antibody is a very potent strain-specific HIV-neutralizing antibody raised against gp120, the envelope glycoprotein of HIV-1. This antibody recognizes the V3 loop of gp120, which is a major neutralizing determinant of the virus. The antibody–peptide interactions, involving aromatic and negatively charged residues of the antibody 0.5β, were studied by NMR and double-mutant cycles. A deuterated V3 peptide and a Fab containing deuterated aromatic amino acids were used to assign these interactions to specific V3 residues and to the amino acid type and specific chain of the antibody by NOE difference spectroscopy. Electrostatic interactions between negatively charged residues of the antibody Fv and peptide residues were studied by mutagenesis of both antibody and peptide residues and double-mutant cycles. Several interactions could be assigned unambiguously: F96(L) of the antibody interacts with Pro13 of the peptide, H52(H) interacts with Ile7, Ile9 and Gln10 and D56(H) interacts with Arg11. The interactions of the light-chain tyrosines with Pro13 and Gly14 could be assigned to either Y30a(L) and Y32(L), respectively, or Y32(L) and Y49(L), respectively. Three heavy-chain tyrosines interact with Ile7, Ile20 and Phe17. Several combinations of assignments involving Y32(H), Y53(H), Y96(H) and Y100a(H) may satisfy the NMR and mutagenesis constraints, and therefore at this stage the interactions of the heavy-chain tyrosines were not taken into account. The unambiguous assignments [F96(L), H52(H) and D56(H)] and the two possible assignments of the light-chain tyrosines were used to dock the peptide into the antibody-combining site. The peptide converges to a unique position within the binding site, with the RGPG loop pointing into the center of the groove formed by the antibody complementary determining regions while retaining the β-hairpin conformation and the type-VI RGPG turn [Tugarinov, V., Zvi, A., Levy, R. & Anglister, J. (1999) Nat. Struct. Biol.6, 331–335].

A cis proline turn linking two β-hairpin strands in the solution structure of an antibody-bound HIV-1 IIIB V3 peptide

Solid–state NMR evidence for an antibody–dependent conformation of the V3 loop of HIV–1 gp120

RP135 is a 24-residue peptide corresponding to the principal neutralizing determinant of the envelope glycoprotein gp120 of the human immunodeficiency virus type 1. We have studied the conformation of RP135 in complex with a neutralizing antibody 0.5β raised against gp120 by 2D NMR spectroscopy. The antigenic determinant recognized by this antibody was mapped using a combination of HOHAHA and ROESY measurements, in which resonances of the Fab and the tightly bound peptide residues are eliminated and the mobile residues of the bound peptide are sequentially assigned. We found that residues Ser6 - Thr19 are part of the epitope, while Lys5 and Ile20 are at its boundaries. Difference spectroscopy was applied to study the conformation of the bound peptide representing the epitope within the 52 kDa of the Fab complex. Specific residues of the peptide were deuterated or replaced and the difference between the NOESY spectrum of the complex with the unlabeled residue and the NOESY spectrum of the complex with the modified residue revealed the interactions of the labeled residue both within the peptide and with the Fab fragment. A total of 122 distance restraints derived from the difference spectra enabled the calculation of the structure of the bound peptide. The peptide forms a 10-residue loop, while the two segments flanking this loop interact extensively with each other and possibly form anti-parallel β-strands. The loop conformation could be observed due to the unusual large size (17 residues) of the antigenic determinant recognized by 0.5β.

The principal neutralizing determinant (PND) of human immunodeficiency virus type 1 (HIV-1) is located in the third hypervariable region (V3) of the virus envelope glycoprotein gp120. The conformation of a V3 peptide of HIV-1IIIB bound to the Fab fragment of an anti-gp120 HIV neutralizing antibody, 0.5beta, was studied by 1H NMR spectroscopy. This 18-residue peptide represents the epitope recognized by 0.5beta and encompasses most of the PND. The slow off-rate of the peptide prevents the observation of peptide/Fab interactions as well as intramolecular interactions within the bound peptide by transferred nuclear Overhauser enhancement (TRNOE). To detect and assign interactions within the bound peptide in the 52 kDa complex, NOESY difference spectra were measured using three strategies: (a) deuteration of peptide residues, (b) Arg two head right arrow Lys replacements, and (c) truncation of the peptide antigen. Each difference spectrum was calculated between NOESY spectra measured for two Fab complexes in which the bound peptides differed in their deuteration or in their sequence. The difference spectra revealed numerous interactions between the N-terminus of the epitope (Arg-4, Lys-5, Ser-6, Ile-7, and Ile-9) and its C-terminus (Phe-17, Val-18, Thr-19, and Ile-20). The assigned NOE interactions within the bound peptide were translated into distance restraints that were used to calculate the conformation of the bound peptide by the hybrid distance geometry/simulated annealing method. A total of 39 long-range (residues i - j4), 14 short-range, and 69 intraresidue NOE interactions within the bound peptide have been assigned. Twelve structures without NOE constraint violations were obtained, having a 1.6 A rms deviation for the backbone atoms. The peptide forms a 10-residue loop, while the two segments flanking this loop, KSI and VTI, interact extensively with each other and possibly form antiparallel beta-strands. This loop conformation could be observed due to the unusual large size (17 residues) of the antigenic determinant recognized by 0.5beta.

The 24-amino-acid peptide RP135 (NNTRKSIRIQRGPGRAFVTIGKIG) corresponds in its amino acid sequence to the principal neutralizing determinant of the human immunodeficiency virus type-1, IIIB isolate (HIV-1IIIB, residues 308-331 of the envelope glycoprotein gp120). In order to map the antigenic determinant recognized by 0.5 beta, the complex of RP135 with an anti-gp120 HIV neutralizing antibody, 0.5 beta, which cross reacts with the peptide, was studied by using two-dimensional NMR spectroscopy. A combination of homonuclear Hartmann Hahn two-dimensional experiment and roating-frame Overhauser enhancement spectroscopy of the Fab/peptide complex measured in H2O was used to eliminate the resonances of the Fab and the tightly bound peptide residues and to obtain sequential assignments for those parts of the peptide which retain considerable mobility upon binding. In this manner, a total of 14 residues (Ser6-Thr19) were shown to be part of the antigenic determinant recognized by the antibody 0.5 beta. Lys5 and Ile20 were found to retain considerable mobility in the bound peptide while their amide protons undergo significant change in chemical shift upon binding. This observation suggests that these two residues are at the boundaries of the determinant recognized by the antibody. Competitive binding experiments using truncated peptides strongly support the NMR observations.

Deciphering the cellular immunome of a bacterial pathogen is challenging due to the enormous number of putative peptidic determinants. State-of-the-art prediction methods developed in recent years enable to significantly reduce the number of peptides to be screened, yet the number of remaining candidates for experimental evaluation is still in the range of ten-thousands, even for a limited coverage of MHC alleles. We have recently established a resource-efficient approach for down selection of candidates and enrichment of true positives, based on selection of predicted MHC binders located in high density "hotspots" of putative epitopes. This cluster-based approach was applied to an unbiased, whole genome search of Francisella tularensis CTL epitopes and was shown to yield a 17-25 fold higher level of responders as compared to randomly selected predicted epitopes tested in Kb/Db C57BL/6 mice. In the present study, we further evaluate the cluster-based approach (down to a lower density range) and compare this approach to the classical affinity-based approach by testing putative CTL epitopes with predicted IC(50) values of

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, infects ~8 million annually culminating in ~2 million deaths. Moreover, about one third of the population is latently infected, 10% of which develop disease during lifetime. Current approved prophylactic TB vaccines (BCG and derivatives thereof) are of variable efficiency in adult protection against pulmonary tuberculosis (0%--80%), and directed essentially against early phase infection. It is well established that cellular immunity plays a pivotal role against intracellular bacterial infections, such as tuberculosis. The limited knowledge existing to date on Mtb antigens recognized by T-cells in general and CD8 in particular, motivates an in-depth, genome-scale identification and analysis of potential CTL epitopes, in order to map the immunodominant determinants responsible for shaping the cellular response elicited by Mtb. Such an analysis should allow the design of a synthetic peptide library enriched for genomic regions containing promiscuous HLA-I predicted epitopes. Accordingly, we have developed a comprehensive immunoinformatic analysis for mapping of clusters of CTL epitopes. The 3989 Mtb H37Rv ORF products were scanned for putative 9-11mers binders to 12 HLA-I supertypes, using the ANN-based NetMHC-3.0 program. In order to maximize the number of "promising" epitopes in the library, a comprehensive analysis tool was developed for mapping clusters of CTL binders and visualization of "hotspots" of epitopes. The ~30,000 mapped clusters, containing at least two consecutive, overlapping predicted binders, were subsequently prioritized by employing a ranking strategy based on measures such as epitope density, median binding affinity, allelic coverage and processing. Different scoring schemes were tested, assessing the sensitivity and relative impact of each of the measures employed on the composition of the final 15mer library generated. Finally, a total score was calculated for each cluster, and the top-ranking clusters were selected as the basis for compilation of a final synthetic library of 40,000 15mer peptides. Such a library contains actually a much larger number (~140,000) of putative, promiscuous HLA-I binders, demonstrating the power of the clustering approach in enrichment of potential immune-determinants per library. Validation of these in silico preselected epitopes and antigens awaits ex vivo examination of the peptides in the library for their response with Mtb-specific T cells.

The well-characterized virulence factors of Yersinia pestis the causative agent of plague are those encoded by the virulence plasmids. Previously we have isolated an attenuated Y. pestis transposon insertion mutant in which the chromosomal pcm gene was disrupted. The pcm gene is located within a putative stress response locus that includes the surE, nlpD, and rpoS genes. In this study, we investigated the expression and the role of pcm locus genes in Y. pestis pathogenesis by constructing a set of isogenic surE, pcm, nlpD and rpoS mutants in the fully virulent Kimberley53 strain. We show that the NlpD lipoprotein is the only factor encoded from the pcm locus that is essential for Y. pestis virulence. A chromosomal deletion of the nlpD gene sequence resulted in a drastic reduction in virulence to an LD50 of at least 107 cfu for subcutaneous and airway routes of infection. The mutant was unable to colonize mouse organs following infection. The unsegmented morphology of the nlpD mutant indicates that NlpD is involved in cell separation; however, deletion of nlpD did not affect in vitro growth rate. Trans-complementation experiments with the Y. pestis nlpD gene restored virulence and all other phenotypic defects. Finally, we demonstrate that the nlpD mutant could be used as a very potent live vaccine against bubonic and pneumonic plague.

Classification and differentiation of Bacillus anthracis isolates by genetic markers play an important role in the study of anthrax epidemiology. We have applied a PCR based method – Random Amplification of Polymorphic DNA (RAPD) to identify twenty-five B. anthracis genetic markers. These markers allowed for classification of the studied strains into five different groups. Three selected RAPD markers were cloned and sequenced. Typical integration of the three markers allowed for specific definition of the five RAPD derived genotypes. To test the universal power of these markers to discriminate between diverse B. anthracis strains, the nucleotide sequence of each marker was searched against all available B. anthracis genome sequences (both finished and unfinished). The three markers system could differentiate between strains belonging to the genetic groups Aβ, A1a, A1b, A4 and B1 (as defined by Keim et al., 2000; Maho et al., 2006) and gave rise to a unique combination for group A3, but couldn’t distinguish between the sub groups A3a and A3b. In addition, this system could not distinguish between groups B2 and C (identical three marker combination). In an attempt to improve the resolution of this system we introduced a fourth marker. In silico analysis revealed that the resulting four markers system could now differentiate between group B2 and C, adding a second genotype to groups A1a, A3a and A4. This four marker system could potentially provide an accurate, simple, and inexpensive agarose-based system for classification B. anthracis strains in laboratories involved in research of this bacterium.

A bioinformatic screen of Yersinia pestis genome identified 11 TPS components, carrying the POlypeptide TRansport-Associated (POTRA ) domain, found in other pathogens to be involved in immunogenicity and pathogenicity. All rTpsBs (transporters ) cloned and expressed in Escherichia coli induced a humoral responses in immunized mice. Mice surviving intranasal infection with fully virulent Y. pestis Kimberley53 strain generated sera cross reacting with some recombinants TpsBs. Moreover, at least 3 TpsB polypeptides were directly detected in bacteria isolated following intranasal Y. pestis infection. All in vivo produced TpsBs appear to be truncated, missing about 75–85 amino acids. MALDI-TOF-MS analysis permitted to identify the truncation at a region overlapping a domain of a closely related Bordetella pertussis TpsB (FhaC ), recently proposed to undergo a major conformational change that allows effector translocation.

Immunization of BALB/c mice with vaccinia virus protein A33 (A33VACV) protects mice from intranasal challenge with the WR strain of vaccinia virus or with ectromelia virus making A33 an important candidate to be included in experimental smallpox subunit vaccines. Single vaccination with a recombinant Sindbis virus expressing A33VACV protect mice against lethal VACV-WR and ectromelia virus (ECTV) but not against the closely related cowpox virus (CPXV). Furthermore, even recombinant Sindbis virus expressing the cowpox virus A33 ortholog (A33CPXV) failed to protect either against cowpox or against VACV-WR challenge. Our attempts to map the regions which may account for this differential behavior were directed against a region of difference between the two orthologs. A stretch of 7 amino acids in A33 was mapped as important for protection which contain the following changes in A33CPXV: L112F, Q117K and L118S. This region maps to a single putative prevalent 9-mer CTL epitope with L112 as an essential anchoring residue, and a major target epitope for neutralizing antibodies encompassing L118. Vaccination with A33 harboring these individual substitutions highlighted the crucial role of L118 in induction of protective immunity.

The Variola virus is the causative agent of smallpox disease. Accurate identification of Variola and differential diagnosis between Variola and other vesicle forming (smallpox-like) pathogens are both technically challenging and of great importance. We compiled a list of vesicle forming pathogens that are prone to be misdiagnosed as smallpox. Some of the pathogens (members of the Orthopoxvirus genus) are also genetically highly similar to Variola.

The pathogenesis mechanism and the identity of major virulence factors of the highly infectious human pathogen F. tularensis tularensis, are still poorly characterized. The restricted efficacy of the sole available vaccine (the LVS attenuated strain) and residual toxicity has motivated extensive R&D efforts directed toward the identification of alternative tularemia vaccine formulations based on attenuated mutants, subunit vaccines and T-cell epitope-based vaccines. Identification of vaccine candidates by bioinformatic approaches is mostly driven by the availability of genome sequence data for numerous human-virulent as well as avirulent strains. In an attempt to select for F. tularensis Schu S4 potent antigens, we have developed a strategy based on genome-scale in silico analyses and data mining of published global experimental studies. The compiled information was divided into distinct biologically relevant categories. Protocols for qualitative and quantitative scoring for each of the categories, were developed. Together with implementation of a biological rationale, these served for ranking and prioritization of the putative antigens, providing a basis for subsequent selection of the top-ranking candidates to be evaluated experimentally.

In search for antigens which may form the basis for improved subunit or live attenuated B. anthracis vaccines, extensive genomic, proteomic and serologic analyses coupled with functional screens for surface exposed and/or secreted proteins, were carried out. The screens resulted in selection of over 50 promising novel in-vivo expressed immunogens, classified as S-Layer Homology (SLH) proteins, repeat proteins, hydrolytic enzymes and ABC transporters. DNA vaccination experiments established that most of these novel antigens are indeed able to elicit a strong humoral response. Yet, unlike the major B. anthracis immunogen Protective Antigen (PA), none of the selected immunogens could provide protection against a subsequent virulent B. anthracis strain challenge. When over-expressed in an attenuated non-toxinogenic and non-encapsulated B. anthracis platform strain, at least three of the novel antigens did confer partial protection against a lethal B. anthracis challenge.

The 24 amino acid peptide RP135 corresponds in its amino acid sequence to the principal neutralizing determinant (PND) of the IIIB isolate of HIV-1. Although the sequence of the PND is highly variable, its central part, containing the sequence GPGR, is conserved in most HIV isolates. Using 2D NMR and CD spectroscopy, we have studied the conformation of RP135 and of two shorter versions: one (P547) that includes the GPGR sequence with the N-terminal part of the peptide and the other (P344) that includes GPGR and the C-terminal segment of RP135. In water, the C-terminal part of RP135 was found to exist in several transient turnlike conformations ("nascent helix"). A helical conformation was found to be stabilized by the addition of TFE. A transient turn was observed also in the GPGR sequence, both in water and in aqueous TFE solutions. While no nascent helix conformations could be observed in the N-terminal part of RP135 in water, a helical conformation was partially stabilized by the addition of TFE. The conformations of the two shorter versions of the peptide were similar to those of the corresponding parts of RP135, except that the transient turn in GPGR could not be detected in P547 dissolved in water. The turn in GPGR was previously predicted by Larosa et al. (1990) and was observed by Chandrasekhar et al. (1991) in the PND peptide of HIV-1MN (RP142), which shares only 56% identity with RP135. However, nascent helix conformations were not observed in aqueous solutions of RP142.(ABSTRACT TRUNCATED AT 250 WORDS)

Yersinia pestis is the causative agent of plague. Previously we have isolated an attenuated Y. pestis transposon insertion mutant in which the pcm gene was disrupted. In the present study, we investigated the expression and the role of pcm locus genes in Y. pestis pathogenesis using a set of isogenic surE, pcm, nlpD and rpoS mutants of the fully virulent Kimberley53 strain. We show that in Y. pestis, nlpD expression is controlled from elements residing within the upstream genes surE and pcm. The NlpD lipoprotein is the only factor encoded from the pcm locus that is essential for Y. pestis virulence. A chromosomal deletion of the nlpD gene sequence resulted in a drastic reduction in virulence to an LD(50) of at least 10(7) cfu for subcutaneous and airway routes of infection. The mutant was unable to colonize mouse organs following infection. The filamented morphology of the nlpD mutant indicates that NlpD is involved in cell separation; however, deletion of nlpD did not affect in vitro growth rate. Trans-complementation experiments with the Y. pestis nlpD gene restored virulence and all other phenotypic defects. Finally, we demonstrated that subcutaneous administration of the nlpD mutant could protect animals against bubonic and primary pneumonic plague. Taken together, these results demonstrate that Y. pestis NlpD is a novel virulence factor essential for the development of bubonic and pneumonic plague. Further, the nlpD mutant is superior to the EV76 prototype live vaccine strain in immunogenicity and in conferring effective protective immunity. Thus it could serve as a basis for a very potent live vaccine against bubonic and pneumonic plague.

Background Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), infects ~8 million annually culminating in ~2 million deaths. Moreover, about one third of the population is latently infected, 10% of which develop disease during lifetime. Current approved prophylactic TB vaccines (BCG and derivatives thereof) are of variable efficiency in adult protection against pulmonary TB (0%–80%), and directed essentially against early phase infection. Methods A genome-scale dataset was constructed by analyzing published data of: (1) global gene expression studies under conditions which simulate intra-macrophage stress, dormancy, persistence and/or reactivation; (2) cellular and humoral immunity, and vaccine potential. This information was compiled along with revised annotation/bioinformatic characterization of selected gene products and in silico mapping of T-cell epitopes. Protocols for scoring, ranking and prioritization of the antigens were developed and applied. Results Cross-matching of literature and in silico-derived data, in conjunction with the prioritization scheme and biological rationale, allowed for selection of 189 putative vaccine candidates from the entire genome. Within the 189 set, the relative distribution of antigens in 3 functional categories differs significantly from their distribution in the whole genome, with reduction in the Conserved hypothetical category (due to improved annotation) and enrichment in Lipid and in Virulence categories. Other prominent representatives in the 189 set are the PE/PPE proteins; iron sequestration, nitroreductases and proteases, all within the Intermediary metabolism and respiration category; ESX secretion systems, resuscitation promoting factors and lipoproteins, all within the Cell wall category. Application of a ranking scheme based on qualitative and quantitative scores, resulted in a list of 45 best-scoring antigens, of which: 74% belong to the dormancy/reactivation/resuscitation classes; 30% belong to the Cell wall category; 13% are classical vaccine candidates; 9% are categorized Conserved hypotheticals, all potentially very potent T-cell antigens. Conclusion The comprehensive literature and in silico-based analyses allowed for the selection of a repertoire of 189 vaccine candidates, out of the whole-genome 3989 ORF products. This repertoire, which was ranked to generate a list of 45 top-hits antigens, is a platform for selection of genes covering all stages of M. tuberculosis infection, to be incorporated in rBCG or subunit-based vaccines.

Bacillus anthracis proteins that possess antigenic properties and are able to evoke an immune response were identified by a reductive genomic-serologic screen of a set of in silico-preselected open reading frames (ORFs). The screen included in vitro expression of the selected ORFs by coupled transcription and translation of linear PCR-generated DNA fragments, followed by immunoprecipitation with antisera from B. anthracis- infected animals. Of the 197 selected ORFs, 161 were chromosomal and 36 were on plasmids pXO1 and pXO2, and 138 of the 197 ORFs had putative functional annotations (known ORFs) and 59 had no assigned functions (unknown ORFs). A total of 129 of the known ORFs (93%) could be expressed, whereas only 38 (64%) of the unknown ORFs were successfully expressed. All 167 expressed polypeptides were subjected to immunoprecipitation with the anti- B. anthracis antisera, which revealed 52 seroreactive immunogens, only 1 of which was encoded by an unknown ORF. The high percentage of seroreactive ORFs among the functionally annotated ORFs (37%; 51/129) attests to the predictive value of the bioinformatic strategy used for vaccine candidate selection. Furthermore, the experimental findings suggest that surface-anchored proteins and adhesins or transporters, such as cell wall hydrolases, proteins involved in iron acquisition, and amino acid and oligopeptide transporters, have great potential to be immunogenic. Most of the seroreactive ORFs that were tested as DNA vaccines indeed appeared to induce a humoral response in mice. We list more than 30 novel B. anthracis immunoreactive virulence-related proteins which could be useful in diagnosis, pathogenesis studies, and future anthrax vaccine development.

In a search for novel attenuated vaccine candidates for use againstYersinia pestis, the causative agent of plague, a signature-tagged mutagenesis strategy was used and optimized for a subcutaneously infected mouse model. A library of tagged mutants of the virulentY. pestisKimberley53 strain was generated. Screening of 300 mutants through two consecutive cycles resulted in selection of 16 mutant strains that were undetectable in spleens 48 h postinfection. Each of these mutants was evaluated in vivo by assays for competition against the wild-type strain and for virulence following inoculation of 100 CFU (equivalent to 100 50% lethal doses [LD50] of the wild type). A wide spectrum of attenuation was obtained, ranging from avirulent mutants exhibiting competition indices of 10−5to 10−7to virulent mutants exhibiting a delay in the mean time to death or mutants indistinguishable from the wild type in the two assays. Characterization of the phenotypes and genotypes of the selected mutants led to identification of virulence-associated genes coding for factors involved in global bacterial physiology (e.g.,purH,purK,dnaE, andgreA) or for hypothetical polypeptides, as well as for the virulence regulator genelcrF. One of the avirulent mutant strains (LD50, >107CFU) was found to be disrupted in thepcmlocus, which is presumably involved in the bacterial response to environmental stress. This Kimberley53pcmmutant was superior to the EV76 live vaccine strain because it induced 10- to 100-fold-higher antibody titers to the protective V and F1 antigens and because it conferred efficacious protective immunity.

NMR spectroscopy has become an important tool for structural studies of proteins and macro molecular complexes. In case of an antibody-antigen complex, different approaches are taken to alleviate the size limit, which is beyond NMR capabilities. The method of choice depends on the kinetic properties of the system under investigation and on the antibody fragment available. We have recently studied the conformation of RP135, a 24-residue HIV-1 peptide corresponding to the principal neutralizing determinant of the virus envelop glycoprotein gp120, in complex with 0.5β, a neutralizing antibody raised against gp120. The binding of the peptide to the antibody was too strong to observe TRNOE, therefore 2D-NOESY difference spectroscopy was applied using three strategies: (a) deuteration of specific residues of the peptide; (b) Arg→Lys replacement and; (c) truncation of the peptide antigen. The restraints on interproton distances within the bound peptide were used to calculate its conformation. The peptide forms a 10-residue loop, while the two segments flanking this loop interact extensively with each other and possibly form antiparallel β-strands.

Vaccinia virus protein A33 (A33VACV) plays an important role in protection against orthopoxviruses, and hence is included in experimental multi-subunit smallpox vaccines. In this study we show that single-dose vaccination with recombinant Sindbis virus expressing A33VACV, is sufficient to protect mice against lethal challenge with vaccinia virus WR (VACV-WR) and ectromelia virus (ECTV) but not against cowpox virus (CPXV), a closely related orthopoxvirus. Moreover, a subunit vaccine based on the cowpox virus A33 ortholog (A33CPXV) failed to protect against cowpox and only partially protected mice against VACV-WR challenge. We mapped regions of sequence variation between A33VACV and A33CPXVand analyzed the role of such variations in protection. We identified a single protective region located between residues 104–120 that harbors a putative H-2Kd T cell epitope as well as a B cell epitope - a target for the neutralizing antibody MAb-1G10 that blocks spreading of extracellular virions. Both epitopes in A33CPXV are mutated and predicted to be non-functional. Whereas vaccination with A33VACV did not induce in-vivo CTL activity to the predicted epitope, inhibition of virus spread in-vitro, and protection from lethal VACV challenge pointed to the B cell epitope highlighting the critical role of residue L118 and of adjacent compensatory residues in protection. This epitope’s critical role in protection, as well as its modifications within the orthopoxvirus genus should be taken in context with the failure of A33 to protect against CPXV as demonstrated here. These findings should be considered when developing new subunit vaccines and monoclonal antibody based therapeutics against orthopoxviruses, especially variola virus, the etiologic agent of smallpox.

To increase our understanding of the molecular basis for antibody specificity and for the cross-reactivity of anti-peptide antibodies with native proteins it is important to study the three-dimensional structure of antibody complexes with their peptide antigens. For this purpose it may not be necessary to solve the structure of the whole antibody complex but rather to concentrate on elucidating the combining site structure, the interactions of the antibody with its antigen and the bound peptide conformation. We have developed an NMR methodology based on two-dimensional difference spectrum measurements which extract the information concerning antibody-peptide interactions and intramolecular interactions in the bound ligand from the crowded and unresolved spectrum of the Fab complex. These measurements yield restraints on interproton distances in the complex which are used to dock the peptide into calculated models for the antibodies' combining sites. Comparison of the interactions of three antibodies against a cholera toxin peptide (CTP3), which differ in their cross-reactivity with the toxin, yields information about the size and conformation of antigenic determinants recognized by antibodies, the structure of their combining sites and relationships between antibodies' primary structure, and their interactions with peptide antigens.

The interactions of the peptide RP135a (RKSIRIQRGPGRAFVT), corresponding to residues 311–326 of gp120 of HIV-1IIIB, with the anti-gp120 HIV-1IIIB neutralizing antibody 0.5β were studied by NMR. The NOESY difference spectra measured using specifically deuterated derivatives of the peptide show exclusively the interactions of the deuterated residues both within the bound peptide and with the Fab fragment of the antibody. These measurements reveal hydrophobic interactions within the bound peptide between Ile-4, Ile-6 and Val-15 that create a 12-residue loop with these residues at the base and the conserved GPGR sequence at its top.

Background: The protein 0.5β is a potent strain-specific human immunodeficiency virus type 1 (HIV-1) neutralizing antibody raised against the entire envelope glycoprotein (gp120) of the HIV-1 IIIB strain. The epitope recognized by 0.5β is located within the third hypervariable region (V3) of gp120. Recently, several HIV-1 V3 residues involved in co-receptor utilization and selection were identified. Results: Virtually complete sidechain assignment of the variable fragment (Fv) of 0.5β in complex with the V3 IIIB peptide P1053 (RKSIRIQRGPGRAFVTIG, in single-letter amino acid code) was accomplished and the combining site structure of 0.5β Fv complexed with P1053 was solved using multidimensional nuclear magnetic resonance (NMR). Five of the six complementarity determining regions (CDRs) of the antibody adopt standard canonical conformations, whereas CDR3 of the heavy chain assumes an unexpected fold. The epitope recognized by 0.5β encompasses 14 of the 18 P1053 residues. The bound peptide assumes a β-hairpin conformation with a QRGPGR loop located at the very center of the binding pocket. The Fv and peptide surface areas buried upon binding are 601 A and 743 A 2 , respectively, in the 0.5β Fv–P1053 mean structure. The surface of P1053 interacting with the antibody is more extensive and the V3 peptide orientation in the binding site is significantly different compared with those derived from the crystal structures of a V3 peptide of the HIV-1 MN strain (V3 MN ) complexed to three different anti-peptide antibodies. Conclusions: The surface of P1053 that is in contact with the anti-protein antibody 0.5β is likely to correspond to a solvent-exposed region in the native gp120 molecule. Some residues of this region of gp120 are involved in co-receptor binding, and in discrimination between different chemokine receptors utilized by the protein. Several highly variable residues in the V3 loop limit the specificity of the 0.5β antibody, helping the virus to escape from the immune system. The highly conserved GPG sequence might have a role in maintaining the β-hairpin conformation of the V3 loop despite insertions, deletions and mutations in the flanking regions.

SARS-CoV-2 surface spike protein mediates the viral entry into the host cell and represents the primary immunological target of COVID-19 vaccines as well as post-exposure immunotherapy. Establishment of the highly immunogenic B-cell epitope profile of SARS-CoV-2 proteins in general, and that of the spike protein in particular, may contribute to the development of sensitive diagnostic tools and identification of vaccine` candidate targets. In the current study, the anti-viral antibody response in transgenic K18-hACE-2 mice was examined by implementing an immunodominant epitope mapping approach of the SARS-CoV-2 spike. Serum samples for probing an epitope array covering the entire spike protein were collected from mice following infection with the original SARS-CoV-2 strain as well as the B.1.1.7 Alpha and B.1.351 Beta genetic variants of concern. The analysis resulted in distinction of six linear epitopes common to the humoral response against all virus variants inspected at a frequency of more than 20% of the serum samples. Finally, the universality of the response was probed by cross-protective in vitro experiments using plaque-reducing neutralization tests. The data presented here has important implications for prediction of the efficacy of immune countermeasures against emerging SARS-CoV-2 variants.

A wide range of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing monoclonal antibodies (mAbs) have been reported, most of which target the spike glycoprotein. Therapeutic implementation of these antibodies has been challenged by emerging SARS-CoV-2 variants harboring mutated spike versions. Consequently, re-assessment of previously identified mAbs is of high priority. Four previously selected mAbs targeting non-overlapping epitopes are now evaluated for binding potency to mutated RBD versions, reported to mediate escape from antibody neutralization. In vitro neutralization potencies of these mAbs, and two NTD-specific mAbs, are evaluated against two frequent SARS-CoV-2 variants of concern, the B.1.1.7 Alpha and the B.1.351 Beta. Furthermore, we demonstrate therapeutic potential of three selected mAbs by treatment of K18-human angiotensin-converting enzyme 2 (hACE2) transgenic mice 2 days post-infection with each virus variant. Thus, despite the accumulation of spike mutations, the highly potent MD65 and BL6 mAbs retain their ability to bind the prevalent viral mutants, effectively protecting against B.1.1.7 and B.1.351 variants., Graphical abstract, Novel SARS-CoV-2 antigenic variants jeopardize the efficacy of immunotherapies. Makdasi et al. re-evaluate anti-SARS-CoV-2 Abs previously shown to be highly effective against the original version of the virus. Some of the inspected antibodies retain their neutralization ability and in vivo protective efficacy against various viral variants.