Preclinical efficacy of a cell division protein candidate gonococcal vaccine identified by artificial intelligence.

A safe and effective vaccine is urgently needed to combat the global threat of multidrug-resistant (MDR) Neisseria gonorrhoeae. We screened 26 gonococcal proteins discovered by an artificial intelligence-driven platform called Efficacy Discriminative Educated Network (EDEN) trained to identify novel, protective vaccine antigens against pathogenic bacteria for efficacy in the mouse vaginal colonization model of gonorrhea. Combinations of two to three antigens adjuvanted with GLA-SE (induces TH1 responses) yielded 11 groups that were used to vaccinate mice. An inverse correlation was noted between the complement-dependent bactericidal activity of antisera from each of the 11 groups and the burden of gonococcal colonization. The combination of NGO1549 (FtsN; cell divisome protein) and NGO0265 (predicted cell division protein) most substantially reduced the burden of colonization by MDR strain WHO X. The EDEN prediction score for each group of antigens correlated positively with reductions in overall bacterial burden, providing evidence for its predictive potential. FtsN and NGO0265 administered either individually, in combination, or as a chimeric protein significantly attenuated gonococcal vaginal colonization by all three test strains. IgG in antisera from mice immunized with the chimeric NGO0265-FtsN protein supported the complement-dependent killing of all 50 (100%) gonococcal isolates tested. The efficacy of the chimeric NGO0265-FtsN vaccine required the membrane attack complex (C5b-9) of complement, evidenced by loss of efficacy in complement C9-/- mice. In conclusion, a chimeric molecule comprising NGO0265 and FtsN adjuvanted with GLA-SE elicits IgG with broad anti-gonococcal bactericidal activity, attenuates gonococcal colonization in a complement-dependent manner, and represents a promising gonococcal vaccine candidate.IMPORTANCEVaccines to curb the global spread of multidrug-resistant gonorrhea are urgently needed. Here, 26 vaccine candidates identified by an artificial intelligence-driven platform (Efficacy Discriminative Educated Network[EDEN]) were screened for efficacy in the mouse vaginal colonization model. Complement-dependent bactericidal activity of antisera and the EDEN protective scores both correlated positively with the reduction in overall bacterial colonization burden. NGO1549 (FtsN) and NGO0265, both involved in cell division, displayed the best activity and were selected for further development. Both antigens, when fused to create a chimeric protein, elicited bactericidal antibodies against a wide array of gonococcal isolates and significantly attenuated the duration and burden of gonococcal colonization of mouse vaginas. Protection was abrogated in mice that lacked complement C9, the last step in the formation of the membrane attack complex pore, suggesting complement-dependent bactericidal activity as a mechanistic correlate of protection of the vaccine. FtsN and NGO0265 represent promising vaccine candidates against gonorrhea.

Broadly Protective Multivalent OspA Vaccine against Lyme Borreliosis, Developed Based on Surface Shaping of the C-Terminal Fragment.

The development of vaccines for prevention of diseases caused by pathogenic species can encounter major obstacles if high sequence diversity is observed between individual strains. Therefore, development might be restricted either to conserved antigens, which are often rare, or to multivalent vaccines, which renders the production more costly and cumbersome. In light of this complexity, we applied a structure-based surface shaping approach for the development of a Lyme borreliosis (LB) vaccine suitable for the United States and Europe. The surface of the C-terminal fragment of outer surface protein A (OspA) was divided into distinct regions, based primarily on binding sites of monoclonal antibodies (MAbs). In order to target the six clinically most relevant OspA serotypes (ST) in a single protein, exposed amino acids of the individual regions were exchanged to corresponding amino acids of a chosen OspA serotype. Six chimeric proteins were constructed, and, based on their immunogenicity, four of these chimeras were tested in mouse challenge models. Significant protection could be demonstrated for all four proteins following challenge with infected ticks (OspA ST1, OspA ST2, and OspA ST4) or with in vitro-grown spirochetes (OspA ST1 and OspA ST5). Two of the chimeric proteins were linked to form a fusion protein, which provided significant protection against in vitro-grown spirochetes (OspA ST1) and infected ticks (OspA ST2). This article presents the proof-of-concept study for a multivalent OspA vaccine targeting a wide range of pathogenic LB Borrelia species with a single recombinant antigen for prevention of Lyme borreliosis.

The novel Lyme borreliosis vaccine VLA15 shows broad protection against Borrelia species expressing six different OspA serotypes.

We have previously shown that the Outer surface protein A (OspA) based Lyme borreliosis vaccine VLA15 induces protective immunity in mice. Herein, we report the induction of protective immunity by VLA15 with mouse models using ticks infected with B. burgdorferi (OspA serotype 1), B. afzelii (OspA serotype 2) and B. bavariensis (OspA serotype 4) or with in vitro grown B. garinii (OspA serotype 5 and 6) for challenge. For B. garinii (OspA serotype 3), we have developed a growth inhibition assay using chicken complement and functional antibodies targeting B. garinii (OspA serotype 3) could be demonstrated after immunization with VLA15. Furthermore, following three priming immunizations, a booster dose was administered five months later and the induction of immunological memory could be confirmed. Thus, the antibody titers after the booster dose were increased considerably compared to those after primary immunization. In addition, the half-lives of anti-OspA serotype specific antibodies after administration of the booster immunization were longer than after primary immunization. Taken together, we could show that VLA15 induced protection in mice against challenge with four different clinically relevant Borrelia species (B. burgdorferi, B. afzelii, B. garinii and B. bavariensis) expressing five of the six OspA serotypes included in the vaccine. The protection data is supported by functional assays showing efficacy against spirochetes expressing any of the six OspA serotypes (1 to 6). To our knowledge, this is the first time a Lyme borreliosis vaccine has been able to demonstrate such broad protection in preclinical studies. These new data provide further promise for the clinical development of VLA15 and supports our efforts to provide a new Lyme borreliosis vaccine available for global use.

Characterization and optimization of a novel vaccine for protection against Lyme borreliosis.

Lyme borreliosis (LB) is the most common vector-borne disease in the northern hemisphere and there is no vaccine available for disease prevention. The majority of LB cases in Europe are caused by four different Borrelia species expressing six different OspA serotypes, whereas in the US only one of these serotypes is present. Immunization with the outer surface protein A (OspA) can prevent infection and the C-terminal part of OspA is sufficient for protection against infection transmitted by Ixodes ticks. Here we show that the order of the stabilized monomeric OspA fragments making up the heterodimers in our LB vaccine does not influence the induced immunogenicity and protection. Using bioinformatics analysis (surface electrostatics), we have designed an improved version of an LB vaccine which has an increased immunogenicity for OspA serotype 3 and an optimized expression and purification profile. The OspA heterodimers were highly purified with low amounts of endotoxin, host cell proteins and host cell DNA. All three proteins were at least 85% triacylated which ensured high immunogenicity. The LB vaccine presented here was designed, produced and characterized to a level which warrants further development as a second generation human LB vaccine.

Complete genome sequence of Borrelia afzelii K78 and comparative genome analysis.

The main Borrelia species causing Lyme borreliosis in Europe and Asia are Borrelia afzelii, B. garinii, B. burgdorferi and B. bavariensis. This is in contrast to the United States, where infections are exclusively caused by B. burgdorferi. Until to date the genome sequences of four B. afzelii strains, of which only two include the numerous plasmids, are available. In order to further assess the genetic diversity of B. afzelii, the most common species in Europe, responsible for the large variety of clinical manifestations of Lyme borreliosis, we have determined the full genome sequence of the B. afzelii strain K78, a clinical isolate from Austria. The K78 genome contains a linear chromosome (905,949 bp) and 13 plasmids (8 linear and 5 circular) together presenting 1,309 open reading frames of which 496 are located on plasmids. With the exception of lp28-8, all linear replicons in their full length including their telomeres have been sequenced. The comparison with the genomes of the four other B. afzelii strains, ACA-1, PKo, HLJ01 and Tom3107, as well as the one of B. burgdorferi strain B31, confirmed a high degree of conservation within the linear chromosome of B. afzelii, whereas plasmid encoded genes showed a much larger diversity. Since some plasmids present in B. burgdorferi are missing in the B. afzelii genomes, the corresponding virulence factors of B. burgdorferi are found in B. afzelii on other unrelated plasmids. In addition, we have identified a species specific region in the circular plasmid, cp26, which could be used for species determination. Different non-coding RNAs have been located on the B. afzelii K78 genome, which have not previously been annotated in any of the published Borrelia genomes.

Design and development of a novel vaccine for protection against Lyme borreliosis.

There is currently no Lyme borreliosis vaccine available for humans, although it has been shown that the disease can be prevented by immunization with an OspA-based vaccine (LYMErix). Outer surface protein A (OspA) is one of the dominant antigens expressed by the spirochetes when present in a tick. The Borrelia species causing Lyme borreliosis in Europe express different OspA serotypes on their surface, B. burgdorferi (serotype 1), B. afzelii (serotype 2), B. garinii (serotypes, 3, 5 and 6) and B. bavariensis (serotype 4), while only B. burgdorferi is present in the US. In order to target all these pathogenic Borrelia species, we have designed a multivalent OspA-based vaccine. The vaccine includes three proteins, each containing the C-terminal half of two OspA serotypes linked to form a heterodimer. In order to stabilize the C-terminal fragment and thus preserve important structural epitopes at physiological temperature, disulfide bonds were introduced. The immunogenicity was increased by introduction of a lipidation signal which ensures the addition of an N-terminal lipid moiety. Three immunizations with 3.0 µg adjuvanted vaccine protected mice from a challenge with spirochetes expressing either OspA serotype 1, 2 or 5. Mice were protected against both challenge with infected ticks and in vitro grown spirochetes. Immunological analyses (ELISA, surface binding and growth inhibition) indicated that the vaccine can provide protection against the majority of Borrelia species pathogenic for humans. This article presents the approach which allows for the generation of a hexavalent vaccine that can potentially protect against a broad range of globally distributed Borrelia species causing Lyme borreliosis.

Identification and characterization of Borrelia antigens as potential vaccine candidates against Lyme borreliosis.

The three Borrelia species, Borrelia afzelii, Borrelia burgdorferi and Borrelia garinii are the main species causing the most common tick-borne zoonosis, Lyme borreliosis. By applying a genomic approach relying on human antibodies we have identified 122 antigenic Borrelia proteins associated with Lyme borreliosis, including already known and published protective antigens. The heterogeneity of the Borrelia species causing Lyme borreliosis makes the search for conserved antigens providing broad protection challenging. Using several in vitro assays we narrowed down the selection to 15 vaccine candidates. These antigens were further analyzed for antigenicity and cross-reactivity using sera from mice infected with the three pathogenic Borrelia species. All antigens analyzed showed a high degree of cross-reactivity between the three Borrelia species, essential for providing cross-protection. We also investigated whether mice infected with B. afzelii through tick exposure are primed to mount cytokine responses. For a selection of these antigens, we observed preferentially a pro-inflammatory response in C3H/HeN mice, while in contrast also a type 2 T cell response was seen in the Borrelia-resistant mouse strain BALB/c. Thus, antigens mounting a type 2 or mixed type 2/type 1 T cell response might be preferred vaccine candidates for evaluation in animal models of Lyme borreliosis.

Global ecology and epidemiology of Borrelia garinii spirochetes.

Lyme borreliosis (LB) is a tick-transmitted infectious disease caused by Borrelia burgdorferi sensu lato (s. l.). In Europe, three different Borrelia species are the main causative agents of LB: B. burgdorferi sensu stricto (s.s.), Borrelia afzelii, and Borrelia garinii. The latter depends heavily on birds as its main reservoir hosts. In fact, birds can act both as biological carriers of Borrelia and transporters of infected ticks. The seasonal migration of many bird species not only aid in the spread of B. garinii to new foci but also influence the high level of diversity found within this species. B. garinii have been isolated not only from terrestrial birds in Europe, but also from seabirds worldwide, and homology between isolates in these two different infection cycles suggests an overlap and exchange of strains. In addition, it has been shown that birds can maintain and spread B. garinii genotypes associated with LB in humans. This review article discusses the importance of birds in the ecology and epidemiology of B. garinii spirochetes.

Globally disseminated human pathogenic Escherichia coli of O25b-ST131 clone, harbouring blaCTX-M-15 , found in Glaucous-winged gull at remote Commander Islands, Russia.

With focus on environmental dissemination of antibiotic resistance among clinically relevant bacteria, such as the rising ESBL type of resistance among Escherichia coli, we investigated antibiotic resistance levels in wild birds in the Commander Islands and Kamchatka, Russia. Despite overall low resistance levels in randomly selected E. coli (one from each sample), we found multi-resistant ESBL-producing E. coli harbouring blaCTX-M-14 and blaCTX-M-15 using selective screening. Among these multi-resistant ESBL-producing E. coli we found one blaCTX-M-15 harbouring strain belonging to the O25b-ST131 clone, recognized for its clonal disseminated worldwide as a human pathogen. The potential in acquiring resistant bacteria of human origin, especially highly pathogenic clones, as well as downstream consequences of that, should not be underestimated but further investigated.

Complex population structure of Lyme borreliosis group spirochete Borrelia garinii in subarctic Eurasia.

Borrelia garinii, a causative agent of Lyme borreliosis in Europe and Asia, is naturally maintained in marine and terrestrial enzootic cycles, which primarily involve birds, including seabirds and migratory passerines. These bird groups associate with, correspondingly, Ixodes uriae and Ixodes ricinus ticks, of which the latter species may bite and transmit the infection to humans. Studies of the overlap between these two natural cycles of B. garinii have been limited, in part due to the absence of representative collections of this spirochete's samples, as well as of the lack of reliable measure of the genetic heterogeneity of its strains. As a prerequisite for understanding the epidemiological correlates of the complex maintenance of B. garinii, the present study sought to assess the diversity and phylogenetic relationships of this species' strains from its natural hosts and patients with Lyme borreliosis from subarctic Eurasia. We used sequence typing of the partial rrs-rrl intergenic spacer (IGS) of archived and prospective samples of B. garinii from I. uriae ticks collected predominantly on Commander Islands in North Pacific, as well as on the islands in northern Sweden and arctic Norway. We also typed B. garinii samples from patients with Lyme borreliosis and I. ricinus ticks infesting migratory birds in southern Sweden, or found questing in selected sites on the islands in the Baltic Sea and Lithuania. Fifty-two (68%) of 77 B. garinii samples representing wide geographical range and associated with I. ricinus and infection of humans contributed 12 (60%) of total 20 identified IGS variants. In contrast, the remaining 25 (32%) samples recovered from I. uriae ticks from a few islands accounted for as many as 10 (50%) IGS types, suggesting greater local diversity of B. garinii maintained by seabirds and their ticks. Two IGS variants of the spirochete in common for both tick species were found in I. ricinus larvae from migratory birds, an indication that B. garinii strains are exchanged between different ecological niches. Notably, B. garinii variants associated with I. uriae ticks were found in each of the six clusters, representing two phylogenetic lineages of this species identified among the studied samples. Our findings suggest that B. garinii in subarctic Eurasia comprises two partially overlapping populations with different levels of genetic heterogeneity, presumably, due to distinctive selective pressures on the spirochete in its marine and terrestrial enzootic cycles.

First record of Lyme disease Borrelia in the Arctic.

The epidemiology and ecology of Lyme disease is very complex, and its reported geographical distribution is constantly increasing. Furthermore, the involvement of birds in long distance dispersal and their role as reservoir hosts is now well established. In this study, we have shown that sea birds in the Arctic region of Norway carry Ixodes uriae ticks infected with Lyme disease Borrelia garinii spirochetes. Interestingly, DNA sequencing showed that these isolates are closely related to B. garinii previously isolated from birds, as well as from clinical specimens in northern Europe.

Functional analysis of a lipid galactosyltransferase synthesizing the major envelope lipid in the Lyme disease spirochete Borrelia burgdorferi.

One of the major lipids in the membranes of Borrelia burgdorferi is monogalactosyl diacylglycerol (MGalDAG), a glycolipid recently shown to carry antigenic potency. Herein, it is shown that the gene mgs (TIGR designation bb0454) of B. burgdorferi encodes for the protein bbMGS that, when expressed in Escherichia coli, catalyzes the glycosylation of 1,2-diacylglycerol with specificity for the donor substrate UDP-Gal yielding MGalDAG. Related lipid enzymes were found in many Gram-positive bacteria. The presence of this galactosyltransferase activity and synthesis of a cholesteryl galactoside by another enzyme were verified in B. burgdorferi cell extract. Besides MGalDAG, phosphatidylcholine, phosphatidylglycerol, and cholesterol were also found as major lipids in the cell envelope. The high isoelectric point of bbMGS and clustered basic residues in its amino acid sequence suggest that the enzyme interacts with acidic lipids in the plasma membrane, in agreement with strong enzymatic activation of bbMGS by phosphatidylglycerol. The membrane packing and immunological properties of MGalDAG are likely to be of great importance in vivo.

Migratory passerine birds as reservoirs of Lyme borreliosis in Europe.

To define the role of birds as reservoirs and disseminators of Borrelia spirochetes, we characterized tick infestation and reservoir competence of migratory passerine birds in Sweden. A total of 1,120 immature Ixodes ricinus ticks were removed from 13,260 birds and assayed by quantitative polymerase chain reaction (PCR) for Borrelia, followed by DNA sequencing for species and genotype identification. Distributions of ticks on birds were aggregated, presumably because of varying encounters with ticks along migratory routes. Lyme borreliosis spirochetes were detected in 160 (1.4%) ticks. Borrelia garinii was the most common species in PCR-positive samples and included genotypes associated with human infections. Infestation prevalence with infected ticks was 5 times greater among ground-foraging birds than other bird species, but the 2 groups were equally competent in transmitting Borrelia. Migratory passerine birds host epidemiologically important vector ticks and Borrelia species and vary in effectiveness as reservoirs on the basis of their feeding behavior.

Expression of complement factor H binding immunoevasion proteins in Borrelia garinii isolated from patients with neuroborreliosis.

The Lyme disease-pathogen Borrelia burgdorferi binds the complement inhibitor factor H (FH) to its outer surface protein E- (OspE) and BbA68-families of lipoproteins. In earlier studies, only serum-resistant strains of the genospecies B. burgdorferi sensu stricto or B. afzelii, but not serum-sensitive B. garinii strains, have been shown to bind FH. Since B. garinii often causes neuroborreliosis in man, we have readdressed the interactions of B. garinii with FH. B. garinii 50/97 strain did not express FH-binding proteins. By transforming the B. garinii 50/97 strain with an OspE-encoding gene from complement-resistant B. burgdorferi (ospE-297), its resistance to serum killing could be increased. OspE genes were detected and cloned from the B. garinii BITS, Pistoia and 40/97 strains by PCR and sequencing. The deduced amino acid sequences differed in an N-terminal lysine-rich FH-binding region from OspE sequences of resistant strains. Recombinant B. garinii BITS OspE protein was found to have a considerably lower FH-binding activity than the B. burgdorferi sensu stricto 297 OspE protein P21 (P21-297). Unlike bacteria that had been kept in culture for a long time, neurovirulent B. garinii strains from neuroborreliosis patients were found to express approximately 27-kDa FH-binding proteins. These were not recognized by polyclonal anti-OspE or anti-BbA68 antibodies. We conclude that B. garinii strains carry ospE genes but have a decreased expression of OspE proteins and a reduced ability to bind FH, especially when grown for prolonged periods in vitro. Recently isolated neuroinvasive B. garinii strains, however, can express FH-binding proteins, which may contribute to the virulence of neuroborreliosis-causing B. garinii strains.

Molecular analysis of the channel-forming protein P13 and its paralogue family 48 from different Lyme disease Borrelia species.

The aetiological agent of Lyme disease, Borrelia burgdorferi cycles between its tick vector and mammalian hosts, implying that it can sense different environments and consequently change the expression of genes encoding several surface-associated proteins. The genome of the type strain B. burgdorferi B31 has revealed 175 different gene families. The p13 gene, situated on the chromosome, encodes a channel-forming protein that belongs to the gene family 48 consisting of eight additional paralogous genes. The heterogeneity of the P13 protein from different Lyme disease Borrelia strains was investigated. The predicted surface-exposed domains are the most heterogeneous regions and contain probable epitopes of P13. The membrane-spanning architecture of P13 was determined and a model for the location of this protein in the outer membrane is presented. The transcription of the paralogues of gene family 48 during in vitro culturing and in a mouse infection model was also analysed. The bba01 gene is the only p13 paralogue present in all three Lyme-disease-causing genospecies; it is stable during cultivation in vitro and the BBA01 protein was expressed in all Borrelia strains investigated. Conversely, paralogues bbi31, bbq06 and bbh41 were only detected in B. burgdorferi and the corresponding plasmids harbouring bbi31 and bbh41 were lost during in vitro passage. Finally, p13 and bbi31 are the only members of gene family 48 that are transcribed in mice, suggesting their importance during mammalian infection.