Probing the Role of bba30, a Highly Conserved Gene of the Lyme Disease Spirochete, Throughout the Mouse-Tick Infectious Cycle.

Borrelia burgdorferi, the causative agent of Lyme disease, has a complex and segmented genome consisting of a small linear chromosome and up to 21 linear and circular plasmids. Some of these plasmids are essential as they carry genes that are critical during the life cycle of the Lyme disease spirochete. Among these is a highly conserved linear plasmid, lp54, which is crucial for the mouse-tick infectious cycle of B. burgdorferi. However, the functions of most lp54-encoded open reading frames (ORFs) remain unknown. In this study, we investigate the contribution of a previously uncharacterized lp54 gene during the infectious cycle of B. burgdorferi. This gene, bba30, is conserved in the Borrelia genus but lacks any identified homologs outside the genus. Homology modeling of BBA30 ORF indicated the presence of a nucleic acid binding motif, helix-turn-helix (HTH), near the amino terminus of the protein, suggesting a putative regulatory function. A previous study reported that spirochetes with a transposon insertion in bba30 exhibited a noninfectious phenotype in mice. In the current study, however, we demonstrate that the highly conserved bba30 gene is not required by the Lyme disease spirochete at any stage of the experimental mouse-tick infectious cycle. We conclude that the undefined circumstances under which bba30 potentially confers a fitness advantage in the natural life cycle of B. burgdorferi are not factors of the experimental infectious cycle that we employ.

Infection history of the blood-meal host dictates pathogenic potential of the Lyme disease spirochete within the feeding tick vector.

Lyme disease in humans is caused by several genospecies of the Borrelia burgdorferi sensu lato (s.l.) complex of spirochetal bacteria, including B. burgdorferi, B. afzelii and B. garinii. These bacteria exist in nature as obligate parasites in an enzootic cycle between small vertebrate hosts and Ixodid tick vectors, with humans representing incidental hosts. During the natural enzootic cycle, infected ticks in endemic areas feed not only upon naïve hosts, but also upon seropositive infected hosts. In the current study, we considered this environmental parameter and assessed the impact of the immune status of the blood-meal host on the phenotype of the Lyme disease spirochete within the tick vector. We found that blood from a seropositive host profoundly attenuates the infectivity (>104 fold) of homologous spirochetes within the tick vector without killing them. This dramatic neutralization of vector-borne spirochetes was not observed, however, when ticks and blood-meal hosts carried heterologous B. burgdorferi s.l. strains, or when mice lacking humoral immunity replaced wild-type mice as blood-meal hosts in similar experiments. Mechanistically, serum-mediated neutralization does not block induction of host-adapted OspC+ spirochetes during tick feeding, nor require tick midgut components. Significantly, this study demonstrates that strain-specific antibodies elicited by B. burgdorferi s.l. infection neutralize homologous bacteria within feeding ticks, before the Lyme disease spirochetes enter a host. The blood meal ingested from an infected host thereby prevents super-infection by homologous spirochetes, while facilitating transmission of heterologous B. burgdorferi s.l. strains. This finding suggests that Lyme disease spirochete diversity is stably maintained within endemic populations in local geographic regions through frequency-dependent selection of rare alleles of dominant polymorphic surface antigens.

B-cell epitope of beta toxin of Clostridium perfringens genetically conjugated to a carrier protein: expression, purification and characterization of the chimeric protein.

Beta toxin (btx) is the prime virulence factor for the pathogenesis of Clostridium perfringens type C strain, known to cause necrotic enteritis and enterotoxaemia in mammalian species. The existing vaccines targeting btx are formaldehyde inactivated culture filtrates of Clostridium. These filtrates raise antigenic load in the host leading to nonspecific and poor responses. The present study aimed to overcome these drawbacks and generate a chimeric protein carrying in silico identified B-cell epitope of btx fused with a carrier protein as a vaccine candidate. Using bioinformatic tools, three stretches of amino acids were predicted as putative B-cell epitopes. One of the epitopes spanning 140-156 amino acid residues was genetically conjugated with B-subunit of heat labile enterotoxin (LTB) of Escherichia coli and expressed as a translational fusion in Vibrio cholerae secretory expression system. High level expression of the recombinant fusion protein rLTB-Btx140-156 was obtained and the protein was successfully purified. The recombinant protein retained the native LTB property to pentamerize and bind to GM1 ganglioside receptor of LTB. The antigenicity of both the epitope and the carrier protein was maintained in fusion protein as indicated by immunoblotting against anti-LTB and anti-btx antibody. The rLTB-Btx140-156 fusion protein therefore can be evaluated as a potential vaccine candidate against C. perfringens.

Single-dose VSV-based vaccine protects against Kyasanur Forest disease in nonhuman primates.

Kyasanur Forest disease virus (KFDV) is an endemic arbovirus in western India mainly transmitted by hard ticks of the genus Haemaphysalis. KFDV causes Kyasanur Forest disease (KFD), a syndrome including fever, gastrointestinal symptoms, and hemorrhages. There are no approved treatments, and the efficacy of the only vaccine licensed in India has recently been questioned. Here, we studied the protective efficacy of a vesicular stomatitis virus (VSV)-based vaccine expressing the KFDV precursor membrane and envelope proteins (VSV-KFDV) in pigtailed macaques. VSV-KFDV vaccination was found to be safe and elicited strong humoral and cellular immune responses. A single-dose vaccination reduced KFDV loads and pathology and protected macaques from KFD-like disease. Furthermore, VSV-KFDV elicited cross-reactive neutralizing immune responses to Alkhurma hemorrhagic fever virus, a KFDV variant found in Saudi Arabia.

Ebola Virus Glycoprotein Domains Associated with Protective Efficacy.

Ebola virus (EBOV) is the cause of sporadic outbreaks of human hemorrhagic disease in Africa, and the best-characterized virus in the filovirus family. The West African epidemic accelerated the clinical development of vaccines and therapeutics, leading to licensure of vaccines and antibody-based therapeutics for human use in recent years. The most widely used vaccine is based on vesicular stomatitis virus (VSV) expressing the EBOV glycoprotein (GP) (VSV-EBOV). Due to its favorable immune cell targeting, this vaccine has also been used as a base vector for the development of second generation VSV-based vaccines against Influenza, Nipah, and Zika viruses. However, in these situations, it may be beneficial if the immunogenicity against EBOV GP is minimized to induce a better protective immune response against the other foreign immunogen. Here, we analyzed if EBOV GP can be truncated to be less immunogenic, yet still able to drive replication of the vaccine vector. We found that the EBOV GP glycan cap and the mucin-like domain are both dispensable for VSV-EBOV replication. The glycan cap, however, appears critical for mediating a protective immune response against lethal EBOV challenge in mice.

A live-attenuated viral vector vaccine protects mice against lethal challenge with Kyasanur Forest disease virus.

Kyasanur Forest disease virus (KFDV) is a tick-borne flavivirus endemic in India known to cause severe hemorrhagic and encephalitic disease in humans. In recent years, KFDV has spread beyond its original endemic zone raising public health concerns. Currently, there is no treatment available for KFDV but a vaccine with limited efficacy is used in India. Here, we generated two new KFDV vaccine candidates based on the vesicular stomatitis virus (VSV) platform. We chose the VSV-Ebola virus (VSV-EBOV) vector either with the full-length or a truncated EBOV glycoprotein as the vehicle to express the precursor membrane (prM) and envelope (E) proteins of KFDV (VSV-KFDV). For efficacy testing, we established a mouse disease model by comparing KFDV infections in three immunocompetent mouse strains (BALB/c, C57Bl/6, and CD1). Both vaccine vectors provided promising protection against lethal KFDV challenge in the BALB/c model following prime-only prime-boost and immunizations. Only prime-boost immunization with VSV-KFDV expressing full-length EBOV GP resulted in uniform protection. Hyperimmune serum derived from prime-boost immunized mice protected naïve BALB/c mice from lethal KFDV challenge indicating the importance of antibodies for protection. The new VSV-KFDV vectors are promising vaccine candidates to combat an emerging, neglected public health problem in a densely populated part of the world.

Molecular docking analysis of Clostridium perfringens beta toxin model with potential inhibitors from the ZINC database.

Beta toxin from Clostridium perfringens after being secreted in gut is capable of causing necrotic enteritis in humans and several other animal species and does not respond to routinely used antibiotics. Therefore, there is a need to design an effective inhibitor for the Clostridium perfringens beta toxin (CPB) using cutting edge drug discovery technologies. Hence, potential CPB inhibitors were identified using computer aided screening of compounds from the ZINC database. Further, we document the molecular docking analysis of Clostridium perfringens beta toxin model (that revealed 4 binding pockets, A-D) with the identified potential inhibitors. We show that ZINC291192 [N-[(1-methylindol-3-yl) methyl eneamino]-7,10-dioxabicyclo[4.4.0]deca-2,4,11-triene-8- carboxamide] has optimal binding features with calculated binding energy of -10.38 kcal/mol and inhibition constant of 24.76 nM for further consideration.

Alkhurma haemorrhagic fever virus causes lethal disease in IFNAR-/- mice.

ABSTRACTAlkhurma haemorrhagic fever virus (AHFV), a tick-borne flavivirus closely related to Kyasanur Forest disease virus, is the causative agent of a severe, sometimes fatal haemorrhagic/encephalitic disease in humans. To date, there are no specific treatments or vaccines available to combat AHFV infections. A challenge for the development of countermeasures is the absence of a reliable AHFV animal disease model for efficacy testing. Here, we used mice lacking the type I interferon (IFN) receptor (IFNAR-/-). AHFV strains Zaki-2 and 2003 both caused uniform lethality in these mice after intraperitoneal injection, but strain 2003 seemed more virulent with a median lethal dose of 0.4 median tissue culture infectious doses (TCID50). Disease manifestation in this animal model was similar to case reports of severe human AHFV infections with early generalized signs leading to haemorrhagic and neurologic complications. AHFV infection resulted in early high viremia followed by high viral loads (<108 TCID50/g tissue) in all analyzed organs. Despite systemic viral replication, virus-induced pathology was mainly found in the spleen, lymph nodes, liver and heart. This uniformly lethal AHFV disease model will be instrumental for pathogenesis studies and countermeasure development against this neglected zoonotic pathogen.

Kyasanur Forest Disease and Alkhurma Hemorrhagic Fever Virus-Two Neglected Zoonotic Pathogens.

Kyasanur Forest disease virus (KFDV) and Alkhurma hemorrhagic fever virus (AHFV) are tick-borne flaviviruses that cause life-threatening hemorrhagic fever in humans with case fatality rates of 3-5% for KFDV and 1-20% for AHFV, respectively. Both viruses are biosafety level 4 pathogens due to the severity of disease they cause and the lack of effective countermeasures. KFDV was discovered in India and is restricted to parts of the Indian subcontinent, whereas AHFV has been found in Saudi Arabia and Egypt. In recent years, both viruses have spread beyond their original endemic zones and the potential of AHFV to spread through ticks on migratory birds is a public health concern. While there is a vaccine with limited efficacy for KFDV used in India, there is no vaccine for AHFV nor are there any therapeutic concepts to combat infections with these viruses. In this review, we summarize the current knowledge about pathogenesis, vector distribution, virus spread, and infection control. We aim to bring attention to the potential public health threats posed by KFDV and AHFV and highlight the urgent need for the development of effective countermeasures.

Immunization with recombinant fusion of LTB and linear epitope (40-62) of epsilon toxin elicits protective immune response against the epsilon toxin of Clostridium perfringens type D.

Epsilon toxin (Etx) produced by Clostridium perfringens types B and D, a major causative agent of enterotoxaemia causes significant economic losses to animal industry. Conventional vaccines against these pathogens generally employ formalin-inactivated culture supernatants. However, immunization with the culture supernatant and full length toxin subjects the animal to antigenic load and often have adverse effect due to incomplete inactivation of the toxins. In the present study, an epitope-based vaccine against Clostridium perfringens Etx, comprising 40-62 amino acid residues of the toxin in translational fusion with heat labile enterotoxin B subunit (LTB) of E. coli, was evaluated for its protective potential. The ability of the fusion protein rLTB.Etx40-62 to form pentamers and biologically active holotoxin with LTA of E. coli indicated that the LTB present in the fusion protein retained its biological activity. Antigenicity of both the components in the fusion protein was retained as anti-fusion protein antisera detected both the wild type Etx and LTB in Western blot analysis. Immunization of BALB/c mice with the fusion protein resulted in a significant increase in all isotypes, predominantly IgG1, IgG2a and IgG2b. Anti-fusion protein antisera neutralized the cytotoxicity of epsilon toxin both in vitro and in vivo. Thus, the results demonstrate the potential of rLTB.Etx40-62 as a candidate vaccine against C. perfringens.

Identification of glutamate ABC-Transporter component in Clostridium perfringens as a putative drug target.

Clostridium perfringens is an anaerobic pathogen known to cause vast number of diseases in mammals and birds. Various toxins and hydrolysing enzymes released by the organism are responsible for the necrosis of soft tissues. Due to serious safety issues associated with current vaccines against C. perfringens, there is a need for new drug or vaccine targets. C. perfringens is extremely dependent on its host for nutrition which can be targeted for vaccine development or drug design. Therefore, it is of interest to identify the unique transport systems used by C. perfringens involved in uptake of essential amino acids that are synthesized by the host, so that therapeutic agents can be designed to target the specific transport systems. Use of bioinformatics tools resulted in the identification of a protein component of the glutamate transport system that is not present in the host. Analysis of the conservation profile of the protein domain indicated it to be a glutamate binding protein which also stimulates the ATPase activity of ATP Binding Cassettes (ABC) transporters. Homology modelling of the protein showed two distinct lobes, which is a characteristic of substrate binding proteins. This suggests that the carboxylates of glutamate might be stabilized by electrostatic interactions with basic residues as is observed with other binding proteins. Hence, the homology model of this potential drug target can be employed for in silico docking studies by suitable inhibitors.