Development of a Multivalent Subunit Vaccine against Tularemia Using Tobacco Mosaic Virus (TMV) Based Delivery System

Francisella tularensisis a facultative intracellular pathogen, and is the causative agent of a fatal human disease known as tularemia. F. tularensis is classified as a Category A Biothreat agent by the CDC based on its use in bioweapon programs by several countries in the past and its potential to be used as an agent of bioterrorism. No licensed vaccine is currently available for prevention of tularemia. In this study, we used a novel approach for development of a multivalent subunit vaccine against tularemia by using an efficient tobacco mosaic virus (TMV) based delivery platform. The multivalent subunit vaccine was formulated to contain a combination of F. tularensis protective antigens: OmpA-like protein (OmpA), chaperone protein DnaK and lipoprotein Tul4 from the highly virulent F. tularensisSchuS4 strain. Two different vaccine formulations and immunization schedules were used. The immunized mice were challenged with lethal (10xLD100) doses of F. tularensisLVS on day 28 of the primary immunization and observed daily for morbidity and mortality. Results from this study demonstrate that TMV can be used as a carrier for effective delivery of multiple F. tularensisantigens. TMV-conjugate vaccine formulations are safe and multiple doses can be administered without causing any adverse reactions in immunized mice. Immunization with TMV-conjugated F. tularensisproteins induced a strong humoral immune response and protected mice against respiratory challenges with very high doses of F. tularensis LVS. This study provides a proof-of-concept that TMV can serve as a suitable platform for simultaneous delivery of multiple protective antigens of F. tularensis. Refinement of vaccine formulations coupled with TMV-targeting strategies developed in this study will provide a platform for development of an effective tularemia subunit vaccine as well as a vaccination approach that may broadly be applicable to many other bacterial pathogens

Preclinical testing of a vaccine candidate against tularemia.

Tularemia is caused by a gram-negative, intracellular bacterial pathogen, Francisella tularensis (Ft). The history weaponization of Ft in the past has elevated concerns that it could be used as a bioweapon or an agent of bioterrorism. Since the discovery of Ft, three broad approaches adopted for tularemia vaccine development have included inactivated, live attenuated, or subunit vaccines. Shortcomings in each of these approaches have hampered the development of a suitable vaccine for prevention of tularemia. Recently, we reported an oxidant sensitive mutant of Ft LVS in putative EmrA1 (FTL_0687) secretion protein. The emrA1 mutant is highly sensitive to oxidants, attenuated for intramacrophage growth and virulence in mice. We reported that EmrA1 contributes to oxidant resistance by affecting the secretion of antioxidant enzymes SodB and KatG. This study investigated the vaccine potential of the emrA1 mutant in prevention of respiratory tularemia caused by Ft LVS and the virulent SchuS4 strain in C57BL/6 mice. We report that emrA1 mutant is safe and can be used at an intranasal (i. n.) immunization dose as high as 1x106 CFU without causing any adverse effects in immunized mice. The emrA1 mutant is cleared by vaccinated mice by day 14-21 post-immunization, induces minimal histopathological lesions in lungs, liver and spleen and a strong humoral immune response. The emrA1 mutant vaccinated mice are protected against 1000-10,000LD100 doses of i.n. Ft LVS challenge. Such a high degree of protection has not been reported earlier against respiratory challenge with Ft LVS using a single immunization dose with an attenuated mutant generated on Ft LVS background. The emrA1 mutant also provides partial protection against i.n. challenge with virulent Ft SchuS4 strain in vaccinated C57BL/6 mice. Collectively, our results further support the notion that antioxidants of Ft may serve as potential targets for development of effective vaccines for prevention of tularemia

Antibody Responses in Mice Immunized with TMV-Monoconjugate and TMV-Multiconjugate Vaccines using Schedule I of Immunization.

<p><i>Francisella</i> specific total IgG, IgG1, IgG2a and IgG2b levels on day 28 in serum samples of C57BL/6 mice immunized with TMV-monoconjugate and TMV-multiconjugate vaccine using Schedule I were determined using an ELISA. Serum samples obtained from naïve mice or those inoculated with TMV alone were used as controls. The data are represented as Mean ±S.D. of absorbance values measured at 450nm. Table shows comparison of antibody titers between groups of mice vaccinated with these vaccine formulations.</p

Immunization of Mice with TMV-Multiconjugate Vaccine Induces Antibody Responses Capable of Recognizing both Native and Recombinant OmpA, DnaK and Tul4 Proteins.

<p><b>(A)</b> Serum collected on day 28 post-immunization from C57BL/6 mice immunized with TMV-multiconjugate vaccine (Schedule II) was pooled (n = 4) and blotted against <i>F</i>. <i>tularensis</i> LVS and SchuS4 lysates. <b>(B)</b> Pooled serum from C57BL/6 mice (n = 4) immunized either with TMV-multiconjugate vaccine, or 100 CFU of <i>F</i>. <i>tularensis</i> LVS were collected on day 28 post immunization and blotted against purified recombinant OmpA, DnaK and Tul4 proteins. Sera from mice inoculated with TMV alone were used as controls.</p

Immunization Schedules I and II.

<p><b>(A)</b> C57BL/6 mice were immunized intranasally (i.n.) either with TMV-monoconjugate (60 μg/mouse) or TMV-multiconjugate vaccine formulations (20 μg each of OmpA-TMV; DnaK-TMV and Tul4-TMV conjugates. Total 60 μg/mouse) and booster vaccinations were administered i.n. using dosages similar to those for primary immunization on days 7 and 14 of the post-primary immunization (Schedule I). <b>(B)</b> Alternatively, mice were administered TMV-multiconjugate vaccines with booster immunizations i.n. on day 5 and 14 and subcutaneously (s.c.) on days 7 and 14 post-primary immunization (Schedule II). The dosages used were similar to those described for TMV-multiconjugate vaccine in <b>A</b>. Mice inoculated with TMV (30 μg/mouse) in a manner similar to the vaccinated groups were kept as controls.</p

A human-serum-free medium can induce more infectious P. falciparum gametocytes than a conventional human-serum-containing medium

Abstract Malaria remains a global health problem, and the standard membrane feeding assay (SMFA) is a key functional assay for development of new interventions to stop malaria transmission from human to mosquito. For SMFA, media with ~ 10% of human serum has been used for infectious gametocyte cultures, however, there are multiple challenges to obtain a suitable human serum. Here we show a human-serum-free culture medium (HSF), which was a mixture of two stem cell culture media and AlbuMAX, supported infectious gametocyte growth. Moreover, the HSF-induced gametocytes elicited significantly higher numbers of oocysts compared to gametocytes cultured with conventional human serum medium (Conv). While some caution is required when comparing percent transmission reducing activity data generated from HSF-SMFA and Conv-SMFA, the HSF method can facilitate the establishment of gametocyte cultures or SMFA by bypassing the need for human serum. Thus, this study will support future development of P. falciparum transmission-blocking interventions

Expression and Purification of Recombinant DnaK, OmpA and Tul4 Proteins of <i>F</i>. <i>tularensis</i> SchuS4.

<p>Purification of recombinant OmpA, DnaK and Tul4 proteins of <i>F</i>. <i>tularensis</i> SchuS4 proteins was confirmed by SDS-PAGE and western blot analysis using anti-His antibodies.</p

OmpA, DnaK and Tul4 Specific Antibody Responses in Mice Immunized with TMV-Monoconjugate and TMV-Multiconjugate Vaccines using Schedule I of Immunization.

<p><i>F</i>. <i>tularensis</i> SchuS4 recombinant proteins OmpA, DnaK and Tul4 specific IgG antibody levels on day 28 in serum samples of C57BL/6 mice immunized with TMV-monoconjugate and TMV-multiconjugate vaccine using Schedule I were determined by ELISA. Serum samples obtained from naïve mice or those inoculated with TMV alone were used as controls. The data are represented as Mean ±S.D. of absorbance values measured at 450nm. Table shows comparison of antibody titers between groups of mice vaccinated with these vaccine formulations.</p

OmpA, DnaK and Tul4 Specific Antibody Responses in Mice Immunized with TMV-Multiconjugate Vaccines using Schedule I and II of Immunization.

<p><i>F</i>. <i>tularensis</i> SchuS4 recombinant proteins OmpA, DnaK and Tul4 specific IgG, antibody levels on day 28 in serum samples of C57BL/6 mice immunized with TMV-multiconjugate vaccine using Schedule II were determined by ELISA. The plates were coated with recombinant <i>F</i>. <i>tularensis</i> SchuS4 proteins. Serum samples obtained from naïve mice or those inoculated with TMV alone were used as controls. The data are represented as Mean ±S.D. of absorbance values measured at 450nm. The comparisons are shown with the data obtained from mice immunized with TMV-multiconjugate vaccine using schedule I (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130858#pone.0130858.g007" target="_blank">Fig 7</a>). Table shows comparison of antibody titers between groups of mice vaccinated with Schedule I and II vaccination regimens.</p

Vaccine Formulations.

<p>Two different vaccine formulations were used. In the first vaccine formulation all three recombinant proteins OmpA, DnaK and Tul4 were conjugated to a single TMV virion (TMV-monoconjugate vaccine). The second vaccine formulation contained each recombinant protein of <i>F</i>. <i>tularensis</i> conjugated individually to TMV and then mixed in equal concentrations to generate a TMV-multiconjugate vaccine.</p