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. 2011 Jun 24;29(29-30):4839-47.
doi: 10.1016/j.vaccine.2011.04.066. Epub 2011 May 5.

Extended safety and efficacy studies of a live attenuated double leucine and pantothenate auxotroph of Mycobacterium tuberculosis as a vaccine candidate

Samantha L Sampson  1 , Keith G Mansfield, Angela Carville, D Mitchell Magee, Teresa Quitugua, Elizabeth W Howerth, Barry R Bloom, Mary K Hondalus
Affiliations

Extended safety and efficacy studies of a live attenuated double leucine and pantothenate auxotroph of Mycobacterium tuberculosis as a vaccine candidate

Samantha L Sampson et al. Vaccine. .

Abstract

We have previously described the development of a live, fully attenuated Mycobacterium tuberculosis (Mtb) vaccine candidate strain with two independent attenuating auxotrophic mutations in leucine and pantothenate biosynthesis. In the present work, those studies have been extended to include testing for protective efficacy in a long-term guinea pig survival model and safety testing in the highly tuberculosis susceptible Rhesus macaque. To model the safety of the ΔleuD ΔpanCD strain in HIV-infected human populations, a Simian immunodeficiency virus (SIV)-infected Rhesus macaque group was included. Immunization with the non-replicating ΔleuD ΔpanCD conferred long-term protection against challenge with virulent M. tuberculosis equivalent to that afforded by BCG as measured by guinea pig survival. In safety studies, clinical, hematological and bacteriological monitoring of both SIV-positive and SIV-negative Rhesus macaques immunized with ΔleuD ΔpanCD, revealed no vaccine-associated adverse effects. The results support the further development of the ΔleuD ΔpanCD strain as a viable tuberculosis (TB) vaccine candidate.

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Figures

FIG. 1
FIG. 1. Mycobacterial antigen-specific DTH Response in guinea pigs
Guinea pigs were immunized with 5.3 × 106 CFU BCG-P or 3.1 × 105 CFU ΔleuD ΔpanCD or remained unimmunized. Seven weeks after immunization, DTH responses were determined by intradermal injection of 40 TU PPD or 1 ug rESAT-6, followed by measurement of the diameter of induration 24h later. Mean induration diameter (±SD) elicited by PPD injection was 16.2 ± 1.8 mm for BCG-P-immunized guinea pigs and 9.0 ± 3.7 mm for ΔleuD ΔpanCD–immunized animals. In response to rESAT-6, the mean induration in ΔleuD ΔpanCD-immunized animals was 7.8 ± 1.1 mm, whereas BCG-P immunized animals did not show any positive responses. No response to either antigen preparation was observed in naïve animals (n= 5 animals per group). *, p<0.01, and **, p<0.001, as determined by one-way ANOVA applying Bonferroni s post-test.
FIG. 2
FIG. 2. Protection of guinea pigs against aerosol challenge with M. tuberculosis
Seven weeks following immunization with BCG-P or ΔleuD ΔpanCD, immunized animals and naive controls were challenged with 33 CFU M. tuberculosis. Survival of infected guinea pigs was followed for up to 452 days post-challenge. Naive animals succumbed to challenge with a median survival time (MST) of 154 days, whereas ΔleuD ΔpanCD- and BCG-immunized guinea pigs survived significantly longer (MST 446 d and 452 d, respectively). *, p<0.0001 as determined by Logrank test comparisons of survival curves, n=11 animals per experimental group.
FIG.3
FIG.3. SIV RNA Viral load in SIV-infected, ΔleuD ΔpanCD-immunized Rhesus macaques
A group of 4 Rhesus macaques were infected with SIVmac251 (time indicated by arrow on X-axis), prior to immunization with ΔleuD ΔpanCD 2 weeks later (Week 0 on graph). SIV viral RNA was extracted from plasma at multiple time points following infection, and genomic copy equivalents (copy eq.) were enumerated using quantitative real-time RT-PCR. Individual macaque identification numbers are indicated on graph.
FIG. 4
FIG. 4. Clinical evaluation of ΔleuD ΔpanCD-immunized SIV-positive and SIV-negative Rhesus macaques
SIVmac251-positive (filled black symbols) and SIVmac251-negative (open grey symbols) macaques were immunized with ΔleuD ΔpanCD and monitored for 1 year post-immunization. (A) Body weight over time. (B) Erythrocyte sedimentation rate (ESR) over time. Arrows on X-axes indicate time of SIV administration. Solid lines highlight 2 SIV-infected macaques Mm503-02 and Mm466-99 euthanized due to excessive weight loss at 9 weeks and 52 weeks post-immunization, respectively.
FIG. 5
FIG. 5. Mycobacterial antigen-specific immune responses in ΔleuD ΔpanCD-immunized SIV-positive and SIV-negative Rhesus macaques
Immune responses to mycobacterial antigens were measured in SIVmac251-positive (filled black symbols) and SIVmac251-negative (open grey symbols) immunized with ΔleuD ΔpanCD. (A) Delayed type hypersensitivity (DTH) responses were scored at 24 h following injection of Old Mammalian Tuberculin (MOT) into the eyelid. (B) IFN-γ production in response to stimulation of whole blood with bovine PPD was measured using the Primagam ELISA- based kit. Y-axis values are expressed as OD450 readings for Bovine-PPD stimulated vs nil antigen control stimulated wells, with values above 0.1 considered to be positive. (C) Numbers of IFN-γ-producing cells in response to stimulation of PBMCs with PPD were measured using the ELISPOT. Arrows on X-axes indicate time of SIV administration.
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