Evaluation of a temperature-restricted, mucosal tuberculosis vaccine in guinea pigs

Abstract

Tuberculosis (TB) is currently the leading cause of death in humans by a single infectious agent, Mycobacterium tuberculosis. The Bacillus Calmette-Guérin (BCG) vaccine prevents pulmonary TB with variable efficacy, but can cause life-threatening systemic infection in HIV-infected infants. In this study, TBvac85, a derivative of Mycobacterium shottsii expressing M. tuberculosis Antigen 85B, was examined as a safer alternative to BCG. Intranasal vaccination of guinea pigs with TBvac85, a naturally temperature-restricted species, resulted in serum Ag85B-specific IgG antibodies. Delivery of the vaccine by this route also induced protection equivalent to intradermal BCG based on organ bacterial burdens and lung pathology six weeks after aerosol challenge with M. tuberculosis strain Erdman. These results support the potential of TBvac85 as the basis of an effective TB vaccine. Next-generation derivatives expressing multiple M. tuberculosis immunogens are in development.

Keywords: Intranasal; Mycobacterium; TBvac85; Tuberculosis; Vaccine.

Fig. 1.. Confirmation of TBvac85 by PCR and immunoblot analyses.

A) Candidates of M. shottsii M175 transformed with plasmid pNBV1-PLCDS30 were screened by PCR for the presence of a 575-bp region of M. tuberculosis fbpB. PCR with pNBVI-PLSCDS30 DNA template served as positive control. B) Cell lysates of M175 and TBvac85 were fractionated (CF: culture filtrate, IF: insoluble fraction, L: cell lysate, SF: soluble fraction) and equal amounts of protein were resolved by 4–12% SDS-PAGE, and examined by western immunoblot using polyclonal anti-Ag85 protein complex antibodies. Sizes of PrecisionPlus Protein Kaleidoscope Prestained Standard bands (MW stds.) are indicated.

Fig. 2.. Guinea pig immune responses and protection by different TBvac85 regimens.

Guinea pigs were sham vaccinated or administered 3 weekly doses of TBvac85 by one of the following regimens: low-dosing by Aerovax (85av-L), medium-dosing by Aerovax (85av-M), medium dosing by Aerogen (85ag-M), high dosing by intranasal instillation (85ii-H). Sera collected from 10 animals per group prior to vaccination (pre) and on Day 40 after prime vaccination (post) were diluted 100-fold and examined by ELISA for antibody responses to Antigen 85B. Results for each vaccine group were normalized to unvaccinated animals. B) On day 50 post-prime vaccination, tuberculin PPD skin tests were performed on 10 animals/group. Induration diameters were measured 24 h later. C) On day 75 post-prime, animals were aerosol-challenged with M, tuberculosis Erdman (10–20 CFU). At 6 weeks post-challenge, animals were euthanized. The lungs from five animals/group were examined for presence of granulomas (white foci) on the surface. Representative samples from each group are shown. D) Histopathology was performed on half of the lung tissues. E) The remaining lung tissues were homogenized and plated on 7H10 medium to enumerate M. tuberculosis CFUs. Data from each group of five animals are expressed logarithmically. The mean log₁₀ CFU standard deviation is indicated (error bars). Mean values that were significantly different between vaccine groups are indicated (*p = 0.01–0.05, **p = 0.001–0.01, ***p = 0.0001–0.001, ****p < 0.0001).

Fig. 3.. Immunological and pathological assessments of TBvac85 dosing in guinea pigs.

Guinea pig groups (N = 5) were intranasally sham-vaccinated (sham), intradermally BCG-vaccinated once (BCGid-1), or immunized with TBvac85 1–3 times by intranasal instillation (85ii-1, 85ii-2, or 85ii-3), or with TBvac85 3 times by Aerogen 5 μm nebulizer (85ag-3). A) Anti-M. tuberculosis Ag85B antibody titers measured by ELISA following 100-fold dilution of sera obtained prior to vaccination (pre) and 4 weeks after the last boosts (post). B) Five and ½ weeks later, the flanks of guinea pigs were shaved and PBS and 2 μg PPD were intradermally injected in separate flanks with a tuberculin syringe; induration was measured 24 h later. Data for PPD responses are shown. No induration was observed at PBS injection sites. C) Three days later, guinea pigs were aerosol infected with 20–40 CFU of M. tuberculosis strain Erdman. Burdens of M. tuberculosis bacteria in the lungs were quantified by CFU counting 5–6 weeks after infection. Data (n = five animals for each group) are expressed logarithmically as the mean log₁₀ CFU standard deviation (SD) (error bars). Significant mean value difference between the vaccine groups are indicated (*p < 0.05; **p < 0.001–0.01, ***p = 0.0001–0.001, and ****p < 0.0001). D) Gross lung pathology of formalin-fixed guinea pig lungs 5–6 weeks post-M. tuberculosis challenge. Granulomas appear as white nodules. Shown are representative images from a total of five per group. E) Histopathology (hematoxylin and eosin staining) of 5 μm-thick slices of lung tissues from panel D. Shown are representative results from a total of five per group.