Rhesus immune responses to SIV Gag expressed by recombinant BCG vectors are independent from pre-existing mycobacterial immunity.

BACKGROUND: A recombinant Mycobacterium bovis BCG (rBCG) vector expressing HIV transgenes is an attractive candidate as a dual vaccine against HIV and TB. However, pre-existing immune responses to mycobacteria may influence immune responses to rBCG. We analyzed data from a rhesus rBCG trial to determine the effect of pre-existing mycobacterial immune responses on the vaccine-induced responses to the vector and expressed transgene. METHODS: Indian-origin rhesus macaques were primed with rBCG expressing simian immunodeficiency virus (SIV) Gag and boosted with attenuated vaccinia NYVAC gag-pol. Mycobacteria responses were measured by Mycobacterium tuberculosis (Mtb) purified protein derivative (PPD) interferon-γ ELISpot and Mtb whole cell lysate (WCL) ELISA. SIV Gag responses were measured by SIV Gag ELISpot and by p11C tetramer binding. RESULTS: Baseline Mtb PPD ELISpot responses and Mtb WCL antibody responses in rhesus macaques overlapped those in human populations. Cellular and antibody responses boosted sharply 4 weeks after rBCG vaccination. Mtb WCL antibody titers at 4 weeks correlated with baseline titers. Primates vaccinated with rBCG developed strong SIV Gag ELISpot and p11C tetramer responses after rBCG prime and NYVAC boost. There were no correlations between the pre-existing mycobacterial immune responses and the SIV Gag T cell responses after vaccination. CONCLUSIONS: Rhesus immune responses to SIV Gag expressed by rBCG vectors were independent from pre-existing anti-mycobacterial immunity. Rhesus macaques may serve as a surrogate for investigations of pre-existing anti-mycobacterial immunity in humans.

The human antibody response to the surface of Mycobacterium tuberculosis.

BACKGROUND: Vaccine-induced human antibodies to surface components of Haemophilus influenzae and Streptococcus pneumonia are correlated with protection. Monoclonal antibodies to surface components of Mycobacterium tuberculosis are also protective in animal models. We have characterized human antibodies that bind to the surface of live M. tuberculosis. METHODS: Plasma from humans with latent tuberculosis (TB) infection (n = 23), active TB disease (n = 40), and uninfected controls (n = 9) were assayed by ELISA for reactivity to the live M. tuberculosis surface and to inactivated M. tuberculosis fractions (whole cell lysate, lipoarabinomannan, cell wall, and secreted proteins). RESULTS: When compared to uninfected controls, patients with active TB disease had higher antibody titers to the surface of live M. tuberculosis (Δ = 0.72 log10), whole cell lysate (Δ = 0.82 log10), and secreted proteins (Δ = 0.62 log10), though there was substantial overlap between the two groups. Individuals with active disease had higher relative IgG avidity (Δ = 1.4 to 2.6) to all inactivated fractions. Surprisingly, the relative IgG avidity to the live M. tuberculosis surface was lower in the active disease group than in uninfected controls (Δ =  -1.53, p = 0.004). Patients with active disease had higher IgG than IgM titers for all inactivated fractions (ratios, 2.8 to 10.1), but equal IgG and IgM titers to the live M. tuberculosis surface (ratio, 1.1). Higher antibody titers to the M. tuberculosis surface were observed in active disease patients who were BCG-vaccinated (Δ = 0.55 log10, p = 0.008), foreign-born (Δ = 0.61 log10, p = 0.004), or HIV-seronegative (Δ = 0.60 log10, p = 0.04). Higher relative IgG avidity scores to the M. tuberculosis surface were also observed in active disease patients who were BCG-vaccinated (Δ = 1.12, p < 0.001) and foreign-born (Δ = 0.87, p = 0.01). CONCLUSIONS/SIGNIFICANCE: Humans with active TB disease produce antibodies to the surface of M. tuberculosis with low avidity and with a low IgG/IgM ratio. Highly-avid IgG antibodies to the M. tuberculosis surface may be an appropriate target for future TB vaccines.

Ultra-low dose of Mycobacterium tuberculosis aerosol creates partial infection in mice.

A murine low dose (LD) aerosol model is commonly used to test tuberculosis vaccines. Doses of 50-400 CFU (24h lung CFU) infect 100% of exposed mice. The LD model measures progression from infection to disease based on organ CFU at defined time points. To mimic natural exposure, we exposed mice to an ultra-low dose (ULD) aerosol. We estimated the presented dose by sampling the aerosol. Female C57BL/6 mice were exposed to Mycobacterium tuberculosis H37Rv aerosol at 1.0, 1.1, 1.6, 5.4, and 11 CFU presented dose, infecting 27%, 36%, 36%, 100%, and 95% of mice, respectively. These data are compatible with a stochastic infection event (Poisson distribution, weighted R(2)=0.97) or with a dose-response relationship (sigmoid distribution, weighted R(2)=0.97). Based on the later assumption, the ID50 was 1.6CFU presented dose (95% confidence interval, 1.2-2.1). We compared organ CFU after ULD and LD aerosols (5.4 vs. 395CFU presented dose). Lung burden was 30-fold lower in the ULD model at 4 weeks (3.4 vs. 4.8 logs, p<0.001) and 18 weeks (≤3.6 vs. 5.0 logs, p=0.01). Mice exposed to ULD aerosols as compared to LD aerosols had greater within-group CFU variability. Exposure to ULD aerosols leads to infection in a subset of mice, and to persistently low organ CFU. The ULD aerosol model may resemble human pulmonary tuberculosis more closely than the standard LD model, and may be used to identify host or bacterial factors that modulate the initial infection event.

Sampling port for real-time analysis of bioaerosol in whole body exposure system for animal aerosol model development.

INTRODUCTION: Multiple factors influence the viability of aerosolized bacteria. The delivery of aerosols is affected by chamber conditions (humidity, temperature, and pressure) and bioaerosol characteristics (particle number, particle size distribution, and viable aerosol concentration). Measurement of viable aerosol concentration and particle size is essential to optimize viability and lung delivery. The Madison chamber is widely used to expose small animals to infectious aerosols. METHODS: A multiplex sampling port was added to the Madison chamber to measure the chamber conditions and bioaerosol characteristics. Aerosols of three pathogens (Bacillus anthracis, Yersinia pestis, and Mycobacterium tuberculosis) were generated under constant conditions and their bioaerosol characteristics were analyzed. Airborne microbes were captured using an impinger or BioSampler. The particle size distribution of airborne microbes was determined using an aerodynamic particle sizer (APS). Viable aerosol concentration, spray factor (viable aerosol concentration/inoculum concentration), and dose presented to the mouse were calculated. Dose retention efficiency and viable aerosol retention rate were calculated from the sampler titers to determine the efficiency of microbe retention in lungs of mice. RESULTS: B. anthracis, Y. pestis, and M. tuberculosis aerosols were sampled through the port. The count mean aerodynamic sizes were 0.98, 0.77, and 0.78 µm with geometric standard deviations of 1.60, 1.90, and 2.37, and viable aerosol concentrations in the chamber were 211, 57, and 1 colony-forming unit (CFU)/mL, respectively. Based on the aerosol concentrations, the doses presented to mice for the three pathogens were 2.5e5, 2.2e4 and 464 CFU. DISCUSSION: Using the multiplex sampling port we determined whether the animals were challenged with an optimum bioaerosol based on dose presented and respirable particle size.

Study of the role of CCR5 in a mouse model of intranasal challenge with Yersinia pestis.

CCR5 is a chemokine receptor used by HIV-1 to enter cells and has recently been found to act as a pathogen associated molecule pattern receptor. Current positive selection for the high frequency of a CCR5-Delta32 allele in humans has been attributed to resistance to HIV, smallpox, and plague infections. Using an intranasal mouse model of Y. pestis infection, we have found that lack of CCR5 does not enhance host resistance to Y. pestis infection and that CCR5-mediated responses might have a protective role. CCR5-/- mice exhibited higher levels of circulating RANTES and MIP-1alpha than those exhibited by wild-type mice at the baseline and throughout the course of Y. pestis infection. High levels of RANTES and MIP-1alpha, which are CCR5 ligands that mediate Natural Killer cell migration, may reflect compensation for the absence of CCR5 signaling.