A low gastric pH mouse model to evaluate live attenuated bacterial vaccines.

The low pH of the stomach serves as a barrier to ingested microbes and must be overcome or bypassed when delivering live bacteria for vaccine or probiotic applications. Typically, the impact of stomach acidity on bacterial survival is evaluated in vitro, as there are no small animal models to evaluate these effects in vivo. To better understand the effect of this low pH barrier to live attenuated Salmonella vaccines, which are often very sensitive to low pH, we investigated the value of the histamine mouse model for this application. A low pH gastric compartment was transiently induced in mice by the injection of histamine. This resulted in a gastric compartment of approximately pH 1.5 that was capable of distinguishing between acid-sensitive and acid-resistant microbes. Survival of enteric microbes during gastric transit in this model directly correlated with their in vitro acid resistance. Because many Salmonella enterica serotype Typhi vaccine strains are sensitive to acid, we have been investigating systems to enhance the acid resistance of these bacteria. Using the histamine mouse model, we demonstrate that the in vivo survival of S. Typhi vaccine strains increased approximately 10-fold when they carried a sugar-inducible arginine decarboxylase system. We conclude that this model will be a useful for evaluating live bacterial preparations prior to clinical trials

Characterization of the Invasive, Multidrug Resistant Non-typhoidal Salmonella Strain D23580 in a Murine Model of Infection.

A distinct pathovar of Salmonella enterica serovar Typhimurium, ST313, has emerged in sub-Saharan Africa as a major cause of fatal bacteremia in young children and HIV-infected adults. D23580, a multidrug resistant clinical isolate of ST313, was previously shown to have undergone genome reduction in a manner that resembles that of the more human-restricted pathogen, Salmonella enterica serovar Typhi. It has since been shown through tissue distribution studies that D23580 is able to establish an invasive infection in chickens. However, it remains unclear whether ST313 can cause lethal disease in a non-human host following a natural course of infection. Herein we report that D23580 causes lethal and invasive disease in a murine model of infection following peroral challenge. The LD50 of D23580 in female BALB/c mice was 4.7 x 10(5) CFU. Tissue distribution studies performed 3 and 5 days post-infection confirmed that D23580 was able to more rapidly colonize the spleen, mesenteric lymph nodes and gall bladder in mice when compared to the well-characterized S. Typhimurium strain SL1344. D23580 exhibited enhanced resistance to acid stress relative to SL1344, which may lend towards increased capability to survive passage through the gastrointestinal tract as well as during its intracellular lifecycle. Interestingly, D23580 also displayed higher swimming motility relative to SL1344, S. Typhi strain Ty2, and the ST313 strain A130. Biochemical tests revealed that D23580 shares many similar metabolic features with SL1344, with several notable differences in the Voges-Proskauer and catalase tests, as well alterations in melibiose, and inositol utilization. These results represent the first full duration infection study using an ST313 strain following the entire natural course of disease progression, and serve as a benchmark for ongoing and future studies into the pathogenesis of D23580

Immunological Characterization of Plant-Based HIV-1 Gag/Dgp41 Virus-Like Particles.

It is widely anticipated that a prophylactic vaccine may be needed to control the HIV/AIDS epidemic worldwide. Despite over two decades of research, a vaccine against HIV-1 remains elusive, although a recent clinical trial has shown promising results. Recent studies have focused on highly conserved domains within HIV-1 such as the membrane proximal external region (MPER) of the envelope glycoprotein, gp41. MPER has been shown to play critical roles in mucosal transmission of HIV-1, though this peptide is poorly immunogenic on its own. Here we provide evidence that plant-produced HIV-1 enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of gp41 comprising the MPER, transmembrane, and cytoplasmic domains (Dgp41) provides an effective platform to display MPER for use as an HIV vaccine candidate. Prime-boost strategies combining systemic and mucosal priming with systemic boosting using two different vaccine candidates (VLPs and CTB-MPR--a fusion of MPER and the B-subunit of cholera toxin) were investigated in BALB/c mice. Serum antibody responses against both the Gag and gp41 antigens were elicited when systemically primed with VLPs. These responses could be recalled following systemic boosting with VLPs. In addition, mucosal priming with VLPs allowed for a boosting response against Gag and gp41 when boosted with either candidate. Importantly, the VLPs also induced Gag-specific CD4 and CD8 T-cell responses. This report on the immunogenicity of plant-based Gag/Dgp41 VLPs may represent an important milestone on the road towards a broadly efficacious and inexpensive subunit vaccine against HIV-1

Survival of strains cultured under non-acid resistance inducing conditions.

<p>Wild-type enteric strains were grown in LB medium to late-log phase under aerobic conditions. <b>(A)</b> Cells were challenged in EG medium (pH 3.0) containing 0.1% casamino acids. Survival during EG medium challenge was assayed hourly for 4 h by plating onto LB agar. Data shown are the mean and SEM of three independent experiments. <b>(B)</b> Mice were either fasted for 6 h (fasted mouse model) or fasted and low gastric pH was induced by histamine injection (histamine mouse model) and then inoculated with 10⁹ CFU of each strain. Sixty min after inoculation, mice were euthanized and the entire small intestine removed and homogenized. Strain survival was assayed by plating onto LB agar containing kanamycin. Data are expressed as the percent of initial inoculum recovered (% survival). The geometric mean and 95% confidence interval of two independent experiments (8 mice total) is depicted.</p

<b>Bacterial strains and plasmids used in this study.</b>

a<p>In genotype descriptions, the subscripted number refers to a composite deletion and insertion of the indicated gene. P, promoter; TT, T4 ip III transcription terminator; <i>ori</i>, origin of replication; Kan^R, kanamycin resistance; Str/Spc^R, streptomycin/spectinomycin resistance; Amp^R, ampicillin resistance.</p

Gastric pH following histamine injection.

<p>Following a 6 h fast, mice were injected at time 0 with 10/kg histamine. The pH of the gastric contents was monitored for 4 h post histamine injection. Data shown are the mean and standard error of the mean of at least five mice per time point.</p

Visualization of the bacterial inoculum in the gastrointestinal tract via light emission.

<p>Low gastric pH was induced in mice by histamine injection prior to inoculation with 1×10⁹ CFU of <i>S.</i> Typhi Ty2(pGEN-<i>luxCDABE</i>). At 30, 60 and 90 min post-inoculation, the gastrointestinal tract was removed and examined for the production of light by luciferase. A visible signal is equivalent to approximately 5×10⁵ CFU. Luminescence is reported as the number of photons detected per s per square cm. Images shown are representative results from groups of seven mice.</p

Effect of arginine decarboxylase on the gastric survival of <i>S.</i> Typhi.

<p>Pairs of attenuated <i>S.</i> Typhi strains differing only in their arginine decarboxylase locus were grown to stationary phase in EGA medium under aerobic conditions. Cells were combined in a 1:1 ratio in PBS containing 1 mM arginine. Low gastric pH was induced by histamine injection in mice fasted for 6 h. Mice were inoculated with 10⁹ CFU of each strain. Sixty min after inoculation, mice were euthanized and the entire small intestine removed and homogenized. Strain survival was assayed by plating onto LB agar containing kanamycin or streptomycin. Data shown are the competitive index of the two strains in each mouse with the geometric mean of two independent experiments (10 mice total) indicated as a solid line.</p

Correlation between in vitro and in vivo survival.

<p>The log₁₀ of the geometric mean number of CFU that survived in vitro challenge at pH 3.0 for 2 h was plotted against the log₁₀ of the geometric mean number of CFU recovered from the intestinal tissue in the <b>(A)</b> histamine mouse model or <b>(B)</b> fasted mouse model. Linear regression was performed on each data set and the r² value of the best-fit line is depicted for each model.</p

Tissue distribution of D23580 and SL1344 in mice following peroral infection.

<p>D23580 or SL1344 cultured to late log phase was administered perorally to 8-week-old female BALB/c mice at 10⁸ CFU per dose. Peyer’s patches (A), mesenteric lymph nodes (B), spleen (C) and gall bladder (D) were excised at 3 and 5 days after peroral infection. Five mice were euthanized at each time point per strain per experiment. The data represent an average of two trials and are presented as the mean of either the CFU per gram of tissue or per total organ (for mesenteric lymph nodes and gall bladder). The horizontal bar indicates geometric means and statistical comparisons were made using the Mann-Whitney test (** indicates p < 0.01; * indicates p < 0.05).</p