Metabolic classification of microbial genomes using functional probes

<p>Abstract</p> <p>Background</p> <p>Microorganisms able to grow under artificial culture conditions comprise only a small proportion of the biosphere's total microbial community. Until recently, scientists have been unable to perform thorough analyses of difficult-to-culture microorganisms due to limitations in sequencing technology. As modern techniques have dramatically increased sequencing rates and rapidly expanded the number of sequenced genomes, in addition to traditional taxonomic classifications which focus on the evolutionary relationships of organisms, classifications of the genomes based on alternative points of view may help advance our understanding of the delicate relationships of organisms.</p> <p>Results</p> <p>We have developed a proteome-based method for classifying microbial species. This classification method uses a set of probes comprising short, highly conserved amino acid sequences. For each genome, <it>in silico </it>translation is performed to obtained its proteome, based on which a probe-set frequency pattern is generated. Then, the probe-set frequency patterns are used to cluster the proteomes/genomes.</p> <p>Conclusions</p> <p>Features of the proposed method include a high running speed in challenge of a large number of genomes, and high applicability for classifying organisms with incomplete genome sequences. Moreover, the probe-set clustering method is sensitive to the metabolic phenotypic similarities/differences among species and is thus supposed potential for the classification or differentiation of closely-related organisms.</p

A Novel Prime and Boost Regimen of HIV Virus-Like Particles with TLR4 Adjuvant MPLA Induces Th1 Oriented Immune Responses against HIV

<div><p>HIV virus-like particles (VLPs) present the HIV envelope protein in its native conformation, providing an ideal vaccine antigen. To enhance the immunogenicity of the VLP vaccine, we sought to improve upon two components; the route of administration and the additional adjuvant. Using HIV VLPs, we evaluated sub-cheek as a novel route of vaccine administration when combined with other conventional routes of immunization. Of five combinations of distinct prime and boost sequences, which included sub-cheek, intranasal, and intradermal routes of administration, intranasal prime and sub-cheek boost (IN+SC) resulted in the highest HIV-specific IgG titers among the groups tested. Using the IN+SC regimen we tested the adjuvant VesiVax Conjugatable Adjuvant Lipid Vesicles (CALV) + monophosphoryl lipid A (MPLA) at MPLA concentrations of 0, 7.5, 12.5, and 25 μg/dose in combination with our VLPs. Mice that received 12.5 or 25 μg/dose MPLA had the highest concentrations of Env-specific IgG2c (20.7 and 18.4 μg/ml respectively), which represents a Th1 type of immune response in C57BL/6 mice. This was in sharp contrast to mice which received 0 or 7.5 μg MPLA adjuvant (6.05 and 5.68 μg/ml of IgG2c respectively). In contrast to IgG2c, MPLA had minor effects on Env-specific IgG1; therefore, 12.5 and 25 μg/dose of MPLA induced the optimal IgG1/IgG2c ratio of 1.3. Additionally, the percentage of germinal center B cells increased significantly from 15.4% in the control group to 31.9% in the CALV + 25 μg MPLA group. These mice also had significantly more IL-2 and less IL-4 Env-specific CD8⁺ T cells than controls, correlating with an increased percentage of Env-specific central memory CD4⁺ and CD8⁺ T cells. Our study shows the strong potential of IN+SC as an efficacious route of administration and the effectiveness of VLPs combined with MPLA adjuvant to induce Env specific Th1-oriented HIV-specific immune responses.</p></div

IgG1 and IgG2c sera titers against VLPs, HIV-1 Bal gp120 Env, and HIV-1 IIIB Pr55 Gag in mice immunized with VLPs.

<p>ELISA plates were coated with 2 μg/ml VLPs, gp120 Env, or Pr55 Gag. IgG1 quantitative ELISA of pooled mouse sera (duplicates repeated in triplicate) specific to (A) VLPs, (B) gp120 Env, and (C) Pr55 Gag. IgG2c quantitative ELISA of pooled mouse sera (duplicates repeated in triplicate) specific to (D) VLPs, (E) gp120 Env, and (F) Pr55 Gag. Ratio of mean concentration of IgG1 to mean concentration of IgG2c for (G) VLPs, (H) gp120 Env, and (I) Pr55 Gag. * <i>p</i><0.05 (1-way ANOVA and Tukey Post-Hoc tests versus control group). Error bars represent mean ± SEM (n = 3); # <i>p</i><0.05 (1-way ANOVA and Tukey Post-Hoc tests versus VLP-only and CALV(0)+VLP groups). & <i>p</i><0.05 (1-way ANOVA and Tukey Post-Hoc tests versus CALV(7.5)+VLP).</p

Specific sera IgG and mucosal IgA titers in mice treated with different prime-boost strategies.

<p>(A) Immunization regimen for evaluation of route of administration. ELISA plates were coated with 2 μg/ml of the indicated target protein. Sera from individual mice at time of sacrifice were diluted 1:100 and specific IgG titers determined against (B) VLPs, (C) gp120 Env, and (D) Pr55 Gag. (E) VLP-specific IgA in mucosal wash (1:20 dilution) from time of sacrifice. (F) IgG1 quantitative ELISA of pooled sera (duplicates repeated in triplicate) specific to VLPs (G) IgG2c quantitative ELISA of pooled sera (duplicates repeated in triplicate) specific to VLPs (H) Ratio of mean IgG1 to mean IgG2c of the indicated immunization groups. Error bars represent mean ± SEM (n = 5 for B, C, D, and E; n = 3 for F and G); * <i>p</i><0.05 (1-way ANOVA and Tukey Post-Hoc tests versus PBS). # <i>p</i><0.05 (1-way ANOVA and Tukey Post-Hoc tests versus all other groups). & <i>p</i><0.05 (1-way ANOVA and Tukey Post-Hoc tests versus SC + ID, SC + IN, and IN + ID).</p

Mucosal IgA from vaginal wash.

<p>ELISA plates were coated with 2 μg/ml of the indicated target protein. Individual vaginal washes were diluted 1:20 in PBS and tested for IgA against (A) VLPs, (B) gp120 Env, and (C) Pr55 Gag. Washes collected before immunization, after intranasal prime, and at time of sacrifice (final) of IgA against (D) VLPs, (E) gp120 Env, and (F) Pr55 Gag. Error bars represent mean ± SEM (n = 8); * <i>p</i><0.05 (2-way ANOVA and Bonferroni Post-Hoc tests versus control and CALV(0)+VLP groups).</p

Intracellular cytokine staining of mouse splenocytes stimulated with HIV-1 Consensus B Env and Gag peptide pools.

<p>(A) Representative dot plot of CD4⁺ T cells expressing IL-2 and IL-4 after stimulation with HIV-1 Consensus B Env and Gag peptide pools. (B) Representative dot plot of CD4⁺ T cells expressing TNF-α and IFN-γ after stimulation with HIV-1 Consensus B Env and Gag peptide pools. (C) Relative levels of IL-2, IL-4, TNF-α, and IFN-γ in CD4⁺ T cells after stimulation with 2 μg/ml Gag peptide pool standardized to PBS control. (D) Relative levels of IL-2, IL-4, TNF-α, and IFN-γ in CD4⁺ T cells after stimulation with 2 μg/ml Env peptide pool standardized to PBS control. (E) Representative dot plot of CD8⁺ T cells expressing IL-2 and IL-4 after stimulation with HIV-1 Consensus B Env and Gag peptide pools. (F) Representative dot plot of CD8⁺ T cells expressing TNF-α and IFN-γ in CD8⁺ T cells after stimulation with HIV-1 Consensus B Env and Gag peptide pools. (G) Relative levels of IL-2, IL-4, TNF-α, and IFN-γ after stimulation with 2 μg/ml Gag peptide pool standardized to PBS control. (H) Relative levels of IL-2, IL-4, TNF-α, and IFN-γ in CD8⁺ T cells after stimulation with 2 μg/ml Env peptide pool standardized to Control. Error bars represent mean ± SEM (n = 6); * <i>p</i><0.05 (2-Way ANOVA and Bonferroni Post-hoc tests versus control group). # <i>p</i><0.05 (2-Way ANOVA and Bonferroni Post-hoc tests versus VLP Only and CALV(0)+VLP groups).</p

Effector (CD44^hi, CD62L^-) and central (CD44^hi, CD62L⁺) memory T cell subsets in mouse CD3e⁺ and either CD4⁺ or CD8a⁺ splenocytes.

<p>(A) Representative dot plot of effector and central memory of CD4⁺ and CD8a⁺ T cells. (B) Percentage of CD4⁺ cells expressing high levels of CD44 and no detectable levels of CD62L in each immunization group. (C) Percentage of CD4⁺ cells expressing high levels of CD44 and CD62L. (D) Percentage of CD8a⁺ cells expressing high levels of CD44 and no detectable levels of CD62L in each immunization group. (E) Percentage of CD8a⁺ cells expressing high levels of CD44⁺ and CD62L⁺ staining. Error bars represent mean ± SEM (n = 6); * indicates p<0.05 (Student’s unpaired t-test when compared to control group).</p