Myeloid Cell Crosstalk Regulates the Efficacy of the DNA/ALVAC/gp120 HIV Vaccine Candidate

Vaccination with DNA-SIV + ALVAC-SIV + gp120 alum results in inflammasome activation, high levels of IL-1β production, emergency myelopoiesis, and the egress of CXCR4+ CD14+ pre-monocytes from bone marrow. Previously we have shown that this vaccine-induced innate monocyte memory is associated with decreased risk of SIVmac251 acquisition. Because IL-1β also promotes the propagation of monocyte-derived suppressor (M-MDSC)-like cells, here we extended our analysis to this negative regulator subset, characterizing its levels and functions in macaques. Interestingly, we found that DNA prime engages M-MDSC-like cells and their levels are positively associated with the frequency of CD14+ classical monocytes, and negatively with the levels of CD16+ monocytes, correlates of decreased and increased risk of SIV acquisition, respectively. Accordingly, M-MDSC frequency, arginase activity, and NO were all associated with decrease of CD8 T cells responses and worse vaccination outcome. DNA vaccination thus induces innate immunity by engaging three subsets of myeloid cells, M-MDSCs, CD14+ innate monocyte memory, and CD16+ monocytes all playing different role in protection. The full characterization of the immunological space created by myeloid cell crosstalk will likely provide clues to improve the efficacy of HIV vaccine candidates

In vivo treatment with insulin-like growth factor 1 reduces CCR5 expression on vaccine-induced activated CD4+ T-cells

<p>Dataset of the publication "In vivo treatment with insulin-like growth factor 1 reduces CCR5 expression on vaccine-induced activated CD4+ T-cells" by Bissa et al. on the journal Vaccines. </p><p>Each folder contains the original files reporting the data used to generate the manuscript.</p><p>For flowcytometry based assays the Flow panel is included in the folders. </p><p>For ELISA based assays the schemes of the plates are included in the folders. </p><p>The excel table "Bissa et al._Vaccines_2023_Animal IDs and viral acquisition" reports the IDs and grouping of the animals together with their viral acquisition</p><p>The excel table "Bissa et al._Vaccines_2023_Master table" reports each data used to generate the figures and supplemental materials included in the publication </p&gt

Construction and characterisation of a recombinant fowlpox virus that expresses the human papilloma virus L1 protein

<p>Abstract</p> <p>Background</p> <p>Human papilloma virus (HPV)-16 is the most prevalent high-risk mucosal genotype. Virus-like-particle (VLP)-based immunogens developed recently have proven to be successful as prophylactic HPV vaccines, but are still too expensive for developing countries. Although vaccinia viruses expressing the HPV-16 L1 protein (HPV-L1) have been studied, fowlpox-based recombinants represent efficient and safer vectors for immunocompromised hosts due to their ability to elicit a complete immune response and their natural host-range restriction to avian species.</p> <p>Methods</p> <p>A new fowlpox virus recombinant encoding HPV-L1 (FP_L1) was engineered and evaluated for the correct expression of HPV-L1 <it>in vitro</it>, using RT-PCR, immunoprecipitation, Western blotting, electron microscopy, immunofluorescence, and real-time PCR assays.</p> <p>Results</p> <p>The FP_L1recombinant correctly expresses HPV-L1 in mammalian cells, which are non-permissive for the replication of this vector.</p> <p>Conclusion</p> <p>This FP_L1recombinant represents an appropriate immunogen for expression of HPV-L1 in human cells. The final aim is to develop a safe, immunogenic, and less expensive prophylactic vaccine against HPV.</p

Identification of antibiotic-resistant Escherichia coli isolated from a municipal wastewater treatment plant

The emergence and diffusion of antibiotic-resistant bacteria has been a major public health problem for many years now. In this study, antibiotic-resistance of coliforms and Escherichia coli were investigated after their isolation from samples collected in a municipal wastewater treatment plant in the Milan area (Italy) along different points of the treatment sequence: inflow to biological treatment; outflow from biological treatment following rapid sand filtration; and outflow from peracetic acid disinfection. The presence of E. coli that showed resistance to ampicillin (AMP) and chloramphenicol (CAF), used as representative antibiotics for the efficacy against Gram-positive and Gram-negative bacteria, was evaluated. After determining E. coli survival using increasing AMP and CAF concentrations, specific single- resistant (AMPR or CAFR) and double-resistant (AMPR/CAFR) strains were identified among E. coli colonies, through amplification of the b-lactamase Tem-1 (bla) and acetyl-transferase catA1 (cat) gene sequences. While a limited number of CAFR bacteria was observed, most AMPR colonies showed the specific resistance genes to both antibiotics, which was mainly due to the presence of the bla gene sequence. The peracetic acid, used as disinfection agent, showed to be very effective in reducing bacteria at the negligible levels of less than 10 CFU/100 mL, compatible with those admitted for the irrigation use of treated waters

CIITA subcellular localisation using confocal microscopy.

<p>The 293T cells were infected with different FP recombinants and analysed using an anti-SIV polyclonal antibody, and anti-HLA-DR and anti-<i>CIITA</i> mAbs. Using anti-SIV serum, the Gp and Env proteins were equally well expressed by FP<i>gp</i> or FP<i>env</i> (C1, E1), as well as by FP<i>gp</i>+FP<i>CIITA</i>_H6 (D1) or FP<i>env</i>+FP<i>CIITA</i>_H6 (F1). After infection with FP<i>CIITA</i>_H6, both HLA-DR and CIITA were expressed (B2, B3). Also, HLA-DR is expressed in cells co-infected with FP<i>gp</i>+FP<i>CIITA</i>_H6 (D2) or FP<i>env</i>+FP<i>CIITA</i>_H6 (F2). As expected, no Gp or Env proteins were present after infection with the FP<i>CIITA</i>_SP or FP<i>CIITA</i>_H6 recombinants (A1, B1), and no HLA-DR or CIITA expression when the cells were infected with FP<i>gp</i> (C2, C3) or FP<i>env</i> (E2, E3). Differential interference contrast (DIC) microscopy of 293T cells is also shown (A5-F5).</p

Expression of the Gp, Env and CIITA proteins by FP recombinants in CEFs and Vero cells.

<p>The cells were infected by the single or double recombinants and examined by WB to determine Gp (A), Env (B) and CIITA (C) protein expression. Gp and Env proteins were expressed following the infection of either CEFs or Vero cells with the FP<i>gpCIITA</i> or FP<i>envCIITA</i> double recombinants (A, B). The expression of CIITA was high when this was driven by the H6 promoter (C). Co-infection of Vero cells with either the FP<i>gp+</i>FP<i>CIITA</i>_H6 or FP<i>env+</i>FP<i>CIITA</i>_H6 recombinants showed high <i>gp</i> and <i>env</i> expression (D). The Gp and Env proteins were detected using a monkey polyclonal anti-SIV serum, and CIITA using the mouse 7-1H mAb. Proteins were revealed using the ECL system. Cells infected with FPwt were used as the negative controls.</p

Analysis of the humoral response.

<p>The anti-Env humoral response was determined using ELISA, with lysed Vero cells previously infected with FP<i>env</i> as the plate-bound antigen. Serum was obtained from all of the mice at T0, T1, T2 and examined. After the second inoculation of T/SA cells, significant increases in the antibody titres were observed in G1, G2 and G3 (T2 <i>vs</i> T1; p <0.001). Also, a significant difference is shown when T/SA cells were infected only with FP<i>env</i> rather than after co-infection with FP<i>env</i>+FP<i>CIITA</i>_H6 (T2, G1 <i>vs</i> G3; p <0.01). The OD₄₅₀ values were expressed after subtracting the T0 values for each mouse. Statistical significances using one-way ANOVA parametric tests and Bonferroni analysis of variance are shown: p <0.01 (**), p <0.001 (***).</p