Assessing the safety, impact and effectiveness of RTS,S/AS01E malaria vaccine following its introduction in three sub-Saharan African countries: methodological approaches and study set-up.

BACKGROUND: Following a 30-year development process, RTS,S/AS01E (GSK, Belgium) is the first malaria vaccine to reach Phase IV assessments. The World Health Organization-commissioned Malaria Vaccine Implementation Programme (MVIP) is coordinating the delivery of RTS,S/AS01E through routine national immunization programmes in areas of 3 countries in sub-Saharan Africa. The first doses were given in the participating MVIP areas in Malawi on 23 April, Ghana on 30 April, and Kenya on 13 September 2019. The countries participating in the MVIP have little or no baseline incidence data on rare diseases, some of which may be associated with immunization, a deficit that could compromise the interpretation of possible adverse events reported following the introduction of a new vaccine in the paediatric population. Further, effects of vaccination on malaria transmission, existing malaria control strategies, and possible vaccine-mediated selective pressure on Plasmodium falciparum variants, could also impact long-term malaria control. To address this data gap and as part of its post-approval commitments, GSK has developed a post-approval plan comprising of 4 complementary Phase IV studies that will evaluate safety, effectiveness and impact of RTS,S/AS01E through active participant follow-up in the context of its real-life implementation. METHODS: EPI-MAL-002 (NCT02374450) is a pre-implementation safety surveillance study that is establishing the background incidence rates of protocol-defined adverse events of special interest. EPI-MAL-003 (NCT03855995) is an identically designed post-implementation safety and vaccine impact study. EPI-MAL-005 (NCT02251704) is a cross-sectional pre- and post-implementation study to measure malaria transmission intensity and monitor the use of other malaria control interventions in the study areas, and EPI-MAL-010 (EUPAS42948) will evaluate the P. falciparum genetic diversity in the periods before and after vaccine implementation. CONCLUSION: GSK's post-approval plan has been designed to address important knowledge gaps in RTS,S/AS01E vaccine safety, effectiveness and impact. The studies are currently being conducted in the MVIP areas. Their implementation has provided opportunities and posed challenges linked to conducting large studies in regions where healthcare infrastructure is limited. The results from these studies will support ongoing evaluation of RTS,S/AS01E's benefit-risk and inform decision-making for its potential wider implementation across sub-Saharan Africa.

Macrophage colony stimulating factor regulation by nuclear factor kappa B: a relevant pathway in human immunodeficiency virus type 1 infected macrophages.

Macrophage colony stimulating factor (M-CSF) is a cytokine that promotes monocyte differentiation and survival. When overexpressed, M-CSF contributes to pathology in a wide variety of diseases, including osteoporosis, obesity, certain human cancers, and in human immunodeficiency virus type 1 (HIV-1) infection, particularly with respect to monocyte/macrophage infection and the development of HIV-1 associated central nervous system disorders. In this study, our aim was to expand the current knowledge of M-CSF regulation, focusing on nuclear factor kappa B (NF-κB), a transcription factor playing a prominent role during inflammation and HIV-1 infection. Our results suggest that tumor necrosis factor alpha (TNF-α) promotes M-CSF secretion in primary macrophages and activates the -1310/+48 bp M-CSF promoter in Mono-Mac 1 cells. Inhibitors of the NF-κB pathway diminish this response. We identified four putative NF-κB and four CCAAT-enhancer-binding protein beta binding sites within the M-CSF promoter. Our findings, using promoter constructs mutated at individual NF-κB sites within the M-CSF promoter region, suggest that these sites are redundant with respect to NF-κB regulation. TNF-α treatment promoted NF-κB p65 binding to the M-CSF promoter in phorbol 12-myristate 13-acetate (PMA) treated U937 cells chronically infected with HIV-1 (U1 cells), but not in PMA treated uninfected U937 cells, suggesting that the presence of HIV-1 increases the NF-κB response. In conclusion, our findings demonstrate that NF-κB induces M-CSF expression on a promoter level via multiple functional NF-κB binding sites and that this pathway is likely relevant in HIV-1 infection of macrophages.

Macrophage colony-stimulating factor in the pathogenesis of HIV infection: potential target for therapeutic intervention.

Macrophage colony stimulating factor (M-CSF) appears to play a major role in promoting and maintaining reservoirs of human immunodeficiency virus type 1 (HIV-1) in infected individuals. HIV-1 infection induces production of M-CSF by macrophages, which in turn promotes further infection of macrophages via increases in CD4 and CCR5 receptors, as well as increases in virus gene expression. M-CSF promotes the ontogeny and survival of macrophages, contributing to both the number and longevity of these infected cells. M-CSF dysregulation promotes the differentiation of monocytes toward macrophages and osteoclasts and at the same time may inhibit differentiation toward dendritic cells, resulting in immune impairment. The potential role of M-CSF in HIV-associated end organ diseases including HIV-associated dementia, HIV-associated nephropathy, and osteoporosis is discussed. This review emphasizes the need for developing M-CSF antagonists for treatment of HIV-1-infected patients.

NnrA is required for full virulence and regulates several Brucella melitensis denitrification genes.

We identified two regulators of denitrification genes in Brucella melitensis 16M: NarR, which regulates the nitrate reductase (nar) operon, and NnrA, which is involved in the expression of the last three reductases of the denitrification pathway (nirK, norB, and nosZ). NnrA is required for virulence in mice and for intracellular resistance to nitric oxide.

Systematic targeted mutagenesis of Brucella melitensis 16M reveals a major role for GntR regulators in the control of virulence.

In order to identify transcriptional regulators involved in virulence gene control in Brucella melitensis, we generated a collection of 88 mutants in the AraC, ArsR, Crp, DeoR, GntR, IclR, LysR, MerR, RpiR, and TetR families of regulators. This collection was named LiMuR (library of mutants for regulators). We developed a method to test several mutants simultaneously in one animal in order to identify those unable to survive. This method, called the plasmid-tagged mutagenesis method, was used to test the residual virulence of mutants after 1 week in a mouse model of infection. Ten attenuated mutants, of which six and three belong to the GntR and LysR families, respectively, were identified and individually confirmed to replicate at lower rates in mice. Among these 10 mutants, only gntR10 and arsR6 are attenuated in cellular models. The LiMuR also allows simple screenings to identify regulators of a particular gene or operon. As a first example, we analyzed the expression of the virB operon in the LiMuR mutants. We carried out Western blottings of whole-cell extracts to analyze the production of VirB proteins using polyclonal antisera against VirB proteins. Four mutants produced small amounts of VirB proteins, and one mutant overexpressed VirB proteins compared to the wild-type strain. In these five mutants, reporter analysis using the virB promoter fused to lacZ showed that three mutants control virB at the transcriptional level. The LiMuR is a resource that will provide straightforward identification of regulators involved in the control of genes of interest.

The Ton system, an ABC transporter, and a universally conserved GTPase are involved in iron utilization by Brucella melitensis 16M.

Brucella spp. are gram-negative intracellular facultative pathogens that are known to produce 2,3-dihydroxybenzoic acid (DHBA), a catechol siderophore that is essential for full virulence in the natural host. The mechanism of DHBA entry into Brucella and other gram-negative bacteria is poorly understood. Using mini-Tn5Kmcat mutagenesis, we created a transposon library of Brucella melitensis 16M and isolated 32 mutants with a defect in iron acquisition or assimilation. Three of these transposon mutants are deficient in utilization of DHBA. Analysis of these three mutants indicated that the ExbB, DstC, and DugA proteins are required for optimal assimilation of DHBA and/or citrate. ExbB is part of the Ton complex, and DstC is a permease homologue of an iron(III) ABC transporter; in gram-negative bacteria these two complexes are involved in the uptake of iron through the outer and inner membranes, respectively. DugA is a new partner in iron utilization that exhibits homology with the bacterial conserved GTPase YchF. Based on this homology, DugA could have a putative regulatory function in iron assimilation in Brucella. None of the three mutants was attenuated in cellular models or in the mouse model of infection, which is consistent with the previous suggestion that DHBA utilization is not required in these models.