Symptomatic Severe Acute Respiratory Syndrome Coronavirus 2 Reinfection of a Healthcare Worker in a Belgian Nosocomial Outbreak Despite Primary Neutralizing Antibody Response.

BACKGROUND: It is currently unclear whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfection will remain a rare event, only occurring in individuals who fail to mount an effective immune response, or whether it will occur more frequently when humoral immunity wanes following primary infection. METHODS: A case of reinfection was observed in a Belgian nosocomial outbreak involving 3 patients and 2 healthcare workers. To distinguish reinfection from persistent infection and detect potential transmission clusters, whole genome sequencing was performed on nasopharyngeal swabs of all individuals including the reinfection case's first episode. Immunoglobulin A, immunoglobulin M, and immunoglobulin G (IgG) and neutralizing antibody responses were quantified in serum of all individuals, and viral infectiousness was measured in the swabs of the reinfection case. RESULTS: Reinfection was confirmed in a young, immunocompetent healthcare worker as viral genomes derived from the first and second episode belonged to different SARS-CoV-2 clades. The symptomatic reinfection occurred after an interval of 185 days, despite the development of an effective humoral immune response following symptomatic primary infection. The second episode, however, was milder and characterized by a fast rise in serum IgG and neutralizing antibodies. Although contact tracing and viral culture remained inconclusive, the healthcare worker formed a transmission cluster with 3 patients and showed evidence of virus replication but not of neutralizing antibodies in her nasopharyngeal swabs. CONCLUSIONS: If this case is representative of most patients with coronavirus disease 2019, long-lived protective immunity against SARS-CoV-2 after primary infection might not be likely.

Confirmed Zika virus infection in a Belgian traveler returning from Guatemala, and the diagnostic challenges of imported cases into Europe.

We report the first laboratory-confirmed Zika virus (ZIKV) infection in a Belgian traveler after a three week holiday in Guatemala, December 2015. This case along with other imported cases into Europe emphases once again the need for accurate diagnostic tools for this rapidly emerging virus. The challenge is to diagnose patients in the acute phase, which appears short, as serological testing is complicated by cross-reactivity, vaccination status and scarce availability of specific ZIKV tests.

CD4 mimetic miniproteins: potent anti-HIV compounds with promising activity as microbicides.

OBJECTIVES: The antiviral activity of CD4 miniproteins was evaluated as potential HIV microbicides, using relevant in vitro models. METHODS: Compounds were tested in a single-cycle HIV-1 pseudovirus assay and against replication competent HIV-1 in co-cultures of monocyte-derived dendritic cells (MO-DC) and CD4+ T cells. Cytotoxic activity was evaluated in an MTT assay. RESULTS: Monomeric miniproteins (M47 and M48) showed 50% effective concentration (EC(50)) values of 79-105 nM against a subtype B, CCR5 co-receptor-using Ba-L pseudovirus. Higher activity was found for the dimeric miniproteins M48D30, M48D50 and M48D100 (EC(50) between 15 and 30 nM), in contrast to the tetrameric miniproteins M48T30, M48T50 and M48T100 (EC(50) between 107 and 377 nM). The hetero-bivalent miniprotein M48-Hep and miniproteins that targeted the Phe-43 cavity on gp120 (M48-U1, M48-U2 and M48-U3) were highly active, with EC(50) values as low as 2 nM for M48-U1. All miniproteins showed high activity against CCR5 or CXCR4 co-receptor-using subtype B and CRF-01_A/E pseudoviruses. Many early M48-based compounds were much less active against subtype C pseudoviruses, whereas M48-U compounds that targeted the Phe-43 cavity were very active against all pseudoviruses, including subtype C. In MO-DC/CD4+ T cell co-cultures with replication-competent HIV-1 Ba-L, EC(50) values ranged between 13 and 1719 nM depending on the miniprotein, with M48-U1, M48-U2 and M48-U3 again being the most potent. Importantly, the latter compounds completely prevented viral replication by treating the cultures from 2 h before until 24 h after infection, at non-toxic concentrations of 66-6564 nM. CONCLUSIONS: These novel CD4 miniproteins might constitute a promising class of HIV microbicides.

A dual chamber model of female cervical mucosa for the study of HIV transmission and for the evaluation of candidate HIV microbicides.

A dual chamber system was established to model heterosexual HIV transmission. Cell-associated, but not cell-free HIV, added to a confluent layer of cervical epithelial cells in the apical chamber, reproducibly infected monocyte-derived dendritic cells (MO-DC) and CD4(+) T cells in the basal compartment. Only minimal epithelial transmigration of HIV-infected mononuclear cells (HIV-PBMCs) was observed. Most evidence points to transepithelial migration of virus, released from HIV-PBMCs after their activation by epithelial cells. We used this model for evaluation of the therapeutic index of various potentially preventive antiviral compounds, including non-nucleoside reverse transcriptase inhibitors (NNRTIs, including UC781 and various diaryltriazines and diarylpyrimidines), poly-anionic entry inhibitors (including PRO2000, cellulose sulphate, dextrane sulphate 5000 and polystyrene sulphonate) and the fusion inhibitor T-20. The epithelium was pre-treated with compound and incubated with HIV-PBMCs for 24 h. Afterwards the apical chamber was removed and MO-DC/CD4(+) T cell co-cultures were further cultured without compound. NNRTIs, including a TMC120 gel, blocked infection of the sub-epithelial targets at sub-micromolar concentrations. Polyanionic entry inhibitors (up to 100 microg/ml) and T-20 (up to 449 microg/ml) failed to inhibit transmission. Moreover, whereas the NNRTIs used interfered with epithelial integrity with cervical epithelium only at very high concentrations, the evaluated entry inhibitors showed toxicity at concentrations that did not prevent infection.

A series of diaryltriazines and diarylpyrimidines are highly potent nonnucleoside reverse transcriptase inhibitors with possible applications as microbicides.

An in vitro model of monocyte-derived dendritic cells (MO-DC) and CD4(+) T cells, representing the primary targets of sexual human immunodeficiency virus (HIV) transmission, was used to evaluate the antiviral and immune suppressive activity of new classes of nonnucleoside reverse transcriptase inhibitors, diaryltriazines (DATAs) and diarylpyrimidines (DAPYs), compared to the reference compounds UC-781 and PMPA. Antiviral activity (as reflected by the 50% effective concentration [EC(50)]) was determined by treating HIV-infected MO-DC/CD4(+)-T-cell cocultures with a dose range of a compound during 14 days, followed by analysis of supernatants in HIV p24 antigen enzyme-linked immunosorbent assay. A limited, 24-h treatment evaluated the compounds as microbicides. Viral rescue was evaluated in a PCR by monitoring proviral DNA in secondary cultures with phytohemagglutinin-interleukin-2 blasts. We determined 50% immunosuppressive concentrations in mixed leukocyte cultures of MO-DC and allogeneic T cells, with compound either continuously present or present only during the first 24 h. The EC(50) values of DATA and DAPY compounds ranged from 0.05 to 3 nM compared to 50 nM for UC-781 and 89 nM for PMPA. When evaluated in the "microbicide" setting, the most potent compounds completely blocked HIV infection at 10 to 100 nM. The immunosuppressive concentrations were well above the EC(50), resulting in favorable therapeutic indices for all compounds tested. The DATA and DAPY compounds described here are more potent than earlier reverse transcriptase inhibitors and show favorable pharmacological profiles in vitro. They could strengthen the antiretroviral armamentarium and might be useful as microbicides.

In vitro evaluation of nonnucleoside reverse transcriptase inhibitors UC-781 and TMC120-R147681 as human immunodeficiency virus microbicides.

The nonnucleoside reverse transcriptase inhibitors UC-781 and TMC120-R147681 (Dapivirine) effectively prevented human immunodeficiency virus (HIV) infection in cocultures of monocyte-derived dendritic cells and T cells, representing primary targets in sexual transmission. Both drugs had a favorable therapeutic index. A 24-h treatment with 1,000 nM UC-781 or 100 nM TMC120-R147681 prevented cell-free HIV infection, whereas 10-fold-higher concentrations blocked cell-associated HIV.