Toward the elimination of hepatitis B: networking to promote the prevention of vertical transmission of hepatitis B virus through population-based interventions and multidisciplinary groups in Africa.

The WHO African Region had 81 million people with chronic hepatitis B in 2019, which remains a silent killer. Hepatitis B virus (HBV), hepatitis delta virus (HDV), and HIV can be transmitted from the mother to child. If the HBV infection is acquired at infancy, it may lead to chronic hepatitis B in 90% of the cases. WHO reports that 6.4 million children under 5 years live with chronic hepatitis B infection worldwide. The prevention of mother-to-child transmission (PMTCT) of HBV is therefore critical in the global elimination strategy of viral hepatitis as we take lessons from PMTCT of HIV programs in Africa. We sought to create a network of multidisciplinary professional and civil society volunteers with the vision to promote cost-effective, country-driven initiatives to prevent the MTCT of HBV in Africa. In 2018, the Mother-Infant Cohort Hepatitis B Network (MICHep B Network) with members from Cameroon, Zimbabwe, and the United Kingdom and later from Chad, Gabon, and Central African Republic was created. The long-term objectives of the network are to organize capacity-building and networking workshops, create awareness among pregnant women, their partners, and the community, promote the operational research on MTCT of HBV, and extend the network activities to other African countries. The Network organized in Cameroon, two "Knowledge, Attitude and Practice" (KAP) surveys, one in-depth interview of 45 health care workers which revealed a high acceptability of the hepatitis B vaccine by families, two in-person workshops in 2018 and 2019, and one virtual in 2021 with over 190 participants, as well as two workshops on grant writing, bioethics, and biostatistics of 30 postgraduate students. Two HBV seroprevalence studies in pregnant women were conducted in Cameroon and Zimbabwe, in which a prevalence of 5.8% and 2.7%, respectively, was reported. The results and recommendations from the MICHep B Network activities could be implemented in countries of the MICHep B Network and beyond, with the goal of providing free birth dose vaccine against hepatitis B in Africa.

Added Value of Mean Blood Pressure and Placental Growth Factor in the Early Detection of Pre-eclampsia among Gabonese Women.

OBJECTIVE: To evaluate the use of the Foetal Medicine Foundation (FMF) algorithm in routine practice for early pre-eclampsia (PE) screening in Libreville. MATERIALS AND METHODS: We conducted a cohort study on pregnant women within their 11-13 + 6 weeks of gestation (WG). We had measured mean blood pressure (MBP), placental growth factor (PlGF), soluble Fms-like tyrosine kinase 1, Uterine Artery Pulsatility Index (UtA-PI) and resistance index (UtA-RI). Statistical analyses were considered significant for P < 0.05. RESULTS: There were 30 participants. At the first quarter (T1), 36.7% of them were at high risk of PE according to the FMF algorithm and were consequently prescribed aspirin (100 mg/d). By the end of the observation period, we have found a 13% incidence of PE. MBP was higher in the higher risk PE group than in the lower risk group as early as the T1 (90 ± 6 vs. 81 ± 6 mmHg; P = 0.0007, threshold is >86 mmHg/area under the curve (AUC) = 0.86; P = 0.0012). It was the same for PlGF (58 ± 24 vs. 88 ± 38 pg/ml; P = 0.03; threshold is <71.98 pg/ml/AUC = 0.73; P = 0.03). At the second quarter (20-27 WG), biochemical markers did not change between the two groups. UtA-RI, UtA-PI and notch were unconclusive individually, but they are still very important for FMF algorithm application. CONCLUSION: Early detection of PE using the FMF algorithm is possible in routine practice in Gabon. MBP and PlGF levels at T1 seem to be very significant. However, the present study must continue to obtain the larger cohorts that would achieve more conclusive statistical analyses.

Allergen sensitization and polysensitization pattern of adults and children in an urban Sub-Saharan African setting (Libreville, Gabon).

Background: It is believed that allergic diseases are increasing in Africa. However, the health sector in Africa has yet to catch up with this paradigm shift. We looked at the number of patients referred to us for allergy testing and investigated allergen sensitization. Methods: A retrospective analysis was done on 97 serum allergen-specific IgE results collected from patients suspected of having allergies in Libreville from 2018 to 2021. Specific IgE responses to 180 allergens were investigated. The general sensitization patterns were analyzed. Also analyzed were sensitization patterns for adults and children. The difference in the IgE-binding allergen positivity rate between groups was calculated by using the chi-square (χ2) test. Results: The allergens most commonly causing sensitization were from mites (65%), barley (48%), peach (48%), dog and/or cat dander (44%), house dust (44%), peanut (39%), tomato (39%), cockroach (37%), crab (36%), garlic and/or onion (34%), rye (34%), egg white (32%), shrimp (32%), kiwi (32%), soya bean (32%), citrus mix (29%), cheese (27%), milk (27%), walnut (27%), ox-eye daisy (24%) and orchard grass (24%). Moreover, 60% of patients (36 of 60) were polysensitized to inhalant allergens, 53% (31 of 58) were polysensitized to food allergens, and 29% (14 of 48) were polysensitized to inhalant and food allergens; 65% of patients (53 of 81) were sensitized to allergens originating from mites, fungi (including Candida albicans, Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum, and Pennicillium notatum), or bacteria (staphylococcal enterotoxin B). Conclusions: The sensitization pattern of allergens in our setting is rich and varied, with a high prevalence of polysensitization.

Unrecognized introduction of SARS-CoV-2 variants of concern to Central Africa: Import and local transmission of B.1.1.7 in Gabon in the very early stage of the variant spread to the African continent.

The rapid spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant of concern with higher infectivity has already resulted in the enormous increase in infection cases worldwide. We report an unrecognized introduction of the variant B.1.1.7 in Gabon in December 2020, which was the initial phase of the variant introduction to Africa. The B.1.1.7 variant was also detected in a hospitalized patient in January 2021, indicating a rapid spread of the variant in Gabon since its first detection. Phylogenetic analysis revealed that the detected B.1.1.7 variants originated from the distinct regions, strongly suggesting that the B.1.1.7 variant had been repeatedly introduced to Gabon since December 2020. These results provide insights on the unrecognized risks of infections with variants of concern, and show the necessity to conduct continuous genomic monitoring for immediate alert and control of novel SARS-CoV-2 variant infections.

Dynamic and features of SARS-CoV-2 infection in Gabon.

In a context where SARS-CoV-2 population-wide testing is implemented, clinical features and antibody response in those infected have never been documented in Africa. Yet, the information provided by analyzing data from population-wide testing is critical to understand the infection dynamics and devise control strategies. We described clinical features and assessed antibody response in people screened for SARS-CoV-2 infection. We analyzed data from a cohort of 3464 people that we molecularly screened for SARS-CoV-2 infection in our routine activity. We recorded people SARS-CoV-2 diagnosis, age, gender, blood types, white blood cells (WBC), symptoms, chronic disease status and time to SARS-CoV-2 RT-PCR conversion from positive to negative. We calculated the age-based distribution of SARS-CoV-2 infection, analyzed the proportion and the spectrum of COVID-19 severity. Furthermore, in a nested sub-study, we screened 83 COVID-19 patients and 319 contact-cases for anti-SARS-CoV-2 antibodies. Males and females accounted for respectively 51% and 49% of people screened. The studied population median and mean age were both 39 years. 592 out of 3464 people (17.2%) were diagnosed with SARS-CoV-2 infection with males and females representing, respectively, 53% and 47%. The median and mean ages of SARS-CoV-2 infected subjects were 37 and 38 years respectively. The lowest rate of infection (8%) was observed in the elderly (aged > 60). The rate of SARS-Cov-2 infection in both young (18-35 years old) and middle-aged adults (36-60 years old) was around 20%. The analysis of SARS-CoV-2 infection age distribution showed that middle-aged adults accounted for 54.7% of SARS-CoV-2 positive persons, followed respectively by young adults (33.7%), children (7.7%) and elderly (3.8%). 68% (N = 402) of SARS-CoV-2 infected persons were asymptomatic, 26.3% (N = 156) had influenza-like symptoms, 2.7% (N = 16) had influenza-like symptoms associated with anosmia and ageusia, 2% (N = 11) had dyspnea and 1% (N = 7) had respiratory failure, which resulted in death. Data also showed that 12% of SARS-CoV-2 infected subjects, had chronic diseases. Hypertension, diabetes, and asthma were the top concurrent chronic diseases representing respectively 58%, 25% and 12% of recorded chronic diseases. Half of SARS-CoV-2 RT-PCR positive patients were cured within 14 days following the initiation of the anti-COVID-19 treatment protocol. 78.3% of COVID-19 patients and 55% of SARS-CoV-2 RT-PCR confirmed negative contact-cases were positive for anti-SARS-CoV-2 antibodies. Patients with severe-to-critical illness have higher leukocytes, higher neutrophils and lower lymphocyte counts contrarily to asymptomatic patients and patients with mild-to-moderate illness. Neutrophilic leukopenia was more prevalent in asymptomatic patients and patients with mild-to-moderate disease for 4 weeks after diagnosis (27.1-42.1%). In Patients with severe-to-critical illness, neutrophilic leukocytosis or neutrophilia (35.6-50%) and lymphocytopenia (20-40%) were more frequent. More than 60% of participants were blood type O. It is also important to note that infection rate was slightly higher among A and B blood types compared with type O. In this African setting, young and middle-aged adults are most likely driving community transmission of COVID-19. The rate of critical disease is relatively low. The high rate of anti-SARS-CoV-2 antibodies observed in SARS-CoV-2 RT-PCR negative contact cases suggests that subclinical infection may have been overlooked in our setting.

Circulating anti-SARS-CoV-2 nucleocapsid (N)-protein antibodies and anti-SARS-CoV-2 spike (S)-protein antibodies in an African setting: herd immunity, not there yet!

OBJECTIVE: Herd immunity is achieved when in a population, immune individuals are in a sufficiently large proportion. Neutralizing antibodies specific to SARS-CoV-2 that are produced following infection or vaccination are critical for controlling the spread of COVID-19. The objective of the present work was to investigate the rate of SARS-CoV-2 natural immunization in Gabonese. RESULTS: One thousand, four hundred and ninety two people were enrolled. The overall prevalence of anti-SARS-CoV-2 antibodies was 36.2%. Moreover, 76.4% of people who developed a humoral response to SARS-CoV-2 produced both anti-SARS-CoV-2 N-protein antibodies and anti-SARS-CoV-2 S-protein antibodies, which correspond to 27.7% of the total population. In infants (0-9 month), children (1-17 years) and adults, the prevalence of anti-SARS-CoV-2 antibodies was relatively the same, between 33 and 37% (any antibody types) and between 25 and 28.6% (neutralizing antibodies). In this African context, one-third (1/3) of the screened population was exposed to SARS-CoV-2 and three-quarter (3/4) of those exposed individuals developed neutralizing antibodies against SARS-CoV-2. This data suggest that herd immunity is not yet to be achieved in Gabon.

Cell Penetrating Peptides Used in Delivery of Therapeutic Oligonucleotides Targeting Hepatitis B Virus.

Peptide Nucleic Acid (PNAs) and small noncoding RNAs including small interfering RNAs (siRNAs) represent a new class of oligonucleotides considered as an alternative therapeutic strategy in the chronic hepatitis B treatment. Indeed, chronic hepatitis B virus (HBV) infection remains a major public health problem worldwide, despite the availability of an effective prophylactic vaccine. Current therapeutic approaches approved for chronic HBV treatment are pegylated-interferon alpha (IFN)-α and nucleos(t)ide analogues (NAs). Both therapies do not completely eradicate viral infection and promote severe side effects. In this context, the development of new effective treatments is imperative. This review focuses on antiviral activity of both PNAs and siRNAs targeting hepatitis B virus. Thus, we briefly present our results on the ability of PNAs to decrease hepadnaviral replication in duck hepatitis B virus (DHBV) model. Interestingly, other oligonucleotides as siRNAs could significantly inhibit HBV antigen expression in transient replicative cell culture. Because the application of these oligonucleotides as new antiviral drugs has been hampered by their poor intracellular bioavailability, we also discuss the benefits of their coupling to different molecules such as the cell penetrating peptides (CPPs), which were used as vehicles to deliver therapeutic agents into the cells.

Developments in Cell-Penetrating Peptides as Antiviral Agents and as Vehicles for Delivery of Peptide Nucleic Acid Targeting Hepadnaviral Replication Pathway.

Alternative therapeutic approaches against chronic hepatitis B virus (HBV) infection need to be urgently developed because current therapies are only virostatic. In this context, cell penetration peptides (CPPs) and their Peptide Nucleic Acids (PNAs) cargoes appear as a promising novel class of biologically active compounds. In this review we summarize different in vitro and in vivo studies, exploring the potential of CPPs as vehicles for intracellular delivery of PNAs targeting hepadnaviral replication. Thus, studies conducted in the duck HBV (DHBV) infection model showed that conjugation of (D-Arg)8 CPP to PNA targeting viral epsilon (ε) were able to efficiently inhibit viral replication in vivo following intravenous administration to ducklings. Unexpectedly, some CPPs, (D-Arg)8 and Decanoyl-(D-Arg)8, alone displayed potent antiviral effect, altering late stages of DHBV and HBV morphogenesis. Such antiviral effects of CPPs may affect the sequence-specificity of CPP-PNA conjugates. By contrast, PNA conjugated to (D-Lys)4 inhibited hepadnaviral replication without compromising sequence specificity. Interestingly, Lactose-modified CPP mediated the delivery of anti-HBV PNA to human hepatoma cells HepaRG, thus improving its antiviral activity. In light of these promising data, we believe that future studies will open new perspectives for translation of CPPs and CPP-PNA based technology to therapy of chronic hepatitis B.

Role of Cell-Penetrating Peptides in Intracellular Delivery of Peptide Nucleic Acids Targeting Hepadnaviral Replication.

Peptide nucleic acids (PNAs) are potentially attractive antisense agents against hepatitis B virus (HBV), although poor cellular uptake limits their therapeutic application. In the duck HBV (DHBV) model, we evaluated different cell-penetrating peptides (CPPs) for delivery to hepatocytes of a PNA-targeting hepadnaviral encapsidation signal (ε). This anti-ε PNA exhibited sequence-specific inhibition of DHBV RT in a cell-free system. Investigation of the best in vivo route of delivery of PNA conjugated to (D-Arg)8 (P1) showed that intraperitoneal injection to ducklings was ineffective, whereas intravenously (i.v.) injected fluorescein-P1-PNA reached the hepatocytes. Treatment of virus carriers with i.v.-administered P1-PNA resulted in a decrease in viral DNA compared to untreated controls. Surprisingly, a similar inhibition of viral replication was observed in vivo as well as in vitro in primary hepatocyte cultures for a control 2 nt mismatched PNA conjugated to P1. By contrast, the same PNA coupled to (D-Lys)4 (P2) inhibited DHBV replication in a sequence-specific manner. Interestingly, only P1, but not P2, displayed anti-DHBV activity in the absence of PNA cargo. Hence, we provide new evidence that CPP-PNA conjugates inhibit DHBV replication following low-dose administration. Importantly, our results demonstrate the key role of CPPs used as vehicles in antiviral specificity of CPP-PNA conjugates.

Therapeutic Potential of Cell Penetrating Peptides (CPPs) and Cationic Polymers for Chronic Hepatitis B.

Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. Because current anti-HBV treatments are only virostatic, there is an urgent need for development of alternative antiviral approaches. In this context, cell-penetrating peptides (CPPs) and cationic polymers, such as chitosan (CS), appear of particular interest as nonviral vectors due to their capacity to facilitate cellular delivery of bioactive cargoes including peptide nucleic acids (PNAs) or DNA vaccines. We have investigated the ability of a PNA conjugated to different CPPs to inhibit the replication of duck hepatitis B virus (DHBV), a reference model for human HBV infection. The in vivo administration of PNA-CPP conjugates to neonatal ducklings showed that they reached the liver and inhibited DHBV replication. Interestingly, our results indicated also that a modified CPP (CatLip) alone, in the absence of its PNA cargo, was able to drastically inhibit late stages of DHBV replication. In the mouse model, conjugation of HBV DNA vaccine to modified CS (Man-CS-Phe) improved cellular and humoral responses to plasmid-encoded antigen. Moreover, other systems for gene delivery were investigated including CPP-modified CS and cationic nanoparticles. The results showed that these nonviral vectors considerably increased plasmid DNA uptake and expression. Collectively promising results obtained in preclinical studies suggest the usefulness of these safe delivery systems for the development of novel therapeutics against chronic hepatitis B.

Potent inhibition of late stages of hepadnavirus replication by a modified cell penetrating peptide.

Cationic cell-penetrating peptides (CPPs) and their lipid domain-conjugates (CatLip) are agents for the delivery of (uncharged) biologically active molecules into the cell. Using infection and transfection assays we surprisingly discovered that CatLip peptides were able to inhibit replication of Duck Hepatitis B Virus (DHBV), a reference model for human HBV. Amongst twelve CatLip peptides we identified Deca-(Arg)8 having a particularly potent antiviral activity, leading to a drastic inhibition of viral particle secretion without detectable toxicity. Inhibition of virion secretion was correlated with a dose-dependent increase in intracellular viral DNA. Deca-(Arg)8 peptide did neither interfere with DHBV entry, nor with formation of mature nucleocapsids nor with their travelling to the nucleus. Instead, Deca-(Arg)8 caused envelope protein accumulation in large clusters as revealed by confocal laser scanning microscopy indicating severe structural changes of preS/S. Sucrose gradient analysis of supernatants from Deca-(Arg)8-treated cells showed unaffected naked viral nucleocapsids release, which was concomitant with a complete arrest of virion and surface protein-containing subviral particle secretion. This is the first report showing that a CPP is able to drastically block hepadnaviral release from infected cells by altering late stages of viral morphogenesis via interference with enveloped particle formation, without affecting naked nucleocapsid egress, thus giving a view inside the mode of inhibition. Deca-(Arg)8 may be a useful tool for elucidating the hepadnaviral secretory pathway, which is not yet fully understood. Moreover we provide the first evidence that a modified CPP displays a novel antiviral mechanism targeting another step of viral life cycle compared to what has been so far described for other enveloped viruses.

Clonidine-induced growth hormone and growth-hormone-releasing hormone release is mediated by tachykinin NK2 receptors in sheep.

Clonidine is an α(2)-adrenergic agonist, historically known to treat high blood pressure. Further studies showed that it could be used in the treatment of neuropsychiatric disorders. Afterwards, it has been reported that clonidine stimulated growth hormone (GH) release in many species including man. Using a transnasal surgery technique in awake sheep that allowed accessing hypothalamopituitary portal vessels, our laboratory previously reported that the injection of clonidine in sheep induced a significant, immediate and short-lasting increase in peripheral GH and portal GH-releasing hormone (GHRH) levels. In this study, we show that the clonidine-induced peripheral GH and portal GHRH increase in sheep appears to be mediated by the tachykinin NK2 receptor.

Substance P stimulates Growth Hormone (GH) and GH-Releasing Hormone (GHRH) secretions through tachykinin NK2 receptors in sheep.

Substance P is ubiquitous undecapeptide belonging to the tachykinins family. It has been found in the hypothalamus and is involved in the hypothalamo-hypophysial axis in several mammals, including human. Previous studies have shown that substance P increases GH secretions in rats and human. In this study, we have shown that intravenously infused substance P in sheep caused an increased level of Growth Hormone (GH) and GH-Releasing Hormone (GHRH), and decreased Somatotropin Release Inhibiting Hormone (SRIH) secretions. GH was obtained from peripheral blood. GHRH and SRIH were directly collected from hypophysial portal blood, using a trans-nasal surgery technique in a vigil sheep that allowed accessing to hypothalamo-hypophysial portal vessels. Hormones assays were performed by radioimmunoassay (RIA). Moreover, we showed that substance P-induced GH and GHRH secretion appears to be mediated by NK2 tachykinin receptors, since it is specifically blocked by a non peptidic tachykinin NK2 receptor antagonist (SR48968, Sanofi, Montpellier, France) whereas a non peptidic tachykinin NK1 antagonist (SR140333, Sanofi, Montpellier, France) failed to modify GH and GHRH hormones secretions.

Rise in gamma interferon expression during resolution of duck hepatitis B virus infection.

Gamma interferon (IFN-gamma) expression plays a crucial role in the control of mammalian hepatitis B virus (HBV) infection. However, the role of duck INF-gamma (DuIFN-gamma) in the outcome of duck HBV (DHBV) infection, a reference model for hepadnavirus replication studies, has not yet been investigated. This work explored the dynamics of DuIFN-gamma expression in liver and peripheral blood mononuclear cells (PBMCs) during resolution of DHBV infection in adolescent ducks in relation to serum and liver markers of virus replication, histological changes and humoral response induction. DHBV infection of 3-week-old ducks resulted in transient expression of intrahepatic preS protein (days 3-14) and mild histological changes. Low-level viraemia was detected only during the first 10 days of infection and was accompanied by early anti-preS antibody response induction. Importantly, a strong increase in intrahepatic DuIFN-gamma RNA was detected by real-time RT-PCR at days 6-14, which coincided with a sharp decrease in both viral DNA and preS protein in the liver. Interestingly, liver DuIFN-gamma expression remained augmented to the end of the follow-up period (day 66) and correlated with portal lymphocyte infiltration and persistence of trace quantities of intrahepatic DHBV DNA in animals that had apparently completely resolved the infection. Moreover, in infected ducks, a moderate increase was detected in the levels of DuIFN-gamma in PBMCs (days 12-14), which coincided with the peak in liver DuIFN-gamma RNA levels. These data reveal that increased DuIFN-gamma expression in liver and PBMCs is concomitant with viral clearance, characterizing the resolution of infection, and provide new insights into the host-virus interactions that control DHBV infection.

Genetic vaccination for production of DNA-designed antibodies specific to Hepadnavirus envelope proteins.

We propose a method of avian antibodies production based on DNA immunization of laying ducks with a plasmid encoding specified antigen, followed by egg collection and purification of egg yolk immunoglobulins (IgY). We have validated this approach in the Duck hepatitis B virus (DHBV) model. We report here that following immunization of female ducks with plasmids encoding DHBV envelope proteins, large amounts (at least 50 mg/egg) of specific antibodies can be obtained from eggs of these ducks. Interestingly, the comparison of different plasmid constructs showed the important differences in their efficacy of specific IgY antibodies induction in the sera and eggs of immunized ducks.

Genetic immunization of ducks for production of antibodies specific to Helicobacter pylori UreB in egg yolks.

Following genetic immunization of laying ducks with a plasmid expressing Helicobacter pylori UreB (large subunit of urease), IgY against UreB were obtained from egg yolks. These polyclonal and monospecific IgY antibodies are of higher-titer and specifically recognize recombinant H. pylori urease purified from Escherichia coli. To our knowledge this is the first report describing generation of IgY antibodies directed against antigens of H. pylori by DNA-based immunization.