Microbial stimulation fully differentiates monocytes to DC-SIGN/CD209(+) dendritic cells for immune T cell areas.

Dendritic cells (DCs), critical antigen-presenting cells for immune control, normally derive from bone marrow precursors distinct from monocytes. It is not yet established if the large reservoir of monocytes can develop into cells with critical features of DCs in vivo. We now show that fully differentiated monocyte-derived DCs (Mo-DCs) develop in mice and DC-SIGN/CD209a marks the cells. Mo-DCs are recruited from blood monocytes into lymph nodes by lipopolysaccharide and live or dead gram-negative bacteria. Mobilization requires TLR4 and its CD14 coreceptor and Trif. When tested for antigen-presenting function, Mo-DCs are as active as classical DCs, including cross-presentation of proteins and live gram-negative bacteria on MHC I in vivo. Fully differentiated Mo-DCs acquire DC morphology and localize to T cell areas via L-selectin and CCR7. Thus the blood monocyte reservoir becomes the dominant presenting cell in response to select microbes, yielding DC-SIGN(+) cells with critical functions of DCs.

Mapping immunological and host receptor binding determinants of SARS-CoV spike protein utilizing the Qubevirus platform.

The motifs involved in tropism and immunological interactions of SARS-CoV spike (S) protein were investigated utilizing the Qubevirus platform. We showed that separately, 14 overlapping peptide fragments representing the S protein (F1-14 of 100 residues each) could be inserted into the C terminus of A1 on recombinant Qubevirus without affecting its viability. Additionally, recombinant phage expression resulted in the surface exposure of different engineered fragments in an accessible manner. The F6 from S425-525 was found to contain the binding determinant of the recombinant human angiotensin-converting enzyme 2, with the shortest active binding motif situated between residues S437-492. Upstream, another fragment, F7, containing an overlapping portion of F6 would not bind to recombinant human angiotensin-converting enzyme 2, confirming that a contiguous stretch of residues could adopt the appropriate structural orientation of F6 as an insertion within the Qubevirus. The F6 (S441-460) and other inserts, including F7/F8 (S601-620) and F10 (S781-800), were demonstrated to contain important immunological determinants through recognition and binding of S protein specific (anti-S) antibodies. An engineered chimeric insert bearing the fusion of all three anti-S reactive epitopes improved substantially the recognition and binding to their cognate antibodies. These results provide insights into humoral immune relevant epitopes and tropism characteristics of the S protein with implications for the development of subunit vaccines or other biologics against SARS-CoV.

Performance of the BD FACSPresto near to patient analyzer in comparison with representative conventional CD4 instruments in Cameroon.

BACKGROUND: In the context of scaling the viral load in resource limited settings, following HIV infected patient's adults and children with CD4+ T-lymphocyte count still very important in settings where the decentralization of treatment still has some challenges. Effective HIV monitoring in these resource-constrained settings needs affordable and reliable CD4+ T lymphocytes enumeration methods. We investigated the validity of a BD FACSPresto POC which is a dedicated system for enumeration that uses immunofluorescent technologies. In this study, we have assessed the sensitivity, specificity and correlation between most representative flow cytometry instruments present in Cameroon with more than 5000 CD4 T cells tests per year including FACSCalibur, FACSCount, and PIMA POC from Becton-Dickinson and ALERE respectively. METHODS: 268 patients aged from 1 to 72 years old were enrolled and included in the study after inform consent. The BD FACSPresto POC CD4+ T cell technology was placed at CIRCB and operated by technician staff. HIV infected patients were from Chantal BIYA international reference Center (CIRCB), Centre de Sante Catholique de NKOLODOM, Centre de Sante Catholique de BIKOP and CASS de Nkolndongo-Yaounde We compared the accuracy of the BD FACSPresto and three existing reference technologies with more than 5000 tests per year like FACSCalibur, FACSCount and PIMA according to the number of CD4 test done per year and their repartition in the country. Bland-Altman method and correlation analysis were used to estimate mean bias and 95% limits of agreement and to compare the methods, including analysis by subgroup of participant gestational age. In addition sensitivity and specificity were determined. Statistical significance was set at P-value < 0.05. RESULTS: The BD FACSPresto POC system has excellent precision, accuracy and linearity for CD4+ T lymphocytes enumeration. Good correlations were obtained between the BD FACSPresto poc system and other single platform methods. Bland-Altman plots showed interchangeability between two machines mean bias BD-FACSPresto vs PIMA = - 126,522(- 161,221 to - 91,822) BD-FACSPresto vs FACSCount = - 38,708 (- 58,935 to - 18,482) and FACSPresto vs FACSCALIBUR = 0.791(- 11,908 to 13,491). Mean difference with Absolute CD4+ T-lymphocyte values obtained from the BD FACSPresto system correlated well with PIMA, FACSCount, and FACSCalibur method with R2 equal to 0.88, 0.92 and 0.968 respectively with P < 0.001 for all. The mean comparison between values obtained from BD FACSPresto with PIMA, FACSCount, and FACSCalibur using paired T test give P = 0.17, P = 0.5 and P = 0.6 respectively meaning that there is no significant differences between values obtained with BD FACSPresto and PIMA, FACSCount or FACSCalibur CD4 enumeration machines. Further analysis revealed close agreement between all the three instruments with no significant difference between the forth methods (P = 0.91). CONCLUSION: This BD-FACSPresto POC system is a simple, robust and reliable system for enumeration of absolute and percentage of CD4+ T-lymphocytes especially suitable for remote areas with limited resources. Having one BD-FACSPresto POC system easy to use, should reduce the cost and thus increase and improved access to CD4 testing for HIV infected patients in resource-constrained countries. BD-FACSPresto POC CD4 will enable reduction in patient time and improve the overall quality of ART service count and may improve test access in remote areas. This technology can allow for greater decentralization and wider access to CD4 testing and ART.

A cross-sectional study of acute dengue infection in paediatric clinics in Cameroon.

BACKGROUND: Dengue fever is the world's fastest spreading mosquito borne viral infection. It is prevalent throughout both subtropical and tropical region, and affects over 128 countries. Dengue virus (DENV) infection poses a serious global public health challenge to three billion people, resulting in approximately 200 million cases of morbidity and 50,000 cases of mortality annually. In Cameroon like in most sub-Saharan African countries, DENV infection occur concurrently with other infectious diseases whose symptoms often overlap, rendering differential diagnosis challenging. This study aims at determining the frequency of acute dengue among febrile children under 15 years attending hospitals in some areas of Cameroon. METHODS: A total of 961 children under the age of 15 were recruited in a cross-sectional study using systematic sampling technique and by selecting each subject out of the three. The study was conducted in 10 public health centers in Cameroon. Demographic data and risk factors of the subjects were obtained using well-structured questionnaires. Dengue virus NS1 antigen, IgM and IgG were analysed using a Tell me fast® Combo Dengue NS1-IgG/IgM Rapid Test. An in-house ELISA test for dengue specific IgM antibody was equally performed for confirmation. Descriptive statistical analysis was performed using Graph pad version 6.0. RESULTS: A prevalence of 6.14% acute dengue virus infection was observed among children with febrile illness with a significant difference (p = 0.0488) between males (4.7%) and females (7.7%). In addition, children who reportedly were unprotected from vectors, showed a comparatively higher prevalence of the disease seropositivity than those practicing protective measures. CONCLUSION: DENV infection therefore is an important cause of fever among children in Cameroon. Thus, there is a need to include differential screening for DENV infections as a tool in the management of fever in children in the country.

Filaria specific antibody response profiling in plasma from anti-retroviral naïve Loa loa microfilaraemic HIV-1 infected people.

BACKGROUND: In West and Central Africa areas of endemic Loa loa infections overlap with regions of high prevalence of human immunodeficiency virus type 1 (HIV-1) infections. Because individuals in this region are exposed to filarial parasites from birth, most HIV-1 infected individuals invariably also have a history of filarial parasite infection. Since HIV-1 infection both depletes immune system and maintains it in perpetual inflammation, this can hamper Loa loa filarial parasite mediated immune modulation, leading to enhanced loaisis. METHODS: In this study we have assessed in plasma from asymptomatic anti-retroviral (ARV) naïve Loa loa microfilaraemic HIV-1 infected people the filarial antibody responses specific to a filariasis composite antigen consisting of Wbgp29-BmR1-BmM14-WbSXP. The antibody responses specific to the filariasis composite antigen was determined by enzyme linked immunosorbent assay (ELISA) in plasma from ARV naïve Loa loa microfilaraemic HIV-1 infected participants. In addition the filarial antigen specific IgG antibody subclass profiles were also determined for both HIV-1 positive and negative people. RESULTS: Both Loa loa microfilaraemic HIV-1 positive and negative individuals showed significantly higher plasma levels of IgG1 (P < 0.0001), IgG2 (P < 0.0001) and IgM (P < 0.0001) relative to amicrofilaraemic participants. A significant increase in IgE (P < 0.0001) was observed exclusively in Loa loa microfilaraemic HIV-1 infected people. In contrast there was a significant reduction in the level of IgG4 (p < 0.0001) and IgG3 (P < 0.0001) in Loa loa microfilaraemic HIV-1 infected individuals. CONCLUSIONS: Loa loa microfilaraemia in ARV naïve HIV-1 infected people through differential reduction of plasma levels of filarial antigen specific IgG3, IgG4 and a significant increase in plasma levels of filarial antigen specific IgE could diminish Loa loa mediated immune-regulation. This in effect can result to increase loaisis mediated immunopathology in antiretroviral naive HIV-1 infected people.

Posttreatment Reactions After Single-Dose Diethylcarbamazine or Ivermectin in Subjects With Loa loa Infection.

Background: Severe adverse reactions have been observed in individuals with Loa loa infection treated with either diethylcarbamazine (DEC), the drug of choice for loiasis, or ivermectin (IVM), which is used in mass drug administration programs for control of onchocerciasis and lymphatic filariasis in Africa. In this study, posttreatment clinical and immunologic reactions were compared following single-dose therapy with DEC or IVM to assess whether these reactions have the same underlying pathophysiology. Methods: Twelve patients with loiasis and microfilarial counts <2000 mf/mL were randomized to receive single-dose DEC (8 mg/kg) or IVM (200 µg/kg). Clinical and laboratory assessments were performed at 4, 8, 24, 48, and 72 hours and 5, 7, 9, and 14 days posttreatment. Results: Posttreatment adverse events were similar following DEC or IVM, but peaked earlier in subjects who received DEC, consistent with a trend toward more rapid and complete microfilarial clearance in the DEC group. After a transient rise (post-IVM) or fall (post-DEC) in the first 24 hours posttreatment, the eosinophil count rose significantly in both groups, peaking at day 5 in the DEC group and day 9 in the IVM group. Serum interleukin 5 levels and eosinophil activation, as assessed by surface expression of CD69 and serum levels of eosinophil granule proteins, were increased posttreatment in both groups. Conclusions: Despite differences in eosinophil and lymphocyte counts during the first 24 hours posttreatment, the overall pattern of hematologic and immunologic changes suggest that posttreatment reactions following DEC and IVM share a common pathophysiology. Clinical Trials Registration: NCT01593722.

Phenotypic characterization of regulatory T cells from antiretroviral-naive HIV-1-infected people.

Regulatory T (Treg) cells play a key role in dampening excessive immune activation. However, antiretroviral therapy (ART) -naive HIV-1 infection maintains the immune system in a sustained state of activation that could alter both Treg cell surface markers and functions. As Treg cell surface markers are directly linked to their functions the overall objective of this study was to determine how ART-naive HIV infection affects the phenotypic properties of Treg cells. Our data showed that in ART-naive HIV-1 infection, Treg cells are dominated by effector (CD45RA+  CD27-  CCR7- CD62L- ) and effector memory (CD45RA-  CD27-  CCR7-  CD62L- ) cells. In contrast Treg cells from HIV-negative individuals were mainly naive (CD45RA+  CD27+  CCR7+  CD62L+ ) and central memory (CD45RA- CD27+  CCR7+  CD62L+ ) cells. Whereas effector and effector memory Treg cells showed enhanced expression of CD39 (P < 0·05), CD73 (P < 0·001), HLA-DR and CD38 (P < 0·001); naive and central memory Treg cells showed a significant reduction in the expression of these markers. Overall Treg cell frequencies within total CD4+ T cells correlated positively with plasmatic HIV-1 viral load. As increased viral load is associated with augmented CD4+ T-cell destruction; this could suggest a resistance of peripheral Treg cells to HIV-1 destruction. Hence the modulation of Treg cell phenotype and frequencies could be considered in designing immunotherapeutic strategies targeting immune system restoration during HIV-1 infection.

Targeting the antigen encoded by adenoviral vectors to the DEC205 receptor modulates the cellular and humoral immune response.

Replication-defective adenoviral vectors have emerged as promising vaccine candidates for diseases relying on strong CD8(+) T-cell responses for protection. In this study, we modified a non-replicative adenoviral vector to selectively deliver, in situ, an encoded ovalbumin (OVA) model antigen to dendritic cells (DCs). Efficient uptake and presentation of OVA was achieved through fusion of the antigen to a single-chain antibody directed against DEC205, an endocytic receptor expressed on DCs. The immunogenicity of the vaccine was thereby enhanced as demonstrated by elevated antibody levels and increased T-cell responses after low-dose vaccination with 10(7) viral particles compared with a non-targeted control. Nevertheless, after immunization with higher doses of the targeted vaccine, the capacity of vaccine-induced CD8(+) T cells to produce the cytokine IL-2 was diminished and the CD8(+) T-cell response was dominated by an effector memory phenotype (CD62L(-)/CD127(+)) in contrast to the effector phenotype (CD62L(-)/CD127(-)) observed after non-targeted antigen delivery. Interestingly, the protective capacity of the non-targeted vaccine was superior to that of the targeted vaccine in an antigen-specific vaccinia virus infection as well as in a tumor challenge model. In the latter, the low dose of the DC-targeted vaccine also conferred partial protection from tumor growth, demonstrating dose-dependent effects of the DC-targeting on the quality of the vaccine-induced immune response. Significant differences could be observed in regard to the antibody pattern, the functional and phenotypic T-cell repertoire, and to the protective capacity.

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.

Dendritic cell targeted HIV gag protein vaccine provides help to a DNA vaccine including mobilization of protective CD8+ T cells.

To improve the efficacy of T cell-based vaccination, we pursued the principle that CD4(+) T cells provide help for functional CD8(+) T cell immunity. To do so, we administered HIV gag to mice successively as protein and DNA vaccines. To achieve strong CD4(+) T cell immunity, the protein vaccine was targeted selectively to DEC-205, a receptor for antigen presentation on dendritic cells. This targeting helped CD8(+) T cell immunity develop to a subsequent DNA vaccine and improved protection to intranasal challenge with recombinant vaccinia gag virus, including more rapid accumulation of CD8(+) T cells in the lung. The helper effect of dendritic cell-targeted protein vaccine was mimicked by immunization with specific MHC II binding HIV gag peptides but not peptides from a disparate Yersinia pestis microbe. CD4(+) helper cells upon adoptive transfer allowed wild-type, but not CD40(-/-), recipient mice to respond better to the DNA vaccine. The transfer also enabled recipients to more rapidly accumulate gag-specific CD8(+) T cells in the lung following challenge with vaccinia gag virus. Thus, complementary prime boost vaccination, in which prime and boost favor distinct types of T cell immunity, improves plasmid DNA immunization, including mobilization of CD8(+) T cells to sites of infection.

Evolutionary Qß Phage Displayed Nanotag Library and Peptides for Biosensing.

We selected a novel biotin-binding peptide for sensing biotin, biotinylated proteins, and nucleotides. From a 15-mer library displayed on the RNA coliphage Qß, a 15-amino acid long peptide (HGHGWQIPVWPWGQG) hereby referred to as a nanotag was identified to selectively bind biotin. The target selection was achieved through panning with elution by infection. The selected peptide was tested as a transducer for an immunogenic epitope of the foot-and-mouth disease virus (FMDV) on Qß phage platform separated by a linker. The biotin-tag showed no significant influence on the affinity of the epitope to its cognate antibody (SD6). The nanotag-bound biotin selectively fused either to the C- or N-terminus of the epitope. The epitope would not bind or recognize SD6 while positioned at the N-terminus of the nanotag. Additionally, the biotin competed linearly with the SD6 antibody in a competitive ELISA. Competition assays using the selected recombinant phage itself as a probe or transducer enable the operationalization of this technology as a biosensor toolkit to sense and quantify SD6 analyte. Herein, the published Strep II nanotag (DVEWLDERVPLVET) was used as a control and has similar functionalities to our proposed novel biotin-tag thereby providing a new platform for developing devices for diagnostic purposes.

Mapping immunological and host receptor binding determinants of SARS-CoV spike protein utilizing the Qubevirus platform.

The motifs involved in tropism and immunological interactions of SARS-CoV spike (S) protein were investigated utilizing the Qubevirus platform. We showed that separately, 14 overlapping peptide fragments representing the S protein (F1-14 of 100 residues each) could be inserted into the C-terminus of A1 on recombinant Qubevirus without affecting its viability. Additionally, recombinant phage expression resulted in the surface exposure of different engineered fragments in an accessible manner. The F6 from S 425-525 , was found to contain the binding determinant of the recombinant human angiotensin converting enzyme 2 (rhACE2), with the shortest active binding motif situated between residues S 437-492 . Upstream, another fragment, F7, containing an overlapping portion of F6 would not bind to rhACE2, confirming not just only that residues were linear but equally also the appropriate structural orientation of F6 upon the Qubevirus. The F6 (S 441-460 ) and other inserts, including F7/F8 (S 601-620 ) and F10 (S 781-800 ), were demonstrated to contain important immunological determinants through recognition and binding of S protein specific (anti-S) antibodies. An engineered chimeric insert bearing the fusion of all three anti-S reactive epitopes, improved substantially the recognition and binding to their cognate antibodies. These results provide insights into humoral immune relevant epitopes and tropism characteristics of the S protein with implications for the development of subunit vaccines or other biologics against SARS-CoV. Significance: Mapping epitopes within the receptor binding domains of viruses which are essential for viral tropism is critical for developing antiviral agents and subunit vaccines. In this study we have engineered the surface of Qubevirus to display a peptide library derived from the SARS-CoV S protein. In biopanning with S protein antibodies, we have identified three peptide fragments (EP1, EP2 and EP3) which reacted selectively with antibodies specific to the S protein. We demonstrated that all recombinant phage displayed peptide fragments both individually and as chimera exposed important immunological epitopes to their cognate antibodies. A peptide fragment F6 situated at S 425-525 , was found containing the binding determinant of the recombinant human angiotensin converting enzyme 2 (rhACE2), with the shortest active binding motif situated between residues S 437-492 . The platform is rapidly to identify epitopes and receptor binding sites within viral receptors found in target host cell. Thus, this platform holds great significance.

Newcastle disease virus expressing a dendritic cell-targeted HIV gag protein induces a potent gag-specific immune response in mice.

Viral vaccine vectors have emerged as an attractive strategy for the development of a human immunodeficiency virus (HIV) vaccine. Recombinant Newcastle disease virus (rNDV) stands out as a vaccine vector since it has a proven safety profile in humans, it is a potent inducer of both alpha interferon (IFN-α) and IFN-ß) production, and it is a potent inducer of dendritic cell (DC) maturation. Our group has previously generated an rNDV vector expressing a codon-optimized HIV Gag protein and demonstrated its ability to induce a Gag-specific CD8(+) T cell response in mice. In this report we demonstrate that the Gag-specific immune response can be further enhanced by the targeting of the rNDV-encoded HIV Gag antigen to DCs. Targeting of the HIV Gag antigen was achieved by the addition of a single-chain Fv (scFv) antibody specific for the DC-restricted antigen uptake receptor DEC205 such that the DEC205 scFv-Gag molecule was encoded for expression as a fusion protein. The vaccination of mice with rNDV coding for the DC-targeted Gag antigen induced an enhanced Gag-specific CD8(+) T cell response and enhanced numbers of CD4(+) T cells and CD8(+) T cells in the spleen relative to vaccination with rNDV coding for a nontargeted Gag antigen. Importantly, mice vaccinated with the DEC205-targeted vaccine were better protected from challenge with a recombinant vaccinia virus expressing the HIV Gag protein. Here we demonstrate that the targeting of the HIV Gag antigen to DCs via the DEC205 receptor enhances the ability of an rNDV vector to induce a potent antigen-specific immune response.

Improved cellular and humoral immune responses in vivo following targeting of HIV Gag to dendritic cells within human anti-human DEC205 monoclonal antibody.

Protein vaccines for T-cell immunity are not being prioritized because of poor immunogenicity. To overcome this hurdle, proteins are being targeted to maturing dendritic cells (DCs) within monoclonal antibodies (mAbs) to DC receptors. To extend the concept to humans, we immunized human immunoglobulin-expressing mice with human DEC205 (hDEC205) extracellular domain. 3D6 and 3G9 mAbs were selected for high-affinity binding to hDEC205. In addition, CD11c promoter hDEC205 transgenic mice were generated, and 3G9 was selectively targeted to DCs in these animals. When mAb heavy chain was engineered to express HIV Gag p24, the fusion mAb induced interferon-γ- and interleukin-2-producing CD4(+) T cells in hDEC205 transgenic mice, if polynocinic polycytidylic acid was coadministered as an adjuvant. The T-cell response was broad, recognizing at least 3 Gag peptides, and high titers of anti-human immunoglobulin G antibody were made. Anti-hDEC205 also improved the cross-presentation of Gag to primed CD8(+) T cells from HIV-infected individuals. In all tests, 3D6 and 3G9 targeting greatly enhanced immunization relative to nonbinding control mAb. These results provide preclinical evidence that in vivo hDEC205 targeting increases the efficiency with which proteins elicit specific immunity, setting the stage for proof-of-concept studies of these new protein vaccines in human subjects.

The efficacy of DNA vaccination is enhanced in mice by targeting the encoded protein to dendritic cells.

DNA vaccines promote an immune response by providing antigen-encoding DNA to the recipient, but the efficacy of such vaccines needs improving. Many approaches have considerable potential but currently induce relatively weak immune responses despite multiple high doses of DNA vaccine. Here, we asked whether targeting vaccine antigens to DCs would increase the immunity and protection that result from DNA vaccines. To determine this, we generated a DNA vaccine encoding a fusion protein comprised of the vaccine antigen and a single-chain Fv antibody (scFv) specific for the DC-restricted antigen-uptake receptor DEC205. Following vaccination of mice, the vaccine antigen was expressed selectively by DCs, which were required for the increased efficacy of MHC class I and MHC class II antigen presentation relative to a control scFv DNA vaccine. In addition, a DNA vaccine encoding an HIV gag p41-scFv DEC205 fusion protein induced 10-fold higher antibody levels and increased numbers of IFN-gamma-producing CD4+ and CD8+ T cells. After a single i.m. injection of the DNA vaccine encoding an HIV gag p41-scFv DEC205 fusion protein, mice were protected from an airway challenge with a recombinant vaccinia virus expressing the HIV gag p41, even with 1% of the dose of nontargeted DNA vaccine. The efficacy of DNA vaccines therefore may be enhanced by inclusion of sequences such as single-chain antibodies to target the antigen to DCs.

The microbial mimic poly IC induces durable and protective CD4+ T cell immunity together with a dendritic cell targeted vaccine.

CD4(+) Th1 type immunity is implicated in resistance to global infectious diseases. To improve the efficacy of T cell immunity induced by human immunodeficiency virus (HIV) vaccines, we are developing a protein-based approach that directly harnesses the function of dendritic cells (DCs) in intact lymphoid tissues. Vaccine proteins are selectively delivered to DCs by antibodies to DEC-205/CD205, a receptor for antigen presentation. We find that polyriboinosinic:polyribocytidylic acid (poly IC) independently serves as an adjuvant to allow a DC-targeted protein to induce protective CD4(+) T cell responses at a mucosal surface, the airway. After two doses of DEC-targeted, HIV gag p24 along with poly IC, responder CD4(+) T cells have qualitative features that have been correlated with protective function. The T cells simultaneously make IFN-gamma, tumor necrosis factor (TNF)-alpha, and IL-2, and in high amounts for prolonged periods. The T cells also proliferate and continue to secrete IFN-gamma in response to HIV gag p24. The adjuvant role of poly IC requires Toll-like receptor (TLR) 3 and melanoma differentiation-associated gene-5 (MDA5) receptors, but its analog poly IC(12)U requires only TLR3. We suggest that poly IC be tested as an adjuvant with DC-targeted vaccines to induce numerous multifunctional CD4(+) Th1 cells with proliferative capacity.

Uniqueness of RNA Coliphage Qß Display System in Directed Evolutionary Biotechnology.

Phage display technology involves the surface genetic engineering of phages to expose desirable proteins or peptides whose gene sequences are packaged within phage genomes, thereby rendering direct linkage between genotype with phenotype feasible. This has resulted in phage display systems becoming invaluable components of directed evolutionary biotechnology. The M13 is a DNA phage display system which dominates this technology and usually involves selected proteins or peptides being displayed through surface engineering of its minor coat proteins. The displayed protein or peptide's functionality is often highly reduced due to harsh treatment of M13 variants. Recently, we developed a novel phage display system using the coliphage Qß as a nano-biotechnology platform. The coliphage Qß is an RNA phage belonging to the family of Leviviridae, a long investigated virus. Qß phages exist as a quasispecies and possess features making them comparatively more suitable and unique for directed evolutionary biotechnology. As a quasispecies, Qß benefits from the promiscuity of its RNA dependent RNA polymerase replicase, which lacks proofreading activity, and thereby permits rapid variant generation, mutation, and adaptation. The minor coat protein of Qß is the readthrough protein, A1. It shares the same initiation codon with the major coat protein and is produced each time the ribosome translates the UGA stop codon of the major coat protein with the of misincorporation of tryptophan. This misincorporation occurs at a low level (1/15). Per convention and definition, A1 is the target for display technology, as this minor coat protein does not play a role in initiating the life cycle of Qß phage like the pIII of M13. The maturation protein A2 of Qß initiates the life cycle by binding to the pilus of the F+ host bacteria. The extension of the A1 protein with a foreign peptide probe recognizes and binds to the target freely, while the A2 initiates the infection. This avoids any disturbance of the complex and the necessity for acidic elution and neutralization prior to infection. The combined use of both the A1 and A2 proteins of Qß in this display system allows for novel bio-panning, in vitro maturation, and evolution. Additionally, methods for large library size construction have been improved with our directed evolutionary phage display system. This novel phage display technology allows 12 copies of a specific desired peptide to be displayed on the exterior surface of Qß in uniform distribution at the corners of the phage icosahedron. Through the recently optimized subtractive bio-panning strategy, fusion probes containing up to 80 amino acids altogether with linkers, can be displayed for target selection. Thus, combined uniqueness of its genome, structure, and proteins make the Qß phage a desirable suitable innovation applicable in affinity maturation and directed evolutionary biotechnology. The evolutionary adaptability of the Qß phage display strategy is still in its infancy. However, it has the potential to evolve functional domains of the desirable proteins, glycoproteins, and lipoproteins, rendering them superior to their natural counterparts.

Evaluation of a collaborative model for successful implementation of a National CD4 enumeration EQA program in Cameroon.

Participation in an EQA program is critical to the quality assurance process. Reliable and precise CD4 T-cells enumeration are essential to improve the clinical management of patients by evaluating the disease progression and by monitoring the effectiveness of ART in HIV-patients. The CIRCB, CD4 reference laboratory, in collaboration with the Canadian QASI-program, recruited sites, distributed and analyzed CD4-panels in 61 sites across Cameroon. A trend and performance analysis in the pre-analytical, analytical and post-analytical phases was performed. Continuous training and corrective actions carried out from 2014 to 2018 increased the number of participating sites from 15 to 61 sites, the number of unacceptable results decreased from 50 to 10%. Specific challenges included errors in pre analytic (17.5%), analytic (77.0%) and post-analytic (5.5%) phases. This EQA requires the application of good laboratory practices, fluidic communication between all the stakeholders, continuous training, application of specific on-site corrective measures, and timely equipment maintenance in order to avoid repetitive errors and to increase laboratory performance. It could be extended to other HIV-1 testing like viral load and EID point-of-care. Partnership with QASI serve as a model for implementation of a successful EQA model for resource limited countries wanting to implement EQA for HIV testing and monitoring in alignment with 90-90-90 targets.

Monkeypox Virus in Nigeria: Infection Biology, Epidemiology, and Evolution.

Monkeypox is a zoonotic disease caused by monkeypox virus (MPXV), which is a member of orthopoxvirus genus. The reemergence of MPXV in 2017 (at Bayelsa state) after 39 years of no reported case in Nigeria, and the export of travelers' monkeypox (MPX) from Nigeria to other parts of the world, in 2018 and 2019, respectively, have raised concern that MPXV may have emerged to occupy the ecological and immunological niche vacated by smallpox virus. This review X-rays the current state of knowledge pertaining the infection biology, epidemiology, and evolution of MPXV in Nigeria and worldwide, especially with regard to the human, cellular, and viral factors that modulate the virus transmission dynamics, infection, and its maintenance in nature. This paper also elucidates the role of recombination, gene loss and gene gain in MPXV evolution, chronicles the role of signaling in MPXV infection, and reviews the current therapeutic options available for the treatment and prevention of MPX. Additionally, genome-wide phylogenetic analysis was undertaken, and we show that MPXV isolates from recent 2017 outbreak in Nigeria were monophyletic with the isolate exported to Israel from Nigeria but do not share the most recent common ancestor with isolates obtained from earlier outbreaks, in 1971 and 1978, respectively. Finally, the review highlighted gaps in knowledge particularly the non-identification of a definitive reservoir host animal for MPXV and proposed future research endeavors to address the unresolved questions.

Dengue virus serological markers among potential blood donors: an evidence of asymptomatic dengue virus transmission in Cameroon.

INTRODUCTION: the risk of dengue virus or its antibodies which can be transmitted through blood transfusion by asymptomatic individuals infected, has been a major concern all over the world. Dengue is an endemic disease in sub-Saharan Africa, particularly in Cameroon. The purpose of this study is to determine the frequency of dengue virus (DENV) infection among potential blood donors at Yaounde Jamot Hospital. METHODS: serum samples were collected from 310 potential adult blood donors aged 18-57 years, who signed a written informed consent and completed the questionnaire between March 2019 and August 2019. This serum is used to screen for the presence of serological markers of DENV infection (NS1, IgM and IgG) using immunochromatographic tests (Zhuhai Encode Medical Engineering Co., Ltd, China). IgM/IgG positive samples were confirmed by enzyme-linked immunosorbent assays (ELISA). RESULTS: the overall prevalence was 24.8% among potential blood donors were subdivided as follows: 4.5% (14/310), 12.3% (38/310) and 6.1% (19/310) showed mono-positivity to DENV-NS1 antigen, anti-DENV IgM and anti-DENV IgG antibodies respectively. 1.9% (6/310) of potential blood donors showed dual positivity to anti-DENV IgM antibodies and anti-DENV IgG antibodies. The presence of DENV-NS1 antigen show asymptomatic viremia of dengue at the time of donation, while the presence of IgG antibodies reflects the high endemicity of dengue disease in the city of Yaoundé. CONCLUSION: these findings demonstrate the high level of risk of the DENV transmission among potential blood donors to needy recipients, underscoring the importance of establishing dengue fever blood screening in different services and blood collection units in Cameroon to improve safety transfusion and control the dissemination of the DENV.