P24 Capsid Research Articles

Limited HIV-associated neuropathologies and lack of immune activation in sub-saharan African individuals with late-stage subtype C HIV-1 infection.

Although previous studies have suggested that subtype B HIV-1 proviruses in the brain are associated with physiological changes and immune activation accompanied with microgliosis and astrogliosis, and indicated that both HIV-1 subtype variation and geographical location might influence the neuropathogenicity of HIV-1 in the brain. The natural course of neuropathogenesis of the most widespread subtype C HIV-1 has not been adequately investigated, especially for people living with HIV (PLWH) in sub-Saharan Africa. To characterize the natural neuropathology of subtype C HIV-1, postmortem frontal lobe and basal ganglia tissues were collected from nine ART-naïve individuals who died of late-stage AIDS with subtype C HIV-1 infection, and eight uninfected deceased individuals as controls. Histological staining was performed on all brain tissues to assess brain pathologies. Immunohistochemistry (IHC) against CD4, p24, Iba-1, GFAP, and CD8 in all brain tissues was conducted to evaluate potential viral production and immune activation. Histological results showed mild perivascular cuffs of lymphocytes only in a minority of the infected individuals. Viral capsid p24 protein was only detected in circulating immune cells of one infected individual, suggesting a lack of productive HIV-1 infection of the brain even at the late-stage of AIDS. Notably, similar levels of Iba-1 or GFAP between HIV + and HIV- brain tissues indicated a lack of microgliosis and astrogliosis, respectively. Similar levels of CD8 + cytotoxic T lymphocyte (CTL) infiltration between HIV + and HIV- brain tissues indicated CTL were not likely to be involved within subtype C HIV-1 infected participants of this cohort. Results from this subtype C HIV-1 study suggest that there is a lack of productive infection and limited neuropathogenesis by subtype C HIV-1 even at late-stage disease, which is in contrast to what was reported for subtype B HIV-1 by other investigators.

An immunoinformatics study reveals a new BoLA-DR-restricted CD4+ T cell epitopes on the Gag protein of bovine leukemia virus

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis (EBL), which has been reported worldwide. The expression of viral structural proteins: surface glycoprotein (gp51) and three core proteins - p15 (matrix), p24 (capsid), and p12 (nucleocapsid) induce a strong humoral and cellular immune response at first step of infection. CD4+ T-cell activation is generally induced by bovine leukocyte antigen (BoLA) region– positive antigen-presenting cells (APC) after processing of an exogenous viral antigen. Limited data are available on the BLV epitopes from the core proteins recognized by CD4+ T-cells. Thus, immunoinformatic analysis of Gag sequences obtained from 125 BLV isolates from Poland, Canada, Pakistan, Kazakhstan, Moldova and United States was performed to identify the presence of BoLA-DRB3 restricted CD4+ T-cell epitopes. The 379 15-mer overlapping peptides spanning the entire Gag sequence were run in BoLA-DRB3 allele-binding regions using a BoLA-DRB- peptide binding affinity prediction algorithm. The analysis identified 22 CD4+ T-cell peptide epitopes of variable length ranging from 17 to 22 amino acids. The predicted epitopes interacted with 73 different BoLA-DRB3 alleles found in BLV-infected cattle. Importantly, two epitopes were found to be linked with high proviral load in PBMC. A majority of dominant and subdominant epitopes showed high conservation across different viral strains, and therefore could be attractive targets for vaccine development.

In-house ELISA protocols for capsid p24 detection of diverse HIV isolates

BackgroundThe capsid p24 (CA-p24) antigen is a component of the viral capsid of human immunodeficiency virus (HIV) that has been commonly used for clinical diagnosis and monitoring of HIV infections in Enzyme-linked Immunosorbent Assays (ELISAs). Commercial CA-p24 ELISAs are widely used in research settings, but these kits are costly and have limited breadth for detecting diverse HIV isolates.MethodsCommercial CA-p24 antibodies were used as capture and detection antibodies. Specific CA-p24 ELISAs were established with these antibodies and tested for the detection of HIV-1 isolates with the aim of developing in-house protocols to recognize HIV-1 infections in vitro for research purposes.ResultsHere we present four protocols for in-house ELISAs to detect HIV CA-p24 using commercial antibodies. The assays were able to detect the CA-p24 antigen of different HIV-1 isolates tested. Comparison between the protocols showed that these in-house ELISAs exhibit high specificity, sensitivity, and reproducibility for CA-p24 quantitation but their reactivity varied per HIV-1 isolate and subtype.ConclusionsThese optimized ELISA protocols represent valuable tools to investigate HIV-1 infections in research facilities at a lower price than commercial CA-p24 kits.

BET Inhibitor JQ1 Attenuates Feline Leukemia Virus DNA, Provirus, and Antigen Production in Domestic Cat Cell Lines.

Feline leukemia virus (FeLV) is a cosmopolitan gammaretrovirus that causes lifelong infections and fatal diseases, including leukemias, lymphomas, immunodeficiencies, and anemias, in domestic and wild felids. There is currently no definitive treatment for FeLV, and while existing vaccines reduce the prevalence of progressive infections, they neither provide sterilizing immunity nor prevent regressive infections that result in viral reservoirs with the potential for reactivation, transmission, and the development of associated clinical diseases. Previous studies of murine leukemia virus (MuLV) established that host cell epigenetic reader bromodomain and extra-terminal domain (BET) proteins facilitate MuLV replication by promoting proviral integration. Here, we provide evidence that this facilitatory effect of BET proteins extends to FeLV. Treatment with the archetypal BET protein bromodomain inhibitor (+)-JQ1 and FeLV challenge of two phenotypically disparate feline cell lines, 81C fibroblasts and 3201 lymphoma cells, significantly reduced FeLV proviral load, total FeLV DNA load, and p27 capsid protein expression at nonlethal concentrations. Moreover, significant decreases in FeLV proviral integration were documented in 81C and 3201 cells. These findings elucidate the importance of BET proteins for efficient FeLV replication, including proviral integration, and provide a potential target for treating FeLV infections.

Deoptimization of FMDV P1 Region Results in Robust Serotype-Independent Viral Attenuation.

Foot-and-mouth disease (FMD), caused by the FMD virus (FMDV), is a highly contagious disease of cloven-hoofed livestock that can have severe economic impacts. Control and prevention strategies, including the development of improved vaccines, are urgently needed to effectively control FMD outbreaks in endemic settings. Previously, we employed two distinct strategies (codon pair bias deoptimization (CPD) and codon bias deoptimization (CD)) to deoptimize various regions of the FMDV serotype A subtype A12 genome, which resulted in the development of an attenuated virus in vitro and in vivo, inducing varying levels of humoral responses. In the current study, we examined the versatility of the system by using CPD applied to the P1 capsid coding region of FMDV serotype A subtype, A24, and another serotype, Asia1. Viruses carrying recoded P1 (A24-P1Deopt or Asia1-P1Deopt) exhibited different degrees of attenuation (i.e., delayed viral growth kinetics and replication) in cultured cells. Studies in vivo using a mouse model of FMD demonstrated that inoculation with the A24-P1Deopt and Asia1-P1Deopt strains elicited a strong humoral immune response capable of offering protection against challenge with homologous wildtype (WT) viruses. However, different results were obtained in pigs. While clear attenuation was detected for both the A24-P1Deopt and Asia1-P1Deopt strains, only a limited induction of adaptive immunity and protection against challenge was detected, depending on the inoculated dose and serotype deoptimized. Our work demonstrates that while CPD of the P1 coding region attenuates viral strains of multiple FMDV serotypes/subtypes, a thorough assessment of virulence and induction of adaptive immunity in the natural host is required in each case in order to finely adjust the degree of deoptimization required for attenuation without affecting the induction of protective adaptive immune responses.

Expression of Bovine Leukaemia Virus (BLV) Gp51 Protein in Blood and Milk Cells of Cows with Leukosis.

Bovine leukaemia virus (BLV) is the retroviral causative agent of enzootic bovine leukosis, the most common neoplastic disease of cattle and a serious problem worldwide. Its diagnosis is commonly by tests for antibodies recognising the p24 capsid protein and structural glycoprotein (gp) 51. With flow cytometry recently having come to veterinary immunology, applications for it may now include BLV. The study determined BLV gp51 expression in blood and milk lymphocytes of naturally infected cows by flow cytometry. Nineteen Polish Black and White Lowland breed cows aged 4-9 years and naturally infected with BLV and ten uninfected counterparts had blood and milk sampled and cultured. The immunological status of the animals was confirmed with ELISA and PCR. Dual-colour flow cytometry analysis was performed with specific monoclonal antibodies for lymphocyte cluster of differentiation (CD) markers and gp51 viral envelope protein and conjugates labelled with fluorescein isothiocyanate or phycoerythrin. Bovine leukaemia virus gp51 was confirmed in lymphocytes by immunofluorescence with anti-gp51 monoclonal antibodies. The gp51 antigen was detected in blood and milk lymphocytes of infected cows, but the percentage of cells expressing it in milk was much lower than in blood. A depleted number of CD4+ lymphocytes, an augmented number of CD8+ lymphocytes, a lower ratio of CD4+ to CD8+ and a proliferation of CD19+ immunoglobulin M+ cells were also found. These proliferated cells were immature, gave no sign of a tendency to differentiation and were characterised by prolonged vitality.

HIV Capsid Protein Genetic Diversity Across HIV-1 Variants and Impact on New Capsid-Inhibitor Lenacapavir.

The HIV p24 capsid protein has an essential, structural, and functional role in the viral replication cycle, being an interesting target for vaccine design, diagnostic tests, and new antiretroviral drugs (ARVs). The HIV-1 variability poses a challenge for the accuracy and efficiency of diagnostic and treatment tools. This study analyzes p24 diversity among HIV-1 variants and within its secondary structure in HIV-1 M, O, P, and N groups. All available HIV-1 p24 nucleotide sequences were downloaded from the Los Alamos HIV Sequence Database, selecting 23,671 sequences belonging to groups O, N, P, and M (9 subtypes, 7 sub-sub types, and 109 circulating recombinant forms or CRFs). Using a bioinformatics tool developed in our laboratory (EpiMolBio program), we analyzed the amino acid conservation compared to the HXB2 subtype B reference sequence and the V-markers, or amino acid changes that were specific for each variant with at least 10 available sequences. We inferred the p24 consensus sequence for HIV-1 and for each group to analyze the overall conservation in p24 main structural regions, reporting the percentage of substitutions per variant affecting the capsid assembly and molecule-binding, including those associated with resistance to the new capsid-inhibitor lenacapavir, and the key residues involved in lenacapavir-p24 interaction, according to the bibliography. Although the overall structure of p24 was highly conserved, the conservation in the secondary structure varied between HIV-1 variants and the type of secondary structure. All HIV-1 variants presented >80% amino acid conservation vs. HXB2 reference sequence, except for group M sub-subtype F1 (69.27%). Mutants affecting the capsid assembly or lenacapavir capsid-binding were found in <1% of the p24 consensus sequence. Our study reports the HIV-1 variants carrying 14 unique single V-markers in 9/38 group M variants and the level of p24 conservation in each secondary structure region among the 4 HIV-1 groups and group M variants, revealing no natural resistance to lenacapavir in any HIV-1 variant. We present a thorough analysis of p24 variability among all HIV-1 variants circulating to date. Since p24 genetic variability can impact the viral replication cycle and the efficacy of new p24-based diagnostic, therapeutic, and vaccine strategies, conservation studies must consider all HIV-1 variants circulating worldwide.

Avoiding the self-nucleation interference: a pH-regulated gold in situ growth strategy to enable ultrasensitive immunochromatographic diagnostics.

Background: Gold nanoparticle-based immunochromatographic assay (AuNP-ICA) has insufficient sensitivity due to its inherent colorimetric signal intensity and low capture efficiency of AuNPs. The metal in situ growth is a common strategy to enhance the sensitivity of AuNP-ICA due to its superior signal amplification potential and simple operation. However, the detection distortion caused by metal self-nucleation during the growth process can seriously affect the accuracy and reproducibility of the strips.Methods: We present a pH-regulated gold in situ growth (GISG) strategy to amplify the colorimetric signal and demonstrate its application in improving the performance of traditional AuNP-ICA. The controllable growth signal amplification is achieved by lowering the pH of the growth solution to weaken the reducibility of hydroxylamine (HA), thus urging the crystallization and growth of Au3+ on the AuNP surface instead of free reduction and self-nucleation. In addition, the mechanism of pH regulation on HA reducibility is elucidated by introducing an electron-donating or electron-withdrawing group to affect the electron density of hydroxyl group.Results: The proposed GISG strategy shows improved sensitivity, low background, robust operation, and good reproducibility. The LOD values of the designed GISG-amplified AuNP-ICA are as low as 0.0198 ng mL-1 for hepatitis B surface antigen and 0.0125 ng mL-1 for HIV-1 capsid p24 antigen, which are lower by about 500- and 70-fold, respectively, than those of the unamplified AuNP-ICA.Conclusions: This method is extended to enable ultrasensitive and rapid diagnosis of viral infections, and has potential as a general signal amplification platform to redefine immunochromatographic diagnostics.

Changes in dynamic and static structures of the HIV-1 p24 capsid protein N-domain caused by amino-acid substitution are associated with its viral viability.

HIV-1 capsid is comprised of over a hundred p24 protein molecules, arranged as either pentamers or hexamers. Three p24 mutants with amino acid substitutions in capsid N-terminal domain protein were examined: G60W (α3-4 loop), M68T (helix 4), and P90T (α4-5 loop), which exhibited no viability for biological activity. One common structural feature of the three p24 N-domain mutants, examined by NMR, was the long-range effect of more β-structures at the β2-strand in the N-terminal region compared with the wild-type. In addition, the presence of fewer helical structures was observed in M68T and P90T, beyond the broad area from helix 1 to the C-terminal part of helix 4. This suggests that both N-terminal beta structures and helices play important roles in the formation of p24 hexamers and pentamers. Next, compared with P90T, we examined cis-conformation or trans-conformation of wild-type adopted by isomerization at G89-P90. Since P90T mutant adopts only a trans-conformation, comparison of chemical shifts and signal intensities between each spectra revealed that the major peaks (about 85%) in the spectrum of wild-type correspond to trans-conformation. Furthermore, it was indicated that the region in cis-conformation (minor; 15%) was more stabilized than that observed in trans-conformation, based on the analyses of heteronuclear Overhauser effect as well as the order-parameter. Therefore, it was concluded that the cis-conformation is more favorable than the trans-conformation for the interaction between the p24 N-terminal domain and cyclophilin-A. This is because HIV-1 with a P90T protein, which adopts only a trans-conformation, is associated with non-viability of biological activity.

Dysregulated Microglial Cell Activation and Proliferation Following Repeated Antigen Stimulation.

Upon reactivation of quiescent neurotropic viruses antigen (Ag)-specific brain resident-memory CD8+ T-cells (bTRM) may respond to de novo-produced viral Ag through the rapid release of IFN-γ, which drives subsequent interferon-stimulated gene expression in surrounding microglia. Through this mechanism, a small number of adaptive bTRM may amplify responses to viral reactivation leading to an organ-wide innate protective state. Over time, this brain-wide innate immune activation likely has cumulative neurotoxic and neurocognitive consequences. We have previously shown that HIV-1 p24 Ag-specific bTRM persist within the murine brain using a heterologous prime-CNS boost strategy. In response to Ag restimulation, these bTRM display rapid and robust recall responses, which subsequently activate glial cells. In this study, we hypothesized that repeated challenges to viral antigen (Ag) (modeling repeated episodes of viral reactivation) culminate in prolonged reactive gliosis and exacerbated neurotoxicity. To address this question, mice were first immunized with adenovirus vectors expressing the HIV p24 capsid protein, followed by a CNS-boost using Pr55Gag/Env virus-like particles (HIV-VLPs). Following the establishment of the bTRM population [>30 days (d)], prime-CNS boost animals were then subjected to in vivo challenge, as well as re-challenge (at 14 d post-challenge), using the immunodominant HIV-1 AI9 CD8+ T-cell epitope peptide. In these studies, Ag re-challenge resulted in prolonged expression of microglial activation markers and an increased proliferative response, longer than the challenge group. This continued expression of MHCII and PD-L1 (activation markers), as well as Ki67 (proliferative marker), was observed at 7, 14, and 30 days post-AI9 re-challenge. Additionally, in vivo re-challenge resulted in continued production of inducible nitric oxide synthase (iNOS) with elevated levels observed at 7, 14 and 30 days post re-challenge. Interestingly, iNOS expression was significantly lower among challenged animals when compared to re-challenged groups. Furthermore, in vivo specific Ag re-challenge produced lower levels of arginase (Arg)-1 when compared with the challenged group. Taken together, these results indicate that repeated Ag-specific stimulation of adaptive immune responses leads to cumulative dysregulated microglial cell activation.

A proposed division of the family Picornaviridae into subfamilies based on phylogenetic relationships and functional genomic organization

The highly diverse virus family Picornaviridae presently comprises 68 approved genera with 158 species plus many unassigned viruses. In order to better match picornavirus taxonomy to the functional and genomic groupings between genera, the establishment of five subfamilies (Caphthovirinae, Kodimesavirinae, Ensavirinae, Paavivirinae and Heptrevirinae) is proposed. The subfamilies are defined by phylogenetic analyses of 3CD (precursor of virus-encoded proteinase and polymerase) and P1 (capsid protein precursor) coding sequences and comprise between 7 and 22 currently approved virus genera. Due to the high within-subfamily and between-subfamily divergences of the picornavirus genera, p-distance estimates are unsuited for the demarcation of subfamilies. Members of the proposed subfamilies typically show some commonalities in their genome organisations, including VP1/2A cleavage mechanisms and possession of leader proteins. Other features, such as internal ribosomal entry site types, are more variable within and between members of genera. Some subfamilies are characterised by homology of proteins 1A, 2A, 2B and 3A encoded by members, which do not belong to the canon of orthologous picornavirus proteins. The proposed addition of a subfamily layer to the taxonomy of picornaviruses provides a valuable additional organisational level to the family that acknowledges the existence of higher-level evolutionary groupings of its component genera.

EXPRESSION OF RECOMBINANT P24 PROTEIN IN ESCHERICHIA COLI FOR SEROLOGICAL DIAGNOSIS OF BOVINE LEUKOSIS

Bovine leukemia is an infectious lymphoproliferative disease in cattle caused by the bovine leukemia virus (BLV). Production losses associated with BLV include increased cull rates, decreased milk production, or reduced fertility. Serological tests are used for diagnosis of BLV, of which the most sensitive is enzyme-linked immunosorbent assay (ELISA). Capsid p24 protein is a diagnostically important antigen of the BLV. The gene of p24 protein was cloned into pET-28 plasmid vector. The recombinant p24 antigen was obtained by using Escherichia coli ArcticExpressRP(DE3)_pET-28/р24 strain. The total yield of the purified p24 protein was 76 mg. Testing of the recombinant p24 protein on control sera showed that the p24 binds to positive control sera and has high antigenic activity. The pilot testing of the recombinant p24 protein on 48 animal samples resulted in 83% positive and 17% negative. The results indicate that the recombinant p24 protein is promising in enzyme-linked immunosorbent assay for the diagnosis of bovine leukemia.

Interaction of porcine circovirus-like virus P1 capsid protein with host proteins

BackgroundPorcine circovirus-like virus P1 is a relatively new kind of virus that is closely related to the post-weaning multisystemic wasting syndrome, congenital tremors, and abortions in swine. The molecular mechanisms of P1 virus infection and pathogenesis are fully unknown. To analyze P1 and its host interactions, we used a yeast two-hybrid (Y2H) assay to identify cellular proteins interacting with the Cap of the P1 virus. In this study, the Cap of the P1 virus exhibited no self-activation and toxicity to yeast cells and was used as bait to screen the Y2H library prepared from the pancreas tissue.ResultsFive cellular proteins (EEP, Ral GDS, Bcl-2-L-12, CPS1, and one not identified) were found to interact with P1 Cap. The interaction between Cap and Ral GDS was confirmed by co-immunoprecipitation.ConclusionsOur data are likely to support the future investigation of the underlying mechanism of P1 infection and pathogenesis.

Identification of a Potential Inhibitor of the FIV p24 Capsid Protein and Characterization of Its Binding Site.

Feline immunodeficiency virus (FIV) is a veterinary infective agent for which there is currently no efficient drug available. Drugs targeting the lentivirus capsid are currently under development for the treatment of human immunodeficiency virus 1 (HIV-1). Here we describe a lead compound that interacts with the FIV capsid. This compound, 696, modulates the in vitro assembly of and stabilizes the assembled capsid protein. To decipher the mechanism of binding of this compound to the protein, we performed the first nuclear magnetic resonance (NMR) assignment of the FIV p24 capsid protein. Experimental NMR chemical shift perturbations (CSPs) observed after the addition of 696 enabled the characterization of a specific binding site for 696 on p24. This site was further analyzed by molecular modeling of the protein:compound interaction, demonstrating a strong similarity with the binding sites of existing drugs targeting the HIV-1 capsid protein. Taken together, we characterized a promising capsid-interacting compound with a low cost of synthesis, for which derivatives could lead to the development of efficient treatments for FIV infection. More generally, our strategy combining the NMR assignment of FIV p24 with NMR CSPs and molecular modeling will be useful for the analysis of future compounds targeting p24 in the quest to identify an efficient treatment for FIV.

Surface Plasmon Resonance Assay for Label-Free and Selective Detection of HIV-1 p24 Protein.

The early detection of the human immunodeficiency virus (HIV) is of paramount importance to achieve efficient therapeutic treatment and limit the disease spreading. In this perspective, the assessment of biosensing assay for the HIV-1 p24 capsid protein plays a pivotal role in the timely and selective detection of HIV infections. In this study, multi-parameter-SPR has been used to develop a reliable and label-free detection method for HIV-1 p24 protein. Remarkably, both physical and chemical immobilization of mouse monoclonal antibodies against HIV-1 p24 on the SPR gold detecting surface have been characterized for the first time. The two immobilization techniques returned a capturing antibody surface coverage as high as (7.5 ± 0.3) × 1011 molecule/cm2 and (2.4 ± 0.6) × 1011 molecule/cm2, respectively. However, the covalent binding of the capturing antibodies through a mixed self-assembled monolayer (SAM) of alkanethiols led to a doubling of the p24 binding signal. Moreover, from the modeling of the dose-response curve, an equilibrium dissociation constant KD of 5.30 × 10−9 M was computed for the assay performed on the SAM modified surface compared to a much larger KD of 7.46 × 10−5 M extracted for the physisorbed antibodies. The chemically modified system was also characterized in terms of sensitivity and selectivity, reaching a limit of detection of (4.1 ± 0.5) nM and an unprecedented selectivity ratio of 0.02.

Use of Synonymous Deoptimization to Derive Modified Live Attenuated Strains of Foot and Mouth Disease Virus.

Foot-and-mouth disease (FMD) is one of the most economically important viral diseases that can affect livestock. In the last 70 years, use of an inactivated whole antigen vaccine has contributed to the eradication of disease from many developed nations. However, recent outbreaks in Europe and Eastern Asia demonstrated that infection can spread as wildfire causing economic and social devastation. Therefore, it is essential to develop new control strategies that could confer early protection and rapidly stop disease spread. Live attenuated vaccines (LAV) are one of the best choices to obtain a strong early and long-lasting protection against viral diseases. In proof of concept studies, we previously demonstrated that “synonymous codon deoptimization” could be applied to the P1 capsid coding region of the viral genome to derive attenuated FMDV serotype A12 strains. Here, we demonstrate that a similar approach can be extended to the highly conserved non-structural P2 and P3 coding regions, providing a backbone for multiple serotype FMDV LAV development. Engineered codon deoptimized P2, P3 or P2, and P3 combined regions were included into the A24Cruzeiro infectious clone optimized for vaccine production, resulting in viable progeny that exhibited different degrees of attenuation in cell culture, in mice, and in the natural host (swine). Derived strains were thoroughly characterized in vitro and in vivo. Our work demonstrates that overall, the entire FMDV genome tolerates codon deoptimization, highlighting the potential of using this technology to derive novel improved LAV candidates.

Controlled copper in situ growth-amplified lateral flow sensors for sensitive, reliable, and field-deployable infectious disease diagnostics

A polyethyleneimine (PEI)-assisted copper in-situ growth (CISG) strategy was proposed as a controlled signal amplification strategy to enhance the sensitivity of gold nanoparticle-based lateral flow sensors (AuNP-LFS). The controlled signal amplification is achieved by introducing PEI as a structure-directing agent to regulate the thermodynamics of anisotropic Cu nanoshell growth on the AuNP surface, thus controlling shape and size of the resultant AuNP@Cu core–shell nanostructures and confining free reduction and self-nucleation of Cu2+ for improved reproducibility and decreased false positives. The PEI-CISG-enhanced AuNP-LFS showed ultrahigh sensitivities with the detection limits of 50 fg mL−1 for HIV-1 capsid p24 antigen and 6 CFU mL−1 for Escherichia coli O157:H7. We further demonstrated its clinical diagnostic efficacy by configuring PEI-CISG into a commercial AuNP-LFS detection kit for SARS-CoV-2 antibody detection. Altogether, this work provides a reliable signal amplification platform to dramatically enhance the sensitivity of AuNP-LFS for rapid and accurate diagnostics of various infectious diseases.

Neutralization capacity of highly divergent type 2 vaccine-derived polioviruses from immunodeficient patients

The use of the oral poliovirus vaccine (OPV) in developing countries has reduced the incidence of poliomyelitis by >99% since 1988 and is the primary tool for global polio eradication. Spontaneous reversions of the vaccine virus to a neurovirulent form can impede this effort. In persons with primary B-cell immunodeficiencies, exposure to OPV can result in chronic infection, mutation, and excretion of immunodeficiency-associated vaccine-derived polioviruses, (iVDPVs). These iVDPVs may have the potential for transmission in a susceptible population and cause paralysis. The extent to which sera from OPV recipients are able to neutralize iVDPVs with varying degrees of antigenic site substitutions is investigated here. We tested sera from a population immunized with a combination vaccine schedule (both OPV and inactivated polio vaccine) against a panel of iVDPVs and found that increases in amino acid substitution in the P1 capsid protein resulted in a decrease in the neutralizing capacity of the sera. This study underscores the importance of maintaining high vaccine coverage in areas of OPV use as well as active surveillance of those known to be immunocompromised.

Standalone operation of an EGOFET for ultra-sensitive detection of HIV

A point-of-care (POC) device to enable de-centralized diagnostics can effectively reduce the time to treatment, especially in case of infectious diseases. However, many of the POC solutions presented so far do not comply with the ASSURED (affordable, sensitive, specific, user-friendly, rapid and robust, equipment free, and deliverable to users) guidelines that are needed to ensure their on-field deployment. Herein, we present the proof of concept of a self-powered platform that operates using the analysed fluid, mimicking a blood sample, for early stage detection of HIV-1 infection. The platform contains a smart interfacing circuit to operate an ultra-sensitive electrolyte-gated field-effect transistor (EGOFET) as a sensor and facilitates an easy and affordable readout mechanism. The sensor transduces the bio-recognition event taking place at the gate electrode functionalized with the antibody against the HIV-1 p24 capsid protein, while it is powered via paper-based biofuel cell (BFC) that extracts the energy from the analysed sample itself. The self-powered platform is demonstrated to achieve detection of HIV-1 p24 antigens in fM range, suitable for early diagnosis. From these developments, a cost-effective digital POC device able to detect the transition from “healthy” to “infected” state at single-molecule precision, with no dependency on external power sources while using minimal components and simpler approach, is foreseen.

Comparative multi-assay evaluation of Determine\u2122 HIV-1/2 Ag/Ab Combo rapid diagnostic tests in acute and chronic HIV infection

In resource-limited or point-of-care settings, rapid diagnostic tests (RDTs), that aim to simultaneously detect HIV antibodies and p24 capsid (p24CA) antigen with high sensitivity, can pose important alternatives to screen for early infections. We evaluated the performance of the antibody and antigen components of the old and novel version of the Determine™ HIV-1/2 Ag/Ab Combo RDTs in parallel to quantifications in a fourth-generation antigen/antibody immunoassay (4G-EIA), p24CA antigen immunoassay (p24CA-EIA), immunoblots, and nucleic acid quantification. We included plasma samples of acute, treatment-naïve HIV-1 infections (Fiebig stages I–VI, subtypes A1, B, C, F, CRF02_AG, CRF02_AE, URF) or chronic HIV-1 and HIV-2 infections. The tests’ antigen component was evaluated also for a panel of subtype B HIV-1 transmitted/founder (T/F) viruses, HIV-2 strains and HIV-2 primary isolates. Furthermore, we assessed the analytical sensitivity of the RDTs to detect p24CA using a highly purified HIV-1NL4-3 p24CA standard. We found that 77% of plasma samples from acutely infected, immunoblot-negative HIV-1 patients in Fiebig stages II–III were identified by the new RDT, while only 25% scored positive in the old RDT. Both RDTs reacted to all samples from chronically HIV-1-infected and acutely HIV-1-infected patients with positive immunoblots. All specimens from chronically infected HIV-2 patients scored positive in the new RDT. Of note, the sensitivity of the RDTs to detect recombinant p24CA from a subtype B virus ranged between 50 and 200 pg/mL, mirrored also by the detection of HIV-1 T/F viruses only at antigen concentrations tenfold higher than suggested by the manufacturer. The RTD failed to recognize any of the HIV-2 viruses tested. Our results indicate that the new version of the Determine™ HIV-1/2 Ag/Ab Combo displays an increased sensitivity to detect HIV-1 p24CA-positive, immunoblot-negative plasma samples compared to the precursor version. The sensitivity of 4G-EIA and p24CA-EIA to detect the major structural HIV antigen, and thus to diagnose acute infections prior to seroconversion, is still superior.