Nanoparticles can be coated with targeting ligands to deliver medical agents to specific cells. Serum protein adsorption affects the binding of nanoparticles to target cells. We hypothesized that serum proteins and target receptors compete for binding to nanoparticles. We tested the serum protein binding affinity of 251 nanoparticle designs. Here, we discovered that the binding affinities of serum proteins and receptors to a nanoparticle determine whether it can bind to target cells. We developed and validated a quantitative metric, the binding ratio, to identify nanoparticle designs that can bind to targets in serum with 90% sensitivity and 88% specificity. Using the binding ratio as a numerical guideline for nanoparticle design enabled us to improve the efficiency of nanoparticle binding to target cellular receptors.

Zoonotic viruses rank among the greatest threats to public health, with urbanization and global warming accelerating their emergence and spread. As the risk of future pandemics grows, innovative tools are needed to deepen our understanding of viral pathogenesis and enhance pandemic preparedness. Nonviral protein cages provide a versatile platform for studying viral mechanisms, virus-host interactions, and designing next-generation therapeutic approaches, making them powerful assets in the fight against viral threats.

The COVID-19 pandemic has prompted an unprecedented global response. In particular, extraordinary efforts have been dedicated toward monitoring and predicting variant emergence due to its huge impact, particularly for vaccine escape. Broadly, we classify such methods into two categories: forward mutation prediction, where phenotypes are first observed and the responsible genotypes traced, and reverse mutation prediction, which starts with selected pathogen genetic profiles and characterizes their associated phenotypes. Reverse mutation prediction strategies have advantages in being able to sample a more complete evolutionary space since sequences that do not yet exist can be sampled. The rapid improvement in the maturity and scale of reverse mutation prediction strategies, such as deep mutational scanning, has led to significant amounts of data for machine learning, with concomitant improvement in the prediction results from computational tools. Such integrated prediction approaches are generalizable and offer significant opportunities for anticipating viral evolution and for pandemic preparedness.

Glycan-mediated recognition is critically involved in a variety of pathophysiological events, so strategies targeting unique glycosylation could offer opportunities for novel disease diagnostics and therapeutics. Herein, we survey the current progress in glycan-binding entities and their biomedical applications. Particularly focusing on biologically promising artificial receptors, including boronate affinity-based molecularly imprinted polymers (MIPs) and anti-glycan aptamers, we summarize significant efforts in the recognition of glycans by MIPs and aptamers with high affinity and exquisite specificity. Furthermore, we highlight successful examples in biomedical fields of antiviral treatment, cancer diagnostics and targeted therapeutics. Finally, we briefly sketch the remaining challenges and future perspectives. Collectively, this review provides significant insights for further exploration of glycan-specific biomimetic materials in the broad biomedical area.

Herpes simplex virus 1 (HSV-1) gD interaction with the host cell receptor nectin-1 triggers the membrane fusion cascade during viral entry. Potent neutralizing antibodies to gD prevent receptor-binding or prevent gD interaction with gH/gL critical for fusion. HSV has many strategies to evade host immune responses. We investigated the ability of virion envelope gC to protect envelope gD from antibody neutralization. HSV-1 lacking gC was more sensitive to neutralization by anti-gD monoclonal antibodies than a wild-type rescuant virus. gD in the HSV-1 gC-null viral envelope had enhanced reactivity to anti-gD antibodies compared to wild type. Soluble nectin-1 bound similar to HSV-1 particles regardless of the presence of gC in the envelope. However, entry of HSV-1 ΔgC was more sensitive to inhibition by soluble nectin-1 receptor. The viral membrane protein composition of HSV-1 ΔgC is equivalent to that of wild type, suggesting that the lack of gC is responsible for the increased reactivity of gD-specific antibodies and the consequent increased susceptibility to neutralization by those antibodies. Together, the results suggest that gC in the HSV-1 envelope shields both receptor-binding domains and gH/gL-interacting domains of gD from neutralizing antibodies, facilitating HSV cell entry.IMPORTANCEHSV-1 causes lifelong infections. There is no vaccine and no cure. Understanding HSV immune evasion strategies is an important goal. HSV-1 gC is a multi-functional envelope glycoprotein. This study suggests that virion gC physically shields neighboring gD from antibodies, including neutralizing monoclonal antibodies. This mechanism may allow HSV to escape immune detection, promoting HSV infection in the host.

During replication of some RNA viruses, defective particles can spontaneously arise and interfere with wild-type (WT) virus replication. Recently, engineered versions of these defective interfering particles (DIPs) have been proposed as an HIV-1 therapeutic. However, DIPs have yet to be reported in people with HIV-1 (PWH). Here, we find DIPs in PWH who have a rare, polyclonal form of non-suppressible viremia (NSV). While antiretroviral therapy (ART) rapidly reduces viremia to undetectable levels, some individuals experience sustained viremia due to virus production from cell clones harboring intact or defective proviruses. We characterized the source of NSV in two PWH who never reached undetectable viral load despite ART adherence. Remarkably, in each participant, we found a diverse set of defective viral genomes all sharing the same fatal deletions. We found that this paradoxical accumulation of mutations by viruses with fatal defects was driven by superinfection with intact viruses, resulting in mobilization of defective genomes and accumulation of additional mutations during untreated infection. We show that these defective proviruses interfere with WT virus replication, conditionally replicate, and, in one case, have an R 0 > 1, enabling in vivo spread. Despite this, clinical outcomes show no evidence of a beneficial effect of these DIPs.

Human immunodeficiency virus type 1 (HIV-1) genome diversification is a key determinant of viral evolution and the pathogenesis of HIV/AIDS. Antiretroviral therapy is non-curative, and in the context of monitoring the latent reservoir, precision tools are needed to detect and enumerate HIV-1 genomes as well as to assess their heterogeneity, replication potential, and predict responses to therapy. Current sequencing-based methodologies are often unable to confirm intact genomes and most cell-based reporters provide limited information pertaining to viral fitness. In this study, we describe dual reporter sensor cells (DRSCs), an imaging-based reporter system designed to detect HIV-1 infection and measure several independent attributes of the virus in a single-cell high-content assay. We show that the DRSC assay can be used to measure infection, viral gene activation kinetics, and quantify viral circumvention of host antiviral responses. Using the DRSCs, we confirmed markedly different functional heterogeneity for vif alleles derived from diverse HIV-1 strains and subtypes affecting both rates of APOBEC3G degradation and the cell cycle. Furthermore, the assay allowed for the delineation of virus co-receptor preference (X4- vs R5-tropism) and visualization of virion assembly. Overall, our study illustrates proof-of-principle for a multivariate imaging-based cell-based system capable of detecting HIV-1 and studying viral genetic variability with greater data richness relative to prior available modalities.

Human immunodeficiency virus type 1 (HIV-1) is highly heterogeneous and constantly mutating. These changes drive immune evasion and can cause treatment efforts to fail. Here, we describe the "dual reporter sensor cell" (DRSC) assay; a novel imaging-based approach that allows for the detection of HIV-1 infection coupled with a multivariate definition of several independent phenotypic aspects of viral genome activity in a single integrated assay. We validate the DRSC system by studying lab-adapted and patient isolate-derived versions of the viral Vif accessory protein, confirming marked differences in the capacity of diverse vif alleles to mediate downregulation of antiviral APOBEC3G proteins and dysregulate the cell cycle.

Lymph node T follicular helper (Tfh) cells and germinal center (GC) B cells are critical to generate potent antibodies but are rarely possible to study in humans. To understand how Tfh/GC B-cell interactions during acute HIV-1 infection (AHI) impact the generation of HIV-specific antibodies, we performed a unique cross-sectional analysis of inguinal lymph node biopsies taken prior to antiretroviral therapy (ART) initiation in AHI. Although total Tfh and GC B cell frequencies did not change during AHI, increased frequencies of proliferating Th1-like CXCR3+ Tfh, CXCR3+ non-GC B cells, and total CXCR3+ GC B cells correlated with gp120-specific IgG antibody levels in AHI. Frequencies of proliferating CXCR3+ Tfh in AHI also correlated with gp120-specific IgG antibody levels after 48 weeks of ART, antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, and increased antibody binding to infected cells after ART. Importantly, while beneficial for antibody development, CXCR3+ Tfh cells were also infected by HIV-1 at higher frequencies than their CXCR3- counterparts and may contribute to the initial dissemination of HIV-1 in follicles. Together, these data suggest that activation of CXCR3+ Tfh cells is associated with induction of the germinal center response and subsequent antibody development, making these cells an important target for future therapeutic interventions.

Early initiation of antiretroviral therapy (ART) is important to limit the seeding of the long-lasting HIV-1 reservoir; however, it also precludes the development of HIV-specific antibodies that can help control the virus if ART is stopped. Antibody development occurs within germinal centers in the lymph node and requires activation of both antigen-specific B cells and T follicular helper cells (Tfh), a specialized CD4+ cell that provides B cell help. To understand how early ART initiation may prohibit antibody development, we analyzed the frequencies and activation status of Tfh and B cells in lymph node biopsies collected in the different stages of acute HIV-1 infection. Our data suggest that decreased antibody development after early ART initiation may be due to limited germinal center development at the time of treatment and that new interventions that target activation of CXCR3+ Tfh may be beneficial to increase long-term HIV-specific antibody levels.

Antiretroviral therapy (ART) inhibits Human Immunodeficiency Virus (HIV) replication to maintain undetectable viral loads in people living with HIV, but does not result in a cure. Due to the significant challenges of lifelong ART for many, there is strong interest in therapeutic strategies that result in cure. Recent clinical trials have shown that administration of broadly neutralizing antibodies (bnAbs) when there is some viremia can lead to ART-free viral control in some people; however, the underlying mechanisms are unclear. Our computational modeling shows that bnAbs administered in the presence of some viremia promote the evolution of autologous antibodies (aAbs) that target diverse epitopes of HIV spike proteins. This "net" of polyclonal aAbs could confer control since evasion of this response would require developing mutations in multiple epitopes. Our results provide a common mechanistic framework underlying recent clinical observations upon bnAb/ART therapy, and they should also motivate and inform new trials.

The development of effective vaccines is crucial for combating current and emerging pathogens. Despite significant advances in the field of vaccine development there remain numerous challenges including the lack of standardized data reporting and curation practices, making it difficult to determine correlates of protection from experimental and clinical studies. Significant gaps in data and knowledge integration can hinder vaccine development which relies on a comprehensive understanding of the interplay between pathogens and the host immune system. In this review, we explore the current landscape of vaccine development, highlighting the computational challenges, limitations, and opportunities associated with integrating diverse data types for leveraging artificial intelligence (AI) and machine learning (ML) techniques in vaccine design. We discuss the role of natural language processing, semantic integration, and causal inference in extracting valuable insights from published literature and unstructured data sources, as well as the computational modeling of immune responses. Furthermore, we highlight specific challenges associated with uncertainty quantification in vaccine development and emphasize the importance of establishing standardized data formats and ontologies to facilitate the integration and analysis of heterogeneous data. Through data harmonization and integration, the development of safe and effective vaccines can be accelerated to improve public health outcomes. Looking to the future, we highlight the need for collaborative efforts among researchers, data scientists, and public health experts to realize the full potential of AI-assisted vaccine design and streamline the vaccine development process.

Acute HIV-1 infection (AHI) is a transient period where the virus causes evident damage to the immune system, including an extensive apoptosis of CD4+ T cells associated with a high level of activation and a major cytokine storm to fight the invading virus. HIV infection establishes persistence by integrating the viral genome into host cell DNA in both replicating and non-replicating forms, effectively hiding from immune surveillance within infected lymphocytes as cellular reservoirs. The measurement of total HIV-1 DNA in peripheral blood mononuclear cells (PBMCs) is a reliable reflection of this reservoir. Initiating treatments during AHI with nucleoside reverse transcriptase inhibitors (NRTIs) and/or integrase strand transfer inhibitors (INSTIs) is essential to alter the dynamics of the global reservoir expansion, and to reduce the establishment of long-lived cellular and tissue reservoirs, while preserving and enhancing specific and non-specific immune responses. Furthermore, some of the patients treated at the AHI stage may become post-treatment controllers and should be informative regarding the mechanism of viral control, so patients treated during AHI are undoubtedly the best candidates to test innovative remission strategies toward a functional cure that could play a pivotal role in long-term HIV control. AHI is characterized by high levels of viral replication, with a significant increase in the risk of HIV transmission. Detecting AHI and initiating early treatment following diagnosis provides a window of opportunity to control the epidemic, particularly in high-risk populations.

The ability of HIV-1 Nef to counteract the host restriction factor SERINC5 and enhance virion infectivity has been well established. However, the impact of long-term within-host Nef evolution on this antagonistic capability remains unclear.

Analysis of longitudinal activity of Nef in antagonizing SERINC5.

We investigated the downregulation activity of Nef against SERINC5 at different stages of infection by analyzing the cognate transmitted/founder, set point, and/or chronic Nef isolates from a cohort of 19 people with either subtype B or C HIV-1.

The Nef isolates from different stages exhibited varying abilities to antagonize SERINC5. Long-term evolution resulted in mutations accumulated in Nef and a decline of Nef-mediated SERINC5 downregulation function in subtype B, but not in subtype C viruses, leading to a rapid reduction in viral load from peak viremia. Furthermore, we identified four polymorphisms of both subtype B and C Nef that are associated with variations in the SERINC5 antagonistic function and viral infectivity. HIV-1 NL4-3 variants encoding Nef E63G, A83G, R105K, or D108E mutants exhibited reduced replication capacity through a SERINC5-dependent mechanism. However, among different subjects, only a small part of naturally occurring mutations at these sites were selected by host T-cell responses, suggesting a limited impact of host T-cell responses on influencing Nef's ability to antagonize SERINC5.

These results highlight the potential contribution of functional variation in Nef to differences in HIV-1 pathogenesis and provide significant implications for understanding the evolutionary interaction between Nef and SERINC5 in vivo .

An alternative to lifelong antiretroviral therapy (ART) is needed to achieve durable control of HIV-1. Here, we show that adeno-associated virus (AAV) delivery of two rhesus macaque antibodies to the simian immunodeficiency virus (SIV) envelope glycoprotein (Env) with potent neutralization and antibody-dependent cellular cytotoxicity can prevent viral rebound in macaques infected with barcoded SIVmac239M after discontinuing suppressive ART. After AAV administration, sustained antibody expression with minimal antidrug antibody responses was achieved in all but one animal. After ART withdrawal, SIV replication rebounded within 2 weeks in all control animals but remained <15 copies per milliliter in plasma for more than a year in four of the eight animals that received AAV vectors encoding Env-specific antibodies. Viral sequences from animals that rebounded with delayed kinetics exhibited restricted clonal diversity and antibody escape mutations in Env. Thus, sustained expression of antibodies with potent antiviral activity can afford durable, ART-free containment of pathogenic SIV infection.

Discovered in 1994 in lesions of an AIDS patient, Kaposi's sarcoma-associated herpesvirus (KSHV) is a member of the gammaherpesvirus subfamily of the Herpesviridae family, which contains a total of nine that infect humans. These viruses all contain a large envelope glycoprotein, glycoprotein B (gB), that is required for viral fusion with host cell membrane to initial infection. Although the atomic structures of five other human herpesviruses in their postfusion conformation and one in its prefusion conformation are known, the atomic structure of KSHV gB has not been reported. Here, we report the first structure of the KSHV gB ectodomain determined by single-particle cryogenic electron microscopy (cryoEM). Despite a similar global fold between herpesvirus gB, KSHV gB possesses local differences not shared by its relatives in other herpesviruses. The glycosylation sites of gB are arranged in belts down the symmetry axis with distinct localization compared to that of other herpesviruses, which occludes certain antibody binding sites. An extended glycan chain observed in domain I (DI), located proximal to the host membrane, may suggest its possible role in host cell attachment. Local flexibility of domain IV (DIV) governed by molecular hinges at its interdomain junctions identifies a means for enabling conformational change. A mutation in the domain III (DIII) central helix disrupts incorporation of gB into KSHV virions despite adoption of a canonical fold in vitro. Taken together, this study reveals mechanisms of structural variability of herpesvirus fusion protein gB and informs its folding and immunogenicity.IMPORTANCEIn 1994, a cancer-causing virus was discovered in lesions of AIDS patients, which was later named Kaposi's sarcoma-associated herpesvirus (KSHV). As the latest discovered human herpesvirus, KSHV has been classified into the gammaherpesvirus subfamily of the Herpesviridae. In this study, we have expressed KSHV gB and employed cryogenic electron microscopy (cryoEM) to determine its first structure. Importantly, our structure resolves some glycans beyond the first sugar moiety. These glycans are arranged in a pattern unique to KSHV, which impacts the antigenicity of KSHV gB. Our structure also reveals conformational flexibility caused by molecular hinges between domains that provide clues into the mechanism behind the drastic change between prefusion and postfusion states.

In this issue of Cell Host & Microbe, Hesselman et al. investigate the relationship between the presence of non-canonical cysteine residues in HIV-1 V1 region of the Envelope glycoprotein and the development of neutralization breadth through population-based analyses.

Decoding the HIV-1 immune response, including its humoral arm, in post-treatment controllers (PTCs) is paramount to unveil immune correlates of viral control, which could help developing novel strategies towards HIV-1 remission. Here, we review novel findings on the humoral response to HIV-1 in PTCs.

New data reveal the heterogeneity of humoral immune profiles in PTCs, principally influenced by viral exposure and dynamics. Stably aviremic PTCs, akin early ART-treated individuals, show minimal antibody B-cell response. Conversely, virally exposed PTCs develop functionally coordinated and effective humoral responses to HIV-1. They can produce antibodies cross-neutralizing heterologous HIV-1 viruses, including broadly neutralizing antibodies (bNAbs) exerting selective immune pressure. PTCs also elicit neutralizing antibodies against contemporaneous autologous viruses presumed to play a major role in sustaining viral suppression.

The immune mechanisms underlying virologic control in PTCs likely involve various immune effectors. Notably, functional HIV-1 humoral responses can generate bNAbs and autologous neutralizing antibodies; however, their exact contribution to maintaining long-term control of plasma viremia and the precise mechanisms driving their induction require further investigation.

For use in prevention and treatment, HIV-1 broadly neutralizing antibodies (bnAbs) have to overcome Env conformational heterogeneity of viral quasispecies and neutralize with constant high potency. Comparative analysis of neutralization data from the CATNAP database revealed a nuanced relationship between bnAb activity and Env conformational flexibility, with substantial epitope-specific variation of bnAb potency ranging from increased to decreased activity against open, neutralization-sensitive Env. To systematically investigate the impact of variability in Env conformation on bnAb potency we screened 126 JR-CSF point mutants for generalized neutralization sensitivity to weakly neutralizing antibodies (weak-nAbs) depending on trimer opening and plasma from people with chronic HIV-1 infection. 23 mutations resulted in a highly neutralization sensitive phenotype, which was associated with de-stabilization of the closed, prefusion conformation. Including 19 of these mutants into a Sensitivity Env mutant panel (SENSE-19), we classified bnAbs according to potency variations in response to trimer opening. To verify that these sensitivity patterns are independent of the in vitro assay system, replication-competent SENSE-19 mutant viruses were tested on primary CD4 T cells. While loss of potency on SENSE-19 was registered for bnAbs from several classes recognizing quaternary epitopes on pre-triggered Env, structural destabilization benefitted MPER bnAbs and other inhibitors known to have post-CD4 attachment neutralization activity. Importantly, for a subset of CD4bs bnAbs, and the interface bnAb PGT151, particularly low potency variation was noted, suggesting that Env conformational tolerance can be achieved but is not the rule. In summary, SENSE-19 screens revealed distinct tolerance levels to Env conformational intermediates between bnAbs that provide mechanistic insights in their function and broaden current neutralization breadth assessments.

Enveloped RNA viruses have been causative agents of major pandemic outbreaks in the recent past. Glycans present on these virus surface proteins are critical for multiple processes during the viral infection cycle. Presence of glycans serves as a key determinant of immunogenicity, but intrinsic heterogeneity, dynamics, and evolutionary shifting of glycans in heavily glycosylated enveloped viruses confounds typical structure-function analysis. Glycosylation sites are also conserved across different viral families, which further emphasizes their functional significance. In this review, we summarize findings regarding structure-function correlation of glycans on enveloped RNA virus proteins.

During the process by which human immunodeficiency virus (HIV-1) enters cells, the envelope glycoprotein (Env) trimer on the virion surface engages host cell receptors. Binding to the receptor CD4 induces Env to undergo transitions from a pretriggered, "closed" (State-1) conformation to more "open" (State 2/3) conformations. Most broadly neutralizing antibodies (bNAbs), which are difficult to elicit, recognize the pretriggered (State-1) conformation. More open Env conformations are recognized by poorly neutralizing antibodies (pNAbs), which are readily elicited during natural infection and vaccination with current Env immunogens. Env heterogeneity likely contributes to HIV-1 persistence by skewing antibody responses away from the pretriggered conformation. The conformationally flexible gp160 Env precursor on the infected cell or virion surface potentially presents multiple pNAb epitopes to the host immune system. Although proteolytic cleavage to produce the functional, mature Env trimer [(gp120/gp41)3] stabilizes State-1, many primary HIV-1 Envs spontaneously sample more open conformations. Here, we establish inducible cell lines that produce replication-defective HIV-1 particles with Env trimers stabilized in a pretriggered conformation. The mature Env is enriched on virus-like particles (VLPs). Using complementary approaches, we estimate an average of 25-50 Env trimers on each VLP. The stabilizing changes in Env limit the natural conformational heterogeneity of the VLP Env trimers, allowing recognition by bNAbs but not pNAbs. These defective VLPs provide a more homogeneous source of pretriggered Env trimers in a native membrane environment. Thus, these VLPs may facilitate the characterization of this functionally important Env conformation and its interaction with the immune system.IMPORTANCEA major impediment to the development of an effective HIV/AIDS vaccine is the inefficiency with which human immunodeficiency virus (HIV-1) envelope glycoproteins elicit antibodies that neutralize multiple virus strains. Neutralizing antibodies recognize a particular shape of the envelope glycoproteins that resides on the viral membrane before the virus engages the host cell. Here, we report the creation of stable cell lines that inducibly produce non-infectious HIV-like particles. The normally flexible envelope glycoprotein spikes on these virus-like particles have been stabilized in a conformation that is recognized by broadly neutralizing antibodies. These virus-like particles allow the study of the envelope glycoprotein conformation, its modification by sugars, and its ability to elicit desired neutralizing antibodies.

An antibody-based HIV-1 vaccine will require the induction of potent cross-reactive HIV-1-neutralizing responses. To demonstrate feasibility toward this goal, we combined vaccination targeting the fusion-peptide site of vulnerability with infection by simian-human immunodeficiency virus (SHIV). In four macaques with vaccine-induced neutralizing responses, SHIV infection boosted plasma neutralization to 45%-77% breadth (geometric mean 50% inhibitory dilution [ID50] ∼100) on a 208-strain panel. Molecular dissection of these responses by antibody isolation and cryo-electron microscopy (cryo-EM) structure determination revealed 15 of 16 antibody lineages with cross-clade neutralization to be directed toward the fusion-peptide site of vulnerability. In each macaque, isolated antibodies from memory B cells recapitulated the plasma-neutralizing response, with fusion-peptide-binding antibodies reaching breadths of 40%-60% (50% inhibitory concentration [IC50] < 50 μg/mL) and total lineage-concentrations estimates of 50-200 μg/mL. Longitudinal mapping indicated that these responses arose prior to SHIV infection. Collectively, these results provide in vivo molecular examples for one to a few B cell lineages affording potent, broadly neutralizing plasma responses.

Human immunodeficiency virus (HIV)-1 exhibits remarkable genetic diversity. For this reason, an effective HIV-1 vaccine must elicit antibodies that can neutralize many variants of the virus. While broadly neutralizing antibodies (bnAbs) have been isolated from HIV-1 infected individuals, a general understanding of the virus-antibody coevolutionary processes that lead to their development remains incomplete. We performed a quantitative study of HIV-1 evolution in two individuals who developed bnAbs. We observed strong selection early in infection for mutations affecting HIV-1 envelope glycosylation and escape from autologous strain-specific antibodies, followed by weaker selection for bnAb resistance later in infection. To confirm our findings, we analyzed data from rhesus macaques infected with viruses derived from the same two individuals. We inferred remarkably similar fitness effects of HIV-1 mutations in humans and macaques. Moreover, we observed a striking pattern of rapid HIV-1 evolution, consistent in both humans and macaques, that precedes the development of bnAbs. Our work highlights strong parallels between infection in rhesus macaques and humans, and it reveals a quantitative evolutionary signature of bnAb development.

Addressing the need for accessible SARS-CoV-2 testing, carboxy-PEG 12-thiol functionalized gold nanoparticles conjugates were developed for rapid point-of-care (POC) detection against SARS-CoV-2 spike protein, pseudo-SARS-CoV-2, and authentic Beta SARS-CoV-2 virus particles. These conjugates leverage gold nanoparticles (AuNPs) as signal transducers, cross-linked to either angiotensin-converting enzyme 2 (ACE2) or SARS-CoV-2 spike protein receptor-binding domain (RBD) antibodies as bioreceptors and showed a distinct color shift from pink to blue. To assess their POC feasibility, the conjugates were integrated into facemasks and breathalyzers, wherein aerosolized SARS-CoV-2 antigens were successfully detected, producing a color change within 10 and 30 minutes for the breathalyzer and facemask prototypes, respectively. Furthermore, we explored quantitative analysis using varying concentrations of SARS-CoV-2 spike protein. Both conjugates demonstrated a linear relationship between blue color intensity and virus concentration, with linear ranges of 0.08-0.6 ng/mL and 0.04-0.5 ng/mL, respectively. Low limits of detection and quantification were also achieved. They exhibited specificity, responding solely to SARS-CoV-2 even in complex matrices containing diverse proteins, including the SARS-CoV-1 spike protein. Precision tests yielded coefficient of variations below 2 %, showcasing their remarkable reproducibility. This work presents a promising approach for rapid, sensitive, and specific POC detection of SARS-CoV-2 paving the way for improved pandemic response and management.

N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (HTCC), a quaternized chitosan derivative, has been shown to exhibit a broad spectrum of antimicrobial activity, especially against bacteria and enveloped viruses. Despite this, molecular docking studies showing its atomic-level mechanisms against these microorganisms are scarce. Here, for the first time, we employed molecular docking analyses to investigate the potential antibacterial activity of HTCC against Staphylococcus aureus and its antiviral activity against human immunodeficiency virus 1 (HIV-1). According to the findings, HTCC exhibited promising antibacterial activity with high binding affinities; however, it had limited antiviral activity. To validate these theoretical outcomes, experimental studies were conducted. Different derivatives of HTCC were synthesized and characterized using NMR, XRD, FTIR, and DLS. The in vitro assays validated the potent antibacterial efficacy of HTCC against S. aureus, whereas the antiviral studies did not show good antiviral activity. However, our research also revealed a promising avenue for further exploration of the antimicrobial activity of HTCC nanoparticles (NPs), since, thus far, no studies have been conducted to show the antiviral activity of HTCC NPs against HIV-1. The nanosized HTCC exhibited superior antiviral performance compared to the parent polymers, with complete (100%) inhibition of HIV-1 viral activity at the highest tested concentration (0.33 mg/mL).

Upon binding to the host cell receptor, CD4, the pretriggered (State-1) conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer undergoes transitions to downstream conformations important for virus entry. State 1 is targeted by most broadly neutralizing antibodies (bNAbs), whereas downstream conformations elicit immunodominant, poorly neutralizing antibody (pNAb) responses. Extraction of Env from the membranes of viruses or Env-expressing cells disrupts the metastable State-1 Env conformation, even when detergent-free approaches like styrene-maleic acid lipid nanoparticles (SMALPs) are used. Here, we combine three strategies to solubilize and purify mature membrane Envs that are antigenically native (i.e., recognized by bNAbs and not pNAbs): (1) solubilization of Env with a novel amphipathic copolymer, Amphipol A18; (2) use of stabilized pretriggered Env mutants; and (3) addition of the State-1-stabilizing entry inhibitor, BMS-806. Amphipol A18 was superior to the other amphipathic copolymers tested (SMA and AASTY 11-50) for preserving a native Env conformation. A native antigenic profile of A18 Env-lipid-nanodiscs was maintained for at least 7 days at 4°C and 2 days at 37°C in the presence of BMS-806 and was also maintained for at least 1 h at 37°C in a variety of adjuvants. The damaging effects of a single cycle of freeze-thawing on the antigenic profile of the A18 Env-lipid-nanodiscs could be prevented by the addition of 10% sucrose or 10% glycerol. These results underscore the importance of the membrane environment to the maintenance of a pretriggered (State-1) Env conformation and provide strategies for the preparation of lipid-nanodiscs containing native membrane Envs.IMPORTANCEThe human immunodeficiency virus (HIV-1) envelope glycoproteins (Envs) mediate virus entry into the host cell and are targeted by neutralizing antibodies elicited by natural infection or vaccines. Detailed studies of membrane proteins like Env rely on purification procedures that maintain their natural conformation. In this study, we show that an amphipathic copolymer A18 can directly extract HIV-1 Env from a membrane without the use of detergents. A18 promotes the formation of nanodiscs that contain Env and membrane lipids. Env in A18-lipid nanodiscs largely preserves features recognized by broadly neutralizing antibodies (bNAbs) and conceals features potentially recognized by poorly neutralizing antibodies (pNAbs). Our results underscore the importance of the membrane environment to the native conformation of HIV-1 Env. Purification methods that bypass the need for detergents could be useful for future studies of HIV-1 Env structure, interaction with receptors and antibodies, and immunogenicity.

HIV-1 alters the dynamics and distribution of tetraspanins, a group of proteins integral to membrane organization, to facilitate both entry and egress. Notably, the tetraspanin CD9 is dysregulated during HIV-1 infection, correlating with multifaceted effects on viral replication. Here, we generated llama-derived nanobodies against CD9 to restrict HIV-1 replication. We immunized llamas with recombinant large extracellular loop of CD9 and identified eight clonally distinct nanobodies targeting CD9, each exhibiting a range of affinities and differential binding to cell surface-expressed CD9. Notably, nanobodies T2C001 and T2C002 demonstrated low nanomolar affinities and exhibited differential sensitivities against endogenous and overexpressed CD9 on the cell surface. Although CD9-directed nanobodies did not impede the early stages of HIV-1 life cycle, they effectively inhibited virus-induced syncytia formation and virus replication in T cells and monocyte-derived macrophages. This discovery opens new avenues for host-targeted therapeutic strategies, potentially augmenting existing antiretroviral treatments for HIV-1.

Antibody-dependent cellular phagocytosis (ADCP) has been implicated in protection against HIV-1. However, methods for measuring ADCP currently rely on the phagocytosis of gp120- or gp41-coated beads that do not reflect physiologically relevant conformations of the viral envelope glycoprotein or the size of a virus-infected cell. We therefore developed a novel approach for measuring ADCP of HIV-infected cells expressing natural conformations of Env. A monocytic cell line (THP-1 cells) or primary human monocytes were incubated with a CD4+ T cell line that expresses eGFP upon HIV-1 infection in the presence of antibodies and ADCP was measured as the accumulation of eGFP+ material by flow cytometry. The internalization of HIV-infected cells by monocytes was confirmed visually by image-capture flow cytometry. Cytoskeletal remodeling, pseudopod formation and phagocytosis were also observed by confocal microscopy. We found that potent broadly neutralizing antibodies (bnAbs), but not non-neutralizing antibodies (nnAbs), mediate efficient phagocytosis of cells infected with either primary or lab-adapted HIV-1. A nnAb to a CD4-inducible epitope of gp120 (A32) failed to enable ADCP of HIV-infected cells but mediated efficient phagocytosis of gp120-coated beads. Conversely, a bnAb specific to intact Env trimers (PGT145) mediated potent ADCP of HIV-infected cells but did not facilitate the uptake of gp120-coated beads. These results underscore the importance of measuring ADCP of HIV-infected cells expressing physiologically relevant conformations of Env and show that most antibodies that are capable of binding to Env trimers on virions to neutralize virus infectivity are also capable of binding to Env on the surface of virus-infected cells to mediate ADCP.

HIV-1 envelope (Env) is the key target for antibodies (Abs) against the virus and thus an important HIV-1 vaccine component. Env is synthesized from a gp160 precursor with a signal peptide (SP) at its N-terminus. This study investigated the influence of the SP on Env antigenicity and immunogenicity.

Env proteins from two HIV-1 isolates, AA05 and AC02, were analyzed as gp120 and gp160 in their native wild-type (WT) forms and as chimeras with swapped SPs (AA05-02 and AC02-05). The WT and chimeric Env were assessed for antigenicity and glycosylation using monoclonal antibodies (mAbs) and glycan probes. Immunogenicity was tested in mice using three vaccine types: gp120 protein, gp120 DNA+gp120 protein, and gp120 DNA+gp160 DNA.

The recombinant AC02 gp120 protein was antigenically superior to AA05 as indicated by higher reactivity with most mAbs tested. When SPs were swapped, the antigenicity of the chimeric gp120s (AA05-02 and AC02-05) resembled that of the gp120s from which the SPs were derived; AA05-02 was similar to AC02 and vice versa. Glycan probe reactivity followed a similar pattern: AA05-02 and AC02 showed similar affinity to high-mannose specific mAbs and lectins. Interestingly, the antigenicity of gp160s showed an opposite pattern; membrane-bound gp160 expressed with the AA05 SP (AA05 and AC02-05) showed greater mAb binding than gp160 with the AC02 SP (AC02 and AA05-02). Mice immunized with gp120 protein showed that AA05-02 induced stronger cross-reactive binding Ab responses than AA05 WT, and AC02 elicited stronger responses than AC02-05, indicating AC02 SP enhanced gp120 immunogenicity. However, when DNA vaccines were included (gp120 DNA+gp120 protein and gp120 DNA+gp160 DNA), the use of heterologous SPs diminished the immunogenicity of the WT immunogens. Among the three vaccine regimens tested, only gp120 DNA+gp160 DNA immunization elicited low-level Tier 2 neutralizing Abs, with AA05 WT inducing Abs with greater neutralization capabilities than AA05-02.

These data demonstrate that the SP can significantly impact the antigenicity and immunogenicity of HIV-1 Env proteins. Hence, while SP swapping is a common practice in constructing Env immunogens, this study highlights the importance of careful consideration of the effects of replacing native SPs on the immunogenicity of Env vaccines.

Despite treatment and other interventions, an effective prophylactic HIV vaccine is still an essential goal in the control of HIV. Inducing robust and long-lasting antibody responses is one of the main targets of an HIV vaccine. The delivery of HIV envelope glycoproteins (Env) using nanoparticle (NP) platforms has been shown to elicit better immunogenicity than soluble HIV Env. In this paper, we describe the development of a nanoparticle-based vaccine decorated with HIV Env using the SpyCatcher/SpyTag system. The Env utilised in this study, CAP255, was derived from a transmitted founder virus isolated from a patient who developed broadly neutralising antibodies. Negative stain and cryo-electron microscopy analyses confirmed the assembly and stability of the mi3 into uniform icosahedral NPs surrounded by regularly spaced CAP255 gp140 Env trimers. A three-dimensional reconstruction of CAP255 gp140 SpyTag-SpyCatcher mi3 clearly showed Env trimers projecting from the centre of each of the pentagonal dodecahedral faces of the NP. To our knowledge, this is the first study to report the formation of SpyCatcher pentamers on the dodecahedral faces of mi3 NPs. To investigate the immunogenicity, rabbits were primed with two doses of DNA vaccines expressing the CAP255 gp150 and a mosaic subtype C Gag and boosted with three doses of the NP-developed autologous Tier 2 CAP255 neutralising antibodies (Nabs) and low levels of heterologous CAP256SU NAbs.

Even during extended periods of effective immunological control, a substantial dynamic of the viral genome can be observed in different cellular compartments in HIV-1 positive individuals, indicating the persistence of active viral reservoirs. To obtain further insights, we studied changes in the proviral as well as in the viral HIV-1 envelope (Env) sequence along with transcriptional, translational and viral outgrowth activity as indicators for viral dynamics and genomic intactness. Our study identified distinct reservoir patterns that either represented highly sequence-diverse HIV-1 populations or only a single / few persisting virus variants. The single dominating variants were more often found in individuals starting ART during early infection phases, indicating that early treatment might limit reservoir diversification. At the same time, more sequence-diverse HIV reservoirs correlated with a poorer immune status, indicated by lower CD4 count, a higher number of regimen changes and more co-morbidities. Furthermore, we noted that in T-cell populations in the peripheral blood, replication-competent HIV-1 is predominantly present in Lymph node homing TN (naïve) and TCM (central memory) T cells. Provirus genomes archived in TTM (transitional memory) and TEM (effector memory) T cells more frequently tended to carry inactivating mutations and, population-wise, possess changes in the genetic diversity. These discriminating properties of the viral reservoir in T-cell subsets may have important implications for new early therapy strategies, underscoring the critical role of early therapy in preserving robust immune surveillance and constraining the viral reservoir.

Viruses utilize host cells at all stages of their life cycle, from the transcription of genes and translation of viral proteins to the release of viral copies. The human immune system counteracts viruses through a variety of complex mechanisms, including both innate and adaptive components. Viruses have an ability to evade different components of the immune system and affect them, leading to disruption. This review covers contemporary knowledge about the virus-induced complex interplay of molecular interactions, including regulation of transcription and translation in host cells resulting in the modulation of immune system functions. Thorough investigation of molecular mechanisms and signaling pathways that are involved in modulating of host immune response to viral infections can help to develop novel approaches for antiviral therapy. In this review, we consider new therapeutic approaches for antiviral treatment. Modern therapeutic strategies for the treatment and cure of human immunodeficiency virus (HIV) are considered in detail because HIV is a unique example of a virus that leads to host T lymphocyte deregulation and significant modulation of the host immune response. Furthermore, peculiarities of some promising novel agents for the treatment of various viral infections are described.

Herpes simplex virus 1 (HSV-1) gD interaction with the host cell receptor nectin-1 triggers the membrane fusion cascade during viral entry. Potent neutralizing antibodies to gD prevent receptor-binding or prevent gD interaction with gH/gL critical for fusion. HSV has many strategies to evade host immune responses. We investigated the ability of virion envelope gC to protect envelope gD from antibody neutralization. HSV-1 lacking gC was more sensitive to neutralization by anti-gD monoclonal antibodies than a wild type rescuant virus. gD in the HSV-1 gC-null viral envelope had enhanced reactivity to anti-gD antibodies compared to wild type. HSV-1 ΔgC binding to the nectin-1 receptor was more readily inhibited by a neutralizing anti-gD monoclonal antibody. HSV-1 ΔgC was also more sensitive to inhibition by soluble nectin-1 receptor. The viral membrane protein composition of HSV-1 ΔgC was equivalent to that of wild type, suggesting that the lack of gC is responsible for the increased reactivity of gD-specific antibodies and the consequent increased susceptibility to neutralization by those antibodies. Together, the results suggest that gC in the HSV-1 envelope shields both receptor-binding domains and gH/gL-interacting domains of gD from neutralizing antibodies, facilitating HSV cell entry.

The pretriggered conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer ((gp120/gp41)3) is targeted by virus entry inhibitors and broadly neutralizing antibodies (bNAbs). The lability of pretriggered Env has hindered its characterization. Here, we produce membrane Env variants progressively stabilized in pretriggered conformations, in some cases to a degree beyond that found in natural HIV-1 strains. Pretriggered Env stability correlated with stronger trimer subunit association, increased virus sensitivity to bNAb neutralization, and decreased capacity to mediate cell-cell fusion and virus entry. For some highly stabilized Env mutants, after virus-host cell engagement, the normally inaccessible gp120 V3 region on an Env intermediate became targetable by otherwise poorly neutralizing antibodies. Thus, evolutionary pressure on HIV-1 Env to maintain trimer integrity, responsiveness to the CD4 receptor, and resistance to antibodies modulates pretriggered Env stability. The strongly stabilized pretriggered membrane Envs reported here will facilitate further characterization of this functionally important conformation.