Although a protective HIV-1 vaccine has not yet been realized, significant progress has been made in vaccine designs that trigger B cell lineages with potential to produce broadly neutralizing antibodies (bnAbs). Advancing these strategies by optimizing vaccine boosting regimens requires early detection of maturing antibodies with neutralizing activity against native envelope glycoprotein (Env) trimers and streamlined strategies to identify antibodies as they begin to manifest desired levels of breadth and potency. Thus, we designed three types of pseudovirus screening panels based on Envs of contemporary HIV-1 isolates to facilitate detection of bnAb lineages that are on favorable trajectories during a vaccination course. The panels were selected from Tier 2 Transmitted Founder Lineage (TFL) HIV-1 Envs from placebo participants in the Antibody Mediated Prevention (AMP) efficacy trials. Using 15 bnAbs to evaluate the neutralization sensitivity of the viruses, we selected 8-member bnAb class-specific panels most sensitive to bnAbs representing their class: V2-apex, V3-glycan, CD4-receptor binding site (CD4bs), Membrane-Proximal External Region (MPER), or fusion peptide (FP). Next, we combined the most sensitive viruses among the class-specific panels to create a 12-virus panel to enable optimal detection of low-titer bnAb activity across epitope specificities. Finally, as HIV-1 continues to evolve greater levels of antigenic diversity and as current global pseudoviruses bnAb panels rely on viruses collected more than twenty years ago, we showed the importance of using contemporary viral panels to assess bnAb breadth and potency and designed a 12-virus panel representative of the spectrum neutralization profiles among AMP placebo viruses. We characterized pseudoviruses bearing each selected Env using standardized human sera to confirm their Tier 2 status and biological relevance. These updated panels enable sensitive screening of neutralization activity in vaccine studies and can also provide a realistic assessment of the expected breadth and potency of maturing responses against contemporary HIV-1 Envs.

BACKGROUND:Passive immunisation with a combination of broadly neutralising monoclonal antibodies (mAbs) presents a potential HIV-1 prevention modality. This study (HVTN 140/HPTN 101) sought to evaluate PGDM1400LS (targeting V3-glycan) administered alone (part A, previously reported), and in combination (part B) with VRC07-523LS (targeting CD4 binding site) and PGT121.414.LS (targeting V2-apex) in healthy adults without HIV-1. METHODS:The study was a phase 1, multicentre, randomised, open-label trial done across the USA (five sites), Kenya (one site), South Africa (four sites), and Zimbabwe (three sites). Part B participants were randomly assigned to five groups (n=16 each), all receiving the three mAbs at months 0 and 4: infusion group T6 (20 mg/kg each mAb intravenously), T7 (20 mg/kg each mAb subcutaneously), T8 (1·4 g each mAb intravenously), T9 (1·4 g each mAb subcutaneously), and T10 (40 mg/kg each mAb intravenously). Primary endpoints were safety and tolerability throughout the study, and pharmacokinetics and neutralising activity on days 0, 3, 6, 28, 56, 112, 168, 224, and 280. Serum mAb concentrations over time were assessed via a validated anti-idiotype binding assay and analysed with two-compartment population pharmacokinetics models. Serum neutralisation titres were assessed by a validated TZM-bl assay. The study is registered at ClinicalTrials.gov (NCT05184452) and is complete. FINDINGS:For the 80 part B participants enrolled from March 21 to Oct 5, 2022, the median age was 27·0 years, with 47·5% females. Most participants had mild-to-moderate solicited local and systemic symptoms. No serious adverse events were reported. On the basis of pharmacokinetics data from part A and part B combined, the estimated elimination half-life of PGDM1400LS was 53 days (51·4-55·3). Subcutaneous administration of PGDM1400LS exhibited bioavailability of 73·0% (67·5-78·5%) relative to intravenous administration of the same dose. PGT121.414.LS had an estimated elimination half-life of 65 days (61·8-68·1) with subcutaneous bioavailability of 77·7% (71·2-84·1), and VRC07-523LS had an estimated elimination half-life of 44 days (41·6-45·7) with a subcutaneous bioavailability of 80·1% (71·4-88·8). Both weight-based and fixed-dose regimens showed similar pharmacokinetic profiles. Observed neutralisation titres were consistent with those predicted on the basis of concentrations and in vitro neutralisation. No treatment-induced anti-drug antibody responses were detected. INTERPRETATION:The mAb combination of PGDM1400LS, PGT121.414.LS, and VRC07-523LS was safe and well tolerated, with no antagonistic pharmacokinetic interactions or loss of neutralisation activity. These findings support further evaluation of this combination in future efficacy trials. FUNDING:National Institute of Allergy and Infectious Diseases-National Institutes of Health.

BackgroundGene therapy and hematopoietic stem cell transplantation (HSCT) have transformed outcomes for severe mucopolysaccharidosis type I (MPS I-H), yet a critical unmet need remains. Children with MPS I-H frequently experience progressive skeletal, cardiac, and other complications despite timely HSCT, largely because enzyme activity cannot be safely and precisely titrated over time. Irreversible genetic modification via integrating vectors offers supra-physiological enzyme levels but carries long-term safety and re-dosing liabilities in patients treated early in life.MethodsWe investigated RNA activation (RNAa) as a precision dosing strategy to enhance endogenous IDUA expression without permanent genome alteration. Using MTL-CEBPA, a small activating RNA that upregulates CEBPA transcription factor, we characterized CEBPA–IDUA relationships in vitro, in vivo, and in legacy clinical samples from cancer patients.ResultsCCAAT enhancer binding protein alpha activation consistently increased IDUA mRNA across A549, IMR90, and mesenchymal stem cells. In wild-type mice, two intravenous MTL-CEBPA doses produced a ∼2-fold, durable increase in bone marrow IDUA mRNA and plasma enzyme activity, sustained for up to 4 weeks. In humanized bone marrow–transplanted MPS I-H mice, repeated dosing with MTL-CEBPA led to an approximately 2-fold increase in circulating IDUA activity compared with controls over the 3-weeks treatment period. The largest apparent separation from controls was observed in the homozygous cohort, although these genotype-specific differences should be interpreted cautiously given the limited subgroup sizes. In cancer patient–derived monocytes, increased CEBPA protein levels correlated with higher IDUA levels (R2 = 0.571). Consistent with this, approximately half of evaluable patients exhibited increased plasma IDUA activity following treatment.ConclusionThese translational data demonstrate that MTL-CEBPA delivers controlled, reversible enhancement of IDUA in the context of HSCT, providing robust pharmacodynamic proof-of-concept rather than definitive evidence of durable efficacy. By enabling titratable enzyme elevation without integrating vectors, RNAa therapeutics address a key unmet need in Hurler syndrome: safe fine-tuning of residual enzyme activity over a patient’s lifetime. With scalable, cost-effective oligonucleotide manufacturing, MTL-CEBPA and related RNAa therapeutics represent a clinically relevant adjuvant strategy for HSCT-treated MPS I-H, with potential for other enzyme deficiency disorders.

Triple-negative breast cancer (TNBC) is a form of breast cancer clarified by low expression of estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER2). For this reason, therapeutics aimed at targeting these receptors are ineffective in cases of TNBC, which leads to a poorer prognosis. Consequently, there is a need for novel therapeutics at targeting this subtype. CCAAT/enhancer-binding protein β (C/EBPβ) is a leucine zipper transcription factor with a traditional function in mammary gland development and macrophage differentiation. In tumors, C/EBPβ is associated with metastatic and chemoresistant forms of breast cancer. Previous efforts at targeting this transcription factor in the tumor have been hampered by off-target effects and low penetrance into the intratumoral space. Furthermore, studies into C/EBPβ knockdown in vitro have been mixed, owing in part to two distinct isoforms that are differentially expressed in healthy and cancerous tissues. Given that the function of C/EBPβ is closely tied to hypoxia factors such as hypoxia-inducible factor-1α, we hypothesized that the hypoxic intratumoral space may be driving specific isoform development and consequently the pro-metastatic phenotype observed clinically. To this end, we have developed an aptamer-small interfering RNA (siRNA) conjugate containing a transferrin receptor 1 (TfR1) aptamer (a receptor activated under hypoxic conditions) linked to a C/EBPβ siRNA. We have measured C/EBPβ's suppression of metastasis in traditional cell culture under hypoxic conditions and in vivo. These results point toward a novel approach to the contradictory role of C/EBPβ as a driver and mediator of metastasis and a potential therapeutic for its treatment.

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, multiple variants escaping preexisting immunity emerged, causing reinfections of previously exposed individuals. Here, we used antigenic cartography to analyze patterns of cross-reactivity among 21 variants and 15 groups of human sera obtained after primary infection with 10 different variants or after messenger RNA (mRNA)–1273 or mRNA-1273.351 vaccination. We found antigenic differences among pre-Omicron variants caused by substitutions at spike-protein positions 417, 452, 484, and 501. Quantifying changes in response breadth over time and with additional vaccine doses, our results show the largest increase between 4 weeks and >3 months after a second dose. We found changes in immunodominance of different spike regions, depending on the variant an individual was first exposed to, with implications for variant risk assessment and vaccine-strain selection.

The Antibody Mediated Prevention trials showed that the broadly neutralizing antibody (bnAb) VRC01 prevented acquisition of human immunodeficiency virus-1 (HIV-1) sensitive to VRC01. Using AMP trial data, here we show that the predicted serum neutralization 80% inhibitory dilution titer (PT 80 ) biomarker—which quantifies the neutralization potency of antibodies in an individual’s serum against an HIV-1 isolate—can be used to predict HIV-1 prevention efficacy. Similar to the results of nonhuman primate studies, an average PT 80 of 200 (meaning a bnAb concentration 200-fold higher than that required to reduce infection by 80% in vitro) against a population of probable exposing viruses was estimated to be required for 90% prevention efficacy against acquisition of these viruses. Based on this result, we suggest that the goal of sustained PT 80 >200 against 90% of circulating viruses can be achieved by promising bnAb regimens engineered for long half-lives. We propose the PT 80 biomarker as a surrogate endpoint for evaluation of bnAb regimens, and as a tool for benchmarking candidate bnAb-inducing vaccines.

Cross-Reactive Neutralization of SARS-CoV-2 Variants An analysis of cross-reactive viral binding and neutralization of emerging SARS-CoV-2 variants, modeled with the use of pseudoviruses, suggests ...

The Omicron variant of SARS-CoV-2 is raising concerns because of its increased transmissibility and potential for reduced susceptibility to antibody neutralization. To assess the potential risk of this variant to existing vaccines, serum samples from mRNA-1273 vaccine recipients were tested for neutralizing activity against Omicron and compared to neutralization titers against D614G and Beta in live virus and pseudovirus assays. Omicron was 41-84-fold less sensitive to neutralization than D614G and 5.3-7.4-fold less sensitive than Beta when assayed with serum samples obtained 4 weeks after 2 standard inoculations with 100 μg mRNA-1273. A 50 μg boost increased Omicron neutralization titers and may substantially reduce the risk of symptomatic vaccine breakthrough infections.

All current vaccines for COVID-19 utilize ancestral SARS-CoV-2 spike with the goal of generating protective neutralizing antibodies. The recent emergence and rapid spread of several SARS-CoV-2 variants carrying multiple spike mutations raise concerns about possible immune escape. One variant, first identified in the United Kingdom (B.1.1.7, also called 20I/501Y.V1), contains eight spike mutations with potential to impact antibody therapy, vaccine efficacy, and risk of reinfection. Here, we show that B.1.1.7 remains sensitive to neutralization, albeit at moderately reduced levels (∼sim;2-fold), by serum samples from convalescent individuals and recipients of an mRNA vaccine (mRNA-1273, Moderna) and a protein nanoparticle vaccine (NVX-CoV2373, Novavax). A subset of monoclonal antibodies to the receptor binding domain (RBD) of spike are less effective against the variant, while others are largely unaffected. These findings indicate that variant B.1.1.7 is unlikely to be a major concern for current vaccines or for an increased risk of reinfection.

A SARS-CoV-2 variant carrying the Spike protein amino acid change D614G has become the most prevalent form in the global pandemic. Dynamic tracking of variant frequencies revealed a recurrent pattern of G614 increase at multiple geographic levels: national, regional, and municipal. The shift occurred even in local epidemics where the original D614 form was well established prior to introduction of the G614 variant. The consistency of this pattern was highly statistically significant, suggesting that the G614 variant may have a fitness advantage. We found that the G614 variant grows to a higher titer as pseudotyped virions. In infected individuals, G614 is associated with lower RT-PCR cycle thresholds, suggestive of higher upper respiratory tract viral loads, but not with increased disease severity. These findings illuminate changes important for a mechanistic understanding of the virus and support continuing surveillance of Spike mutations to aid with development of immunological interventions.

Currently, the most effective and durable therapeutic option for HIV-1 infection is combination antiretroviral therapy (cART). Although cART is powerful and can delay viral evolution of drug resistance for decades, it is associated with limitations, including an inability to eradicate the virus and a potential for adverse effects. Therefore, it is imperative to discover new HIV therapeutic modalities. In this study, we designed, characterized, and evaluated the in vitro potency of 2'-deoxy-2 2'-fluoroarabinonucleotide (FANA) modified antisense oligonucleotides (ASOs) targeting highly conserved regions in the HIV-1 genome. Carrier-free cellular internalization of FANA ASOs resulted in strong suppression of HIV-1 replication in HIV-1-infected human primary cells. In vitro mechanistic studies suggested that the inhibitory effect of FANA ASOs can be attributed to RNase H1 activation and steric hindrance of dimerization. Using 5'-RACE PCR and sequencing analysis, we confirmed the presence of human RNase H1-mediated target RNA cleavage products in cells treated with FANA ASOs. We observed no overt cytotoxicity or immune responses upon FANA ASO treatment. Together, our results strongly suggest that FANA ASOs hold great promise for antiretroviral therapy. The dual ability of FANA ASOs to target RNA by recruiting RNase H1 and/or sterically blocking RNA dimerization further enhances their therapeutic potential.

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in high demand. With a close genetic and behavioral resemblance to humans, small animals such as the mouse are good candidates for human disease models, through which human-like symptoms and responses can be recapitulated. Further, the mouse genetic background can be altered to accommodate diverse demands. The NOD/SCID/IL2rγnull (NSG) mouse is one of the most widely used immunocompromised mouse strains; it allows engraftment with human hematopoietic stem cells and/or human tissues and the subsequent development of a functional human immune system. This is a critical milestone in understanding the prognosis and pathophysiology of human-specific diseases such as HIV/AIDS and aiding the search for a cure. Herein, we report a detailed protocol for generating a humanized NSG mouse model (hu-NSG) by hematopoietic stem cell transplantation into a radiation-conditioned neonatal NSG mouse. The hu-NSG mouse model shows multi-lineage development of transplanted human stem cells and susceptibility to HIV-1 viral infection. It also recapitulates key biological characteristics in response to combinatorial antiretroviral therapy (cART).

The HIV-1 infectious cycle requires viral protein interactions with host factors to facilitate viral replication, packaging, and release. The infectious cycle further requires the formation of viral/host protein complexes with HIV-1 RNA to regulate the splicing and enable nucleocytoplasmic transport. The HIV-1 Rev protein accomplishes the nuclear export of HIV-1 mRNAs through multimerization with intronic cis-acting targets - the Rev response element (RRE). A nucleolar localization signal (NoLS) exists within the COOH-terminus of the Rev arginine-rich motif (ARM), allowing the accumulation of Rev/RRE complexes in the nucleolus. Nucleolar factors are speculated to support the HIV-1 infectious cycle through various other functions in addition to mediating mRNA-independent nuclear export and splicing. We describe an immunoprecipitation method of wild-type (WT) Rev in comparison to Rev nucleolar mutations (deletion and single-point Rev-NoLS mutations) in the presence of HIV-1 replication for mass spectrometry. Nucleolar factors implicated in the nucleocytoplasmic transport (nucleophosmin B23 and nucleolin C23), as well as cellular splicing factors, lose interaction with Rev in the presence of Rev-NoLS mutations. Various other nucleolar factors, such as snoRNA C/D box 58, are identified to lose interaction with Rev mutations, yet their function in the HIV-1 replication cycle remain unknown. The results presented here demonstrate the use of this approach for the identification of viral/host nucleolar factors that maintain the HIV-1 infectious cycle. The concepts used in this approach are applicable to other viral and disease models requiring the characterization of understudied pathways.

[This corrects the article DOI: 10.1371/journal.ppat.1007431.].

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in high demand. With a close genetic and behavioral resemblance to humans, small animals such as the mouse are good candidates for human disease models, through which human-like symptoms and responses can be recapitulated. Further, the mouse genetic background can be altered to accommodate diverse demands. The NOD/SCID/IL2rγnull (NSG) mouse is one of the most widely used immunocompromised mouse strains; it allows engraftment with human hematopoietic stem cells and/or human tissues and the subsequent development of a functional human immune system. This is a critical milestone in understanding the prognosis and pathophysiology of human-specific diseases such as HIV/AIDS and aiding the search for a cure. Herein, we report a detailed protocol for generating a humanized NSG mouse model (hu-NSG) by hematopoietic stem cell transplantation into a radiation-conditioned neonatal NSG mouse. The hu-NSG mouse model shows multi-lineage development of transplanted human stem cells and susceptibility to HIV-1 viral infection. It also recapitulates key biological characteristics in response to combinatorial antiretroviral therapy (cART).

The CD4 binding site (CD4bs) of the HIV-1 envelope glycoprotein is susceptible to multiple lineages of broadly neutralizing antibodies (bnAbs) that are attractive to elicit with vaccines. The CH235 lineage (VH1-46) of CD4bs bnAbs is particularly attractive because the most mature members neutralize 90% of circulating strains, do not possess long HCDR3 regions, and do not contain insertions and deletions that may be difficult to induce. We used virus neutralization to measure the interaction of CH235 unmutated common ancestor (CH235 UCA) with functional Env trimers on infectious virions to guide immunogen design for this bnAb lineage. Two Env mutations were identified, one in loop D (N279K) and another in V5 (G458Y), that acted synergistically to render autologous CH505 transmitted/founder virus susceptible to neutralization by CH235 UCA. Man5-enriched N-glycans provided additional synergy for neutralization. CH235 UCA bound with nanomolar affinity to corresponding soluble native-like Env trimers as candidate immunogens. A cryo-EM structure of CH235 UCA bound to Man5-enriched CH505.N279K.G458Y.SOSIP.664 revealed interactions of the antibody light chain complementarity determining region 3 (CDR L3) with the engineered Env loops D and V5. These results demonstrate that virus neutralization can directly inform vaccine design and suggest a germline targeting and reverse engineering strategy to initiate and mature the CH235 bnAb lineage.

Excessive or inappropriate inflammatory responses can cause serious and even fatal diseases. The CCAAT/enhancer-binding protein alpha (CEBPA) gene encodes C/EBP alpha, a transcription factor that plays a fundamental role in controlling maturation of the myeloid lineage and is also expressed during the late phase of inflammatory responses when signs of inflammation are decreasing. MTL-CEBPA, a small activating RNA targeting for upregulation of C/EBP alpha, is currently being evaluated in a phase 1b trial for treatment of hepatocellular carcinoma. After dosing, subjects had reduced levels of pro-inflammatory cytokines, and we therefore hypothesized that MTL-CEBPA has anti-inflammatory potential. The current study was conducted to determine the effects of C/EBP alpha saRNA - CEBPA-51 - on inflammation in vitro and in vivo after endotoxin challenge. CEBPA-51 led to increased expression of the C/EBP alpha gene and inhibition of pro-inflammatory cytokines in THP-1 monocytes previously stimulated by E. coli-derived lipopolysaccharide (LPS). Treatment with MTL-CEBPA in an LPS-challenged humanized mouse model upregulated C/EBP alpha mRNA, increased neutro-phils, and attenuated production of several key pro-inflammatory cytokines, including TNF-alpha, IL-6, IL-1 beta, and IFN-gamma. In addition, a Luminex analysis of mouse serum revealed that MTL-CEBPA reduced pro-inflammatory cytokines and increased the anti-inflammatory cytokine IL-10. Collectively, the data support further investigation of MTL-CEBPA in acute and chronic inflammatory diseases where this mechanism has pathogenic importance.

Combination antiretroviral therapy fails in complete suppression of HIV-1 due to drug resistance and persistent latency. Novel therapeutic intervention requires knowledge of intracellular pathways responsible for viral replication, specifically those untargeted by antiretroviral drugs. An understudied phenomenon is the nucleolar localization of Rev phosphoprotein, which completes nucleocytoplasmic transport of unspliced/partially spliced HIV mRNA through multimerization with intronic cis-acting targetsthe Rev-response element (RRE). Rev contains a nucleolar localization signal (NoLS) comprising the COOH terminus of the arginine-rich motif for accumulation within nucleolispeculated as the interaction ground for Rev with cellular proteins mediating mRNA-independent nuclear export and splicing. Functionality of Rev nucleolar access during HIV-1 production and infection was investigated in the context of deletion and single-point mutations within Rev-NoLS. Mutations induced upon Rev-NoLS are hypothesized to inactivate the HIV-1 infectious cycle. HIV-1(HXB2) replication ceased with Rev mutations lacking nucleolar access due to loss or replacement of multiple arginine residues. Rev mutations missing single arginine residues remained strictly nucleolar in pattern and participated in proviral production, however, with reduced efficiency. Viral RNA packaging also decreased in efficiency after expression of nucleolar-localizing mutations. These results were observed during propagation of variant HIV-1(NL4-3) containing nucleolar-localizing mutations within the viral backbone (M4, M5, and M6). Lentiviral particles produced with Rev single-point mutations were transducible at extremely low frequency. Similarly, HIV-1(NL4-3) Rev-NoLS variants lost infectivity, unlike virulent WT (wild type) HIV-1(NL4-3). HIV-1(NL4-3) variants were capable of CD4(+) host entry and reverse transcription as WT HIV-1(NL4-3), but lacked ability to complete a full infectious cycle. We currently reveal that viral integration is deregulated in the presence of Rev-NoLS mutations.