Lactobacilli Expressing Broadly Neutralizing Nanobodies against HIV-1 as Potential Vectors for HIV-1 Prophylaxis?

In the absence of an active prophylactic vaccine against HIV-1, passively administered, broadly neutralizing antibodies (bnAbs) identified in some chronically infected persons were shown to prevent HIV-1 infection in animal models. However, passive administration of bnAbs may not be suited to prevent sexual HIV-1 transmission in high-risk cohorts, as a continuous high level of active bnAbs may be difficult to achieve at the primary site of sexual transmission, the human vagina with its acidic pH. Therefore, we used Lactobacillus, a natural commensal in the healthy vaginal microbiome, to express bn nanobodies (VHH) against HIV-1 that we reported previously. After demonstrating that recombinant VHHA6 expressed in E. coli was able to protect humanized mice from mucosal infection by HIV-1Bal, we expressed VHHA6 in a soluble or in a cell-wall-anchored form in Lactobacillus rhamnosus DSM14870. This strain is already clinically applied for treatment of bacterial vaginosis. Both forms of VHHA6 neutralized a set of primary epidemiologically relevant HIV-1 strains in vitro. Furthermore, VHHA6 was still active at an acidic pH. Thus, lactobacilli expressing bn VHH potentially represent an attractive vector for the passive immunization of women in cohorts at high risk of HIV-1 transmission.

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New SHIVs and Improved Design Strategy for Modeling HIV-1 Transmission, Immunopathogenesis, Prevention and Cure

Journal of Virology ◽

10.1128/jvi.00071-21 ◽

2021 ◽

Author(s):

Hui Li ◽

Shuyi Wang ◽

Fang-Hua Lee ◽

Ryan S. Roark ◽

Alex I. Murphy ◽

...

Keyword(s):

T Cells ◽

Neutralizing Antibodies ◽

Large Scale ◽

Rhesus Macaques ◽

Passive Immunization ◽

Design Strategy ◽

Efficient Replication ◽

Hiv 1

Previously, we showed that substitution of HIV-1 Env residue 375-Ser by bulky aromatic residues enhances binding to rhesus CD4 and enables primary HIV-1 Envs to support efficient replication as simian-human immunodeficiency virus (SHIV) chimeras in rhesus macaques (RMs). Here, we test this design strategy more broadly by constructing SHIVs containing ten primary Envs corresponding to HIV-1 subtypes A, B, C, AE and AG. All ten SHIVs bearing wildtype Env375 residues replicated efficiently in human CD4+ T cells, but only one replicated efficiently in primary rhesus cells. This was a subtype AE SHIV that naturally contained His at Env375. Replacement of wildtype Env375 residues by Trp, Tyr, Phe or His in the other nine SHIVs led to efficient replication in rhesus CD4+ T cells in vitro and in vivo. Nine SHIVs containing optimized Env375 alleles were grown large-scale in primary rhesus CD4+ T cells to serve as challenge stocks in preclinical prevention trials. These virus stocks were genetically homogeneous, native-like in Env antigenicity and tier-2 neutralization sensitivity, and transmissible by rectal, vaginal, penile, oral or intravenous routes. To facilitate future SHIV constructions, we engineered a simplified second-generation design scheme and validated it in RMs. Overall, our findings demonstrate that SHIVs bearing primary Envs with bulky aromatic substitutions at Env375 consistently replicate in RMs, recapitulating many features of HIV-1 infection in humans. Such SHIVs are efficiently transmitted by mucosal routes common to HIV-1 infection and can be used to test vaccine efficacy in preclinical monkey trials. Importance SHIV infection of Indian rhesus macaques is an important animal model for studying HIV-1 transmission, prevention, immunopathogenesis and cure. Such research is timely, given recent progress with active and passive immunization and novel approaches to HIV-1 cure. Given the multifaceted roles of HIV-1 Env in cell tropism and virus entry, and as a target for neutralizing and non-neutralizing antibodies, Envs selected for SHIV construction are of paramount importance. Until recently, it has been impossible to strategically design SHIVs bearing clinically relevant Envs that replicate consistently in monkeys. This changed with the discovery that bulky aromatic substitutions at residue Env375 confer enhanced affinity to rhesus CD4. Here, we show that 10 new SHIVs bearing primary HIV-1 Envs with residue 375 substitutions replicated efficiently in RMs and could be transmitted efficiently across rectal, vaginal, penile and oral mucosa. These findings suggest an expanded role for SHIVs as a model of HIV-1 infection.

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Evaluation of HIV-1 latency reversal and antibody-dependent viral clearance by quantification of singly spliced HIV-1 vpu/env mRNA

Journal of Virology ◽

10.1128/jvi.02124-20 ◽

2021 ◽

Author(s):

Hongbo Gao ◽

Ayşe N. Ozantürk ◽

Qiankun Wang ◽

Gray H. Harlan ◽

Aaron J. Schmitz ◽

...

Keyword(s):

T Cells ◽

Neutralizing Antibodies ◽

Ex Vivo ◽

Viral Envelope ◽

Latent Reservoir ◽

Broadly Neutralizing Antibodies ◽

Major Barrier ◽

Hiv 1 ◽

Reversing Agents

The latent reservoir of HIV-1 is a major barrier for viral eradication. Potent HIV-1 broadly neutralizing antibodies (bNabs) have been used to prevent and treat HIV-1 infections in animal models and clinical trials. Combination of bNabs and latency-reversing agents (LRAs) is considered a promising approach for HIV-1 eradication. PCR-based assays that can rapidly and specifically measure singly spliced HIV-1 vpu/env mRNA are needed to evaluate the induction of the viral envelope production at the transcription level and bNab-mediated reservoir clearance. Here we reported a PCR-based method to accurately quantify the production of intracellular HIV-1 vpu/env mRNA. With the vpu/env assay, we determined the LRA combinations that could effectively induce vpu/env mRNA production in CD4+ T cells from ART-treated individuals. None of the tested LRAs were effective alone. A comparison between the quantitative viral outgrowth assay (Q-VOA) and the vpu/env assay showed that vpu/env mRNA production was closely associated with the reactivation of replication-competent HIV-1, suggesting that vpu/env mRNA was mainly produced by intact viruses. Finally, antibody-mediated in vitro killing in HIV-1-infected humanized mice demonstrated that the vpu/env assay could be used to measure the reduction of infected cells in tissues and was more accurate than the commonly used gag-based PCR assay which measured unspliced viral genomic RNA. In conclusion, the vpu/env assay allows convenient and accurate assessment of HIV-1 latency reversal and bNab-mediated therapeutic strategies. Importance HIV-1 persists in individuals on antiretroviral therapy (ART) due to the long-lived cellular reservoirs that contain dormant viruses. Recent discoveries of HIV-1-specific broadly neutralizing antibodies (bNabs) targeting HIV-1 Env protein rekindled the interest in antibody-mediated elimination of latent HIV-1. Latency-reversing agents (LRAs) together with HIV-1 bNabs is a possible strategy to clear residual viral reservoirs, which makes the evaluation of HIV-1 Env expression upon LRA treatment critical. We developed a PCR-based assay to quantify the production of intracellular HIV-1 vpu/env mRNA. Using patient CD4+ T cells, we found that induction of HIV-1 vpu/env mRNA required a combination of different LRAs. Using in vitro, ex vivo and humanized mouse models, we showed that the vpu/env assay could be used to measure antibody efficacy in clearing HIV-1 infection. These results suggest that the vpu/env assay can accurately evaluate HIV-1 reactivation and bNab-based therapeutic interventions.

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HTLV-II down-regulates HIV-1 replication in IL-2–stimulated primary PBMC of coinfected individuals through expression of MIP-1α

Blood ◽

10.1182/blood.v95.9.2760.009k04_2760_2769 ◽

2000 ◽

Vol 95 (9) ◽

pp. 2760-2769 ◽

Cited By ~ 30

Author(s):

Claudio Casoli ◽

Elisa Vicenzi ◽

Andrea Cimarelli ◽

Giacomo Magnani ◽

Paolo Ciancianaini ◽

...

Keyword(s):

T Cells ◽

Neutralizing Antibodies ◽

Ex Vivo ◽

Interleukin 2 ◽

Peripheral Blood Mononuclear ◽

Human T Cell ◽

Cc Chemokines ◽

Kinetics Of ◽

Hiv 1

The influence of human T-cell leukemia/lymphoma virus type II (HTLV-II) in individuals also infected with HIV-1 is poorly understood. To evaluate the reciprocal influence of HTLV-II and HIV-1 infection, primary peripheral blood mononuclear cell (PBMC) cultures from coinfected individuals were established in the presence of interleukin 2 (IL-2). In these cultures, the kinetics of HTLV-II replication always preceded those of HIV-1. Noteworthy, the kinetics of HIV-1 production were inversely correlated to the HTLV-II proviral load in vivo and its replication ex vivo. These observations suggested a potential interaction between the 2 retroviruses. In this regard, the levels of IL-2, IL-6, and tumor necrosis factor- (TNF-) were measured in the same coinfected PBMC cultures. Endogenous IL-2 was not produced, whereas IL-6 and TNF- were secreted at levels compatible with their known ability to up-regulate HIV-1 expression. The HIV-suppressive CC-chemokines RANTES, macrophage inflammatory protein-1 (MIP-1), and MIP-1β were also determined in IL-2–stimulated PBMC cultures. Of interest, their kinetics and concentrations were inversely related to those of HIV-1 replication. Experiments were performed in which CD8+ T cells or PBMCs from HTLV-II monoinfected individuals were cocultivated with CD4+ T cells from HIV-1 monoinfected individuals separated by a semipermeable membrane in the presence or absence of antichemokine neutralizing antibodies. The results indicate that HTLV-II can interfere with the replicative potential of HIV-1 by up-regulating viral suppressive CC-chemokines and, in particular, MIP-1. This study is the first report indicating that HTLV-II can influence HIV replication, at least in vitro, via up-regulation of HIV-suppressive chemokines.

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Autologous IgG antibodies block outgrowth of a substantial but variable fraction of viruses in the latent reservoir for HIV-1

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2020617117 ◽

2020 ◽

Vol 117 (50) ◽

pp. 32066-32077

Author(s):

Lynn N. Bertagnolli ◽

Joseph Varriale ◽

Sarah Sweet ◽

Jacqueline Brockhurst ◽

Francesco R. Simonetti ◽

...

Keyword(s):

T Cell Activation ◽

Neutralizing Antibodies ◽

Cell Activation ◽

Viral Evolution ◽

Treatment Interruption ◽

Latent Reservoir ◽

Env Protein ◽

Autologous Antibodies ◽

Hiv 1

In untreated HIV-1 infection, rapid viral evolution allows escape from immune responses. Viral replication can be blocked by antiretroviral therapy. However, HIV-1 persists in a latent reservoir in resting CD4+ T cells, and rebound viremia occurs following treatment interruption. The reservoir, which is maintained in part by clonal expansion, can be measured using quantitative viral outgrowth assays (QVOAs) in which latency is reversed with T cell activation to allow viral outgrowth. Recent studies have shown that viruses detected in QVOAs prior to treatment interruption often differ from rebound viruses. We hypothesized that autologous neutralizing antibodies directed at the HIV-1 envelope (Env) protein might block outgrowth of some reservoir viruses. We modified the QVOA to reflect pressure from low concentrations of autologous antibodies and showed that outgrowth of a substantial but variable fraction of reservoir viruses is blocked by autologous contemporaneous immunoglobulin G (IgG). A reduction in outgrowth of >80% was seen in 6 of 15 individuals. This effect was due to direct neutralization. We established a phylogenetic relationship between rebound viruses and viruses growing out in vitro in the presence of autologous antibodies. Some large infected cell clones detected by QVOA carried neutralization-sensitive viruses, providing a cogent explanation for differences between rebound virus and viruses detected in standard QVOAs. Measurement of the frequency of reservoir viruses capable of outgrowth in the presence of autologous IgG might allow more accurate prediction of time to viral rebound. Ultimately, therapeutic immunization targeting the subset of variants resistant to autologous IgG might contribute to a functional cure.

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A Novel MVA-Based HIV Vaccine Candidate (MVA-gp145-GPN) Co-Expressing Clade C Membrane-Bound Trimeric gp145 Env and Gag-Induced Virus-Like Particles (VLPs) Triggered Broad and Multifunctional HIV-1-Specific T Cell and Antibody Responses

Viruses ◽

10.3390/v11020160 ◽

2019 ◽

Vol 11 (2) ◽

pp. 160 ◽

Cited By ~ 4

Author(s):

Beatriz Perdiguero ◽

Cristina Sánchez-Corzo ◽

Carlos Sorzano ◽

Lidia Saiz ◽

Pilar Mediavilla ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Vaccine Candidate ◽

Hiv Vaccine ◽

Replication Capacity ◽

Virus Like Particles ◽

Modified Vaccinia Virus Ankara ◽

Clade C ◽

Membrane Bound ◽

Hiv 1

The development of an effective Human Immunodeficiency Virus (HIV) vaccine that is able to stimulate both the humoral and cellular HIV-1-specific immune responses remains a major priority challenge. In this study, we described the generation and preclinical evaluation of single and double modified vaccinia virus Ankara (MVA)-based candidates expressing the HIV-1 clade C membrane-bound gp145(ZM96) trimeric protein and/or the Gag(ZM96)-Pol-Nef(CN54) (GPN) polyprotein that was processed to form Gag-induced virus-like particles (VLPs). In vitro characterization of MVA recombinants revealed the stable integration of HIV-1 genes without affecting its replication capacity. In cells that were infected with Env-expressing viruses, the gp145 protein was inserted into the plasma membrane exposing critical epitopes that were recognized by broadly neutralizing antibodies (bNAbs), whereas Gag-induced VLPs were released from cells that were infected with GPN-expressing viruses. VLP particles as well as purified MVA virions contain Env and Gag visualized by immunoelectron microscopy and western-blot of fractions that were obtained after detergent treatments of purified virus particles. In BALB/c mice, homologous MVA-gp145-GPN prime/boost regimen induced broad and polyfunctional Env- and Gag-specific CD4 T cells and antigen-specific T follicular helper (Tfh) and Germinal Center (GC) B cells, which correlated with robust HIV-1-specific humoral responses. Overall, these results support the consideration of MVA-gp145-GPN vector as a potential vaccine candidate against HIV-1.

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Vaccines and Broadly Neutralizing Antibodies for HIV-1 Prevention

Annual Review of Immunology ◽

10.1146/annurev-immunol-080219-023629 ◽

2020 ◽

Vol 38 (1) ◽

pp. 673-703 ◽

Cited By ~ 12

Author(s):

Kathryn E. Stephenson ◽

Kshitij Wagh ◽

Bette Korber ◽

Dan H. Barouch

Keyword(s):

Clinical Trials ◽

Neutralizing Antibodies ◽

Passive Immunization ◽

Neutralizing Antibody ◽

Broadly Neutralizing Antibodies ◽

Aids Pandemic ◽

Broadly Neutralizing Antibody ◽

Early Phase Clinical Trials ◽

Hiv 1 ◽

Review Current

Development of improved approaches for HIV-1 prevention will likely be required for a durable end to the global AIDS pandemic. Recent advances in preclinical studies and early phase clinical trials offer renewed promise for immunologic strategies for blocking acquisition of HIV-1 infection. Clinical trials are currently underway to evaluate the efficacy of two vaccine candidates and a broadly neutralizing antibody (bNAb) to prevent HIV-1 infection in humans. However, the vast diversity of HIV-1 is a major challenge for both active and passive immunization. Here we review current immunologic strategies for HIV-1 prevention, with a focus on current and next-generation vaccines and bNAbs.

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Genomic and Functional Analysis of Emerging Virulent and Multidrug-Resistant Escherichia coli Lineage Sequence Type 648

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00243-19 ◽

2019 ◽

Vol 63 (6) ◽

Cited By ~ 17

Author(s):

Katharina Schaufler ◽

Torsten Semmler ◽

Lothar H. Wieler ◽

Darren J. Trott ◽

Johann Pitout ◽

...

Keyword(s):

Escherichia Coli ◽

High Risk ◽

Multidrug Resistant ◽

Phenotypic Analysis ◽

Content Type ◽

Extended Spectrum Beta Lactamase ◽

Niche Adaptation ◽

High Level

ABSTRACT The pathogenic extended-spectrum-beta-lactamase (ESBL)-producing Escherichia coli lineage ST648 is increasingly reported from multiple origins. Our study of a large and global ST648 collection from various hosts (87 whole-genome sequences) combining core and accessory genomics with functional analyses and in vivo experiments suggests that ST648 is a nascent and generalist lineage, lacking clear phylogeographic and host association signals. By including large numbers of ST131 (n = 107) and ST10 (n = 96) strains for comparative genomics and phenotypic analysis, we demonstrate that the combination of multidrug resistance and high-level virulence are the hallmarks of ST648, similar to international high-risk clonal lineage ST131. Specifically, our in silico, in vitro, and in vivo results demonstrate that ST648 is well equipped with biofilm-associated features, while ST131 shows sophisticated signatures indicative of adaption to urinary tract infection, potentially conveying individual ecological niche adaptation. In addition, we used a recently developed NFDS (negative frequency-dependent selection) population model suggesting that ST648 will increase significantly in frequency as a cause of bacteremia within the next few years. Also, ESBL plasmids impacting biofilm formation aided in shaping and maintaining ST648 strains to successfully emerge worldwide across different ecologies. Our study contributes to understanding what factors drive the evolution and spread of emerging international high-risk clonal lineages.

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Impact of Y143 HIV-1 Integrase Mutations on Resistance to Raltegravir In Vitro and In Vivo

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01075-09 ◽

2009 ◽

Vol 54 (1) ◽

pp. 491-501 ◽

Cited By ~ 51

Author(s):

Olivier Delelis ◽

Sylvain Thierry ◽

Frédéric Subra ◽

Françoise Simon ◽

Isabelle Malet ◽

...

Keyword(s):

Viral Genome ◽

Effective Concentration ◽

Coding Region ◽

Delayed Emergence ◽

Infected Cells ◽

High Level ◽

Emergence Of Resistance ◽

Hiv 1

ABSTRACT Integrase (IN), the HIV-1 enzyme responsible for the integration of the viral genome into the chromosomes of infected cells, is the target of the recently approved antiviral raltegravir (RAL). Despite this drug's activity against viruses resistant to other antiretrovirals, failures of raltegravir therapy were observed, in association with the emergence of resistance due to mutations in the integrase coding region. Two pathways involving primary mutations on residues N155 and Q148 have been characterized. It was suggested that mutations at residue Y143 might constitute a third primary pathway for resistance. The aims of this study were to investigate the susceptibility of HIV-1 Y143R/C mutants to raltegravir and to determine the effects of these mutations on the IN-mediated reactions. Our observations demonstrate that Y143R/C mutants are strongly impaired for both of these activities in vitro. However, Y143R/C activity can be kinetically restored, thereby reproducing the effect of the secondary G140S mutation that rescues the defect associated with the Q148R/H mutants. A molecular modeling study confirmed that Y143R/C mutations play a role similar to that determined for Q148R/H mutations. In the viral replicative context, this defect leads to a partial block of integration responsible for a weak replicative capacity. Nevertheless, the Y143 mutant presented a high level of resistance to raltegravir. Furthermore, the 50% effective concentration (EC50) determined for Y143R/C mutants was significantly higher than that obtained with G140S/Q148R mutants. Altogether our results not only show that the mutation at position Y143 is one of the mechanisms conferring resistance to RAL but also explain the delayed emergence of this mutation.

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